The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

Recombinant human IL-37 inhibited endometriosis development in a mouse model through increasing Th1/Th2 ratio by inducing the maturation of dendritic cells.

BACKGROUND: Endometriosis is a serious reproductive and general health consequences. Recombinant human IL-37 (rhIL-37) is an inhibitor of inflammation. METHODS: ELISA assay was performed to detect the concentration of cytokines. Flow cytometry was used to analyze cell proportion. Besides, qRT-PCR and western blotting assay were used to detect the level of gene and protein, respectively. Transwell co-culture system was used for the co-culture of dendritic cells (DCs) and CD4+T cells. RESULTS: Our data showed that rhIL-37 inhibited the development of ectopic lesions in the mice with endometriosis, increased Th1/Th2 ratio and induced DCs maturation. The co-culture system of DCs and CD4+T cells demonstrated that rhIL-37 increased Th1/Th2 cell ratio through promoting DCs maturation. Moreover, the expression of IL-4 in the DCs derived from healthy mice was inhibited by rhIL-37 treatment. rhIL-37 increased Th1/Th2 cell ratio through inhibiting IL-4 in DCs. Subsequently, our results proved that rhIL-37 promoted the maturation of DCs via inhibiting phosphorylation of STAT3. Activation of STAT3 could reverse rhIL-37-induced maturation of DCs. CONCLUSION: Overall, rhIL-37 could protect against endometriosis through increasing the ratio of Th1/Th2 cells via inducing DCs maturation and inhibiting IL-4 expression in the DCs. Furthermore, rhIL-37 induced DCs maturation by inhibiting STAT3 phosphorylation. Our data confirmed the protective effect of rhIL-37 in endometriosis. These data may provide a novel idea for the treatment of the disease.

Histopathology and ARID1A Expression in Endometriosis- Associated Ovarian Carcinoma (EAOC) Carcinogenesis Model with Endometrial Autoimplantation and DMBA Induction.

BACKGROUND: Ovarian carcinoma is one of the most deadly malignancies in the gynecologic field. The cause is not yet known, and the clinical symptoms are not specific. Endometrioid carcinoma and ovarian clear cell carcinoma can originate from endometriosis and are known as endometriosis-related ovarian carcinoma (EAOC). Development of EAOC experimental animal models is needed for basic research and clinical preparation of human tissue tests. This study aimed to determine the role of the ARID1A gene mutation in the carcinogenetic process of EAOC in experimental animal models induced with DMBA. METHODS: In this study, the EAOC experimental model was developed using the autoimplantation technique and DMBA induction. This study involved placebo surgery mice (sham), endometrial autoimplantation, and a combination of endometrial autoimplantation and DMBA induction, which were sacrificed at weeks 5, 10, and 20, respectively. Histopathological assessment and immunohistochemical ARID1A staining with an assessment of positive percentages were carried out on 200 cells. RESULTS: This study produced 1 (20%) atypical endometriosis and 1 (20%) clear cell carcinoma at implantation and after 10 weeks of DMBA induction, and 100% endometrioid carcinoma in the DMBA-induced group. ARID1A staining did not show any significant difference (p = 0.313) in all groups. CONCLUSION: The combination of endometrial autoimplantation techniques and DMBA induction in the ovary produced atypical endometriosis, clear cell carcinoma, and endometrioid carcinoma, where time is an important factor. There was no significant difference in ARID1A expression between the treatment and control groups.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Endometrial autotransplantation in rabbits: Potential for fertility restoration in severe Asherman's syndrome.

OBJECTIVE: Uterine transplantation is now considered a feasible treatment for women with absolute uterine factor infertility and has been successfully performed for a woman with Asherman's syndrome (AS). The endometrium is a clinically and histologically distinct entity from the surrounding myometrium. Endometrial transplantation (ETx) may offer a less invasive option, with less immunogenic impact, to restore fertility in women with severe AS. The objective of this study was to assess the feasibility of ETx by evaluating surgical and reproductive outcomes following endometrial autotransplantation in a rabbit model. STUDY DESIGN: A longitudinal study assessing surgical, biochemical, radiological, reproductive and histological outcomes following endometrial autotransplantation in ten New Zealand white rabbits. RESULTS: Ten procedures were performed, including 8 endometrial auto-transplants (ETx) and 2 endometrial resections (ER), to control against endometrial regeneration. Eight procedures were successful, whereas two rabbits from the ETx group died intra-operatively. Three rabbits were euthanised at 48, 72 and 96 h post-operatively to assess gross and histological appearances. Two rabbits, one from the ETx group and one from the ER group, died four weeks and eight weeks post-operatively. Three rabbits subsequently underwent two cycles of in-vitro fertilization. The first cycle resulted in an implantation rate of 57% in the un-operated uteri. In two rabbits who underwent ETx, an implantation rate of 28.6% was seen. In the second cycle, an implantation rate of 61.9 % (13 implantations) was observed in the control uteri. In the two ETx females, an implantation rate of 14.3 % was seen. No pregnancies were seen in either cycle in the animals who underwent ER. Despite successful implantations in both cycles in the ETx rabbits, no livebirths were achieved. Following death or euthanasia there was gross and microscopic evidence of viable endometrium following ETx, but not following ER. CONCLUSION: This study has revealed, for the first time, the feasibility of ETx with gross and microscopic evidence of viable endometrium, and the demonstration of clinical pregnancies. Whilst further studies are essential, and the achievement of successful livebirths fundamental, ETx may offer a potential fertility restoring opportunity for women with severe, treatment refractory cases of AS.

Inhibition of KIF20A by BKS0349 reduces endometriotic lesions in a xenograft mouse model.

Several studies have suggested a possible etiological association between ovarian endometriosis and ovarian cancer. Evidence has shown that KIF20A overexpression might confer a malignant phenotype to ovarian tumors by promoting proliferation and inhibiting apoptosis. However, no data about the role of KIF20A in endometriosis have been described. In this study, the human endometrium (n = 4) was transfected by mCherry adenovirus and intraperitoneally implanted in mice. Subsequently, mice were divided in three groups (n = 8/group) that were treated with Vehicle, BKS0349 (KIF20A-antagonist) or cabergoline (dopamine receptor agonist) for 21 days. mCherry-labeled endometriotic lesions were monitored over time using the IVIS Imaging System. Mice were sacrificed 72 h after the last administration; proliferation was evaluated by immunohistochemistry and apoptosis by TUNEL. CCND1 gene expression (G1 phase-related gene) was measured by qRT-PCR. A significant reduction in mCherry-fluorescent signal was observed in the BKS0349 group after treatment ended (D24) compared with D0 (P-value = 0.0313). Moreover, the mCherry signal on D24 showed a significant decrease in the BKS0349 group compared with controls (P-value = 0.0303), along with significant size reduction of endometriotic lesions observed in the BKS0349 group compared with control on D24 (P-value = 0.0006). Functional studies showed a significant reduction in proliferating cells in the BKS0349-treated group compared with controls (P-value = 0.0082). In addition, CCND1 expression was decreased in the BKS0349 group compared with control (P-value = 0.049) at D24 and a significant increase in apoptotic cells among endometriotic lesions in BKS0349-treated mice was observed compared with control (P-value = 0.0317). Based on these findings, we concluded that BKS0349 induces apoptosis and inhibits cell proliferation, reducing endometriotic lesion size and suggesting KIF20A inhibition by BKS0349 as a novel therapeutic treatment for endometriosis.

Dose-Dependent Decreased Fertility in Response to the Burden of Endometriosis in a Murine Model.

Endometriosis is a gynecological disease caused by the growth of endometrial cells outside the uterus leading to inflammation, pelvic pain, and infertility. The relationship between the amount of ectopic uterine tissue growth and the severity of symptoms is still unclear. The presence or degree of pain and infertility does not correlate with the stage of disease as currently defined. Here, we report a clear dose-response relationship between the amount of ectopic tissue transplanted and the reproductive outcomes in a murine model of endometriosis. Endometriosis was induced in mice using various amounts of transplanted uterine tissue. Four groups of mice consisted of a sham surgery control or those transplanted with 1, 2, or 4 endometrial segments of 5 mm each. Pregnancy rates were significantly lower in those transplanted with 2 or 4 segments compared to sham or the 1 segment groups. We demonstrate that infertility does correlate with the extent of active disease. Current clinical staging systems do not account for disease activity and may inappropriately weight sequela of disease. Early recognition and treatment in women may help to minimize the effect of endometriosis on fertility. Here, we describe a mouse model of endometriosis and infertility that may be useful to elucidate the mechanisms of infertility in endometriosis.

Human predecidual stromal cells are mesenchymal stromal/stem cells and have a therapeutic effect in an immune-based mouse model of recurrent spontaneous abortion.

BACKGROUND: Human decidual stromal cells (DSCs) are involved in the maintenance and development of pregnancy, in which they play a key role in the induction of immunological maternal-fetal tolerance. Precursors of DSCs (preDSCs) are located around the vessels, and based on their antigen phenotype, previous studies suggested a relationship between preDSCs and mesenchymal stromal/stem cells (MSCs). This work aimed to further elucidate the MSC characteristics of preDSCs. METHODS: We established 15 human preDSC lines and 3 preDSC clones. Physiological differentiation (decidualization) of these cell lines and clones was carried out by in vitro culture with progesterone (P4) and cAMP. Decidualization was confirmed by the change in cellular morphology and prolactin (PRL) secretion, which was determined by enzyme immunoassay of the culture supernatants. We also studied MSC characteristics: (1) In mesenchymal differentiation, under appropriate culture conditions, these preDSC lines and clones differentiated into adipocytes, osteoblasts, and chondrocytes, and differentiation was confirmed by cytochemical assays and RT-PCR. (2) The expression of stem cell markers was determined by RT-PCR. (3) Cloning efficiency was evaluated by limited dilution. (4) Immunoregulatory activity in vivo was estimated in DBA/2-mated CBA/J female mice, a murine model of immune-based recurrent abortion. (5) Survival of preDSC in immunocompetent mice was analyzed by RT-PCR and flow cytometry. RESULTS: Under the effect of P4 and cAMP, the preDSC lines and clones decidualized in vitro: the cells became rounder and secreted PRL, a marker of physiological decidualization. PreDSC lines and clones also exhibited MSC characteristics. They differentiated into adipocytes, osteoblasts, and chondrocytes, and preDSC lines expressed stem cell markers OCT-4, NANOG, and ABCG2; exhibited a cloning efficiency of 4 to 15%; significantly reduced the embryo resorption rate (P < 0.001) in the mouse model of abortion; and survived for prolonged periods in immunocompetent mice. The fact that 3 preDSC clones underwent both decidualization and mesenchymal differentiation shows that the same type of cell exhibited both DSC and MSC characteristics. CONCLUSIONS: Together, our results confirm that preDSCs are decidual MSCs and suggest that these cells are involved in the mechanisms of maternal-fetal immune tolerance.

VEGF Receptor 1-Expressing Macrophages Recruited from Bone Marrow Enhances Angiogenesis in Endometrial Tissues.

Angiogenesis is critical in maintenance of endometrial tissues. Here, we examined the role of VEGF receptor 1 (VEGFR1) signaling in angiogenesis and tissue growth in an endometriosis model. Endometrial fragments were implanted into the peritoneal wall of mice, and endometrial tissue growth and microvessel density (MVD) were determined. Endometrial fragments from wild-type (WT) mice grew slowly with increased angiogenesis determined by CD31+ MVD, peaking on Day 14. When tissues from WT mice were transplanted into VEGFR1 tyrosine kinase-knockout mice, implant growth and angiogenesis were suppressed on Day 14 compared with growth of WT implants in a WT host. The blood vessels in the implants were not derived from the host peritoneum. Immunostaining for VEGFR1 suggested that high numbers of VEGFR1+ cells such as macrophages were infiltrated into the endometrial tissues. When macrophages were deleted with Clophosome N, both endometrial tissue growth and angiogenesis were significantly suppressed. Bone marrow chimera experiments revealed that growth and angiogenesis in endometrial implants were promoted by host bone marrow-derived VEGFR1+/CD11b+ macrophages that accumulated in the implants, and secreted basic fibroblast growth factor (bFGF). A FGF receptor kinase inhibitor, PD173047 significantly reduced size of endometrial tissues and angiogenesis. VEGFR1 signaling in host-derived cells is crucial for growth and angiogenesis in endometrial tissue. Thus, VEGFR1 blockade is a potential treatment for endometriosis.

Macrophages infiltrating endometriosis-like lesions exhibit progressive phenotype changes in a heterologous mouse model.

Endometriotic lesion development involves complex interactions between endometrial tissue, the peritoneum and immune cells. Macrophages are essential in this process; however their precise roles are not defined. To investigate whether infiltrating macrophages acquire functionally different phenotypes during lesion development, human endometrial tissues were grafted into immunodeficient mice expressing macrophage-specific green fluorescent protein (GFP). Although the numbers of GFP-positive macrophages were similar in lesions 4, 7, 10 and 14 days after grafting, their surface markers changed over time. Inflammatory markers MHC class II (MHC II) and iNOS were present on 36% and 41% of macrophages respectively early in lesion development at day 4, whereas abundance of tissue remodelling markers peaked later, with arginase 1 most highly expressed on 57% of macrophages at day 7 and scavenger receptor A (CD204) on 66% of macrophages at day 14. This is consistent with a transition from classical M1 macrophage activity to an alternate M2 profile, which correlates to histological hallmarks of initially acute inflammation followed by tissue remodelling during lesion development. This progressive shift in phenotype is likely to be relevant to the mechanisms by which macrophages are central players in endometriosis-like lesion development.

Experimental Endometriosis: Review of the Literature Through a Century and the Iranian Experience.

Although endometriosis is not a newly discovered disease, it has still remained enigmatic. Understanding the pathophysiology of this disease is still one of the challenges of gynecology and reproductive medicine. Therefore, the subject of endometriosis treatment faces many questions to be answered. Even though this is not considered to be a rare disease, testing novel treatments on subjects is definitely contrary to medical ethics. This is where importance of animal studies is emphasized. History of animal studies on endometriosis dates back to the time when the young John Sampson presented his theory of retrograde flow of menstruation to the gynecologic society of his time to explain the mechanism of endometriosis. However, most of the medical society of that time favored metaplasia as the main mechanism over the new theory. In order to prove Sampson's theory, animal models were used to induce endometriosis, and that was the first study of experimental endometriosis. Nowadays, although no one uses animals to evaluate Sampson theory of endometriosis, however, experimental endometriosis is widely used to study many different aspects of the disease from pathogenesis to possible options for treatments. Also, since then, various animals and different techniques have been proposed and so there is a huge body of literature on experimental endometriosis. Contrary to many countries, Iranian medical societies have neglected animal models for endometriosis until recently. This review article aims to go through the prominent articles on the subject and introduce different animals and methods to its readers and have a special look at Iranian literature on experimental endometriosis.

Endometrial regeneration using cell sheet transplantation techniques in rats facilitates successful fertilization and pregnancy.

OBJECTIVE: To regenerate functional endometrium tissue using "cell sheet" techniques as a regenerative medicine approach to address endometrial disorders causing female factor infertility. DESIGN: In vivo experimental study. SETTING: Preclinical surgical and biomedical research laboratories. ANIMAL(S): Green fluorescent protein (GFP) transgenic rats [SD-Tg (CAG-EGFP) rats] and nude rats (F344/NJcl-rnu/rnu). INTERVENTION(S): GFP-positive rat uterine-derived cells as cell sheets were transplanted into resected rat uterine endometrial sites. Transplanted cell sheet areas were then analyzed using macroscopic observations and histological analysis including immunohistochemistry. Subsequently, crossbreeding was performed to establish fertility and confirm pregnancy in the rat-regenerated uterus. MAIN OUTCOME MEASURE(S): Morphologic and biochemical markers of regenerated endometrium and establishment of pregnancy in otherwise sterile animals. RESULT(S): After cell sheet transplantation, regenerated endometrium was confirmed as GFP-positive tissue engraftment both visually and under histological analysis. After crossbreeding, GFP-positive tissue areas and living fetuses were observed in the transplantation group. CONCLUSION(S): Cell sheet transplantation can regenerate endometrial tissue with histological structure and physiological function supporting pregnancy similar to normal endometrial tissue. Translation of this endometrial cell sheet transplantation method to human patients with endometrial disorders could yield a novel therapy for uterine infertility.

The Fate of Autologous Endometrial Mesenchymal Stromal Cells After Application in the Healthy Equine Uterus.

Because of their distinct differentiation, immunomodulatory, and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relation to the uterus but also for regeneration of other tissues. This study reports the fate of endometrial MSCs following intrauterine application in mares. Stromal cell fractions were isolated from endometrial biopsies taken from seven reproductively healthy mares, expanded, and fluorescence labeled in culture. Phosphate-buffered saline (PBS) or MSCs (15 × 106) were autologously infused into each uterine horn during early diestrus and subsequently tracked by fluorescence microscopy and flow cytometry of endometrial biopsies and blood samples taken periodically after infusion. The inflammatory response to cell infusion was monitored in endometrial cytology samples. MSCs were detected in endometrial sections at 6, 12, and 24 h, but not later (7 or 14 days), after cell infusion. Cells were in all cases located in the uterine lumen, never within the endometrial tissue. No fluorescence signal was detected in blood samples at any time point after infusion. Cytology analyses showed an increase in % of polymorphonuclear neutrophils between 1 and 3 h after uterine infusion with either MSCs or PBS and a further increase by 6 h only in mares infused with PBS. In summary, endometrial MSCs were detected in the uterine lumen for up to 24 h after infusion, but did not migrate into the healthy endometrium. Moreover, MSCs effectively attenuated the inflammatory response to uterine infusion. We conclude that endometrial MSCs obtained from routine uterine biopsies could provide a safe and effective cell source for treatment of inflammatory conditions of the uterus and potentially other tissues.

Feasibility analysis of treating severe intrauterine adhesions by transplanting menstrual blood-derived stem cells.

Intrauterine adhesions (IUA) are associated with the loss of stem cells in the endometrium. Menstrual blood­derived stem cells (MenSCs) can be isolated from the menstrual blood and differentiated into endometrial cells. To check the transplantation feasibility of MenSCs for the treatment of severe IUA, MenSCs were isolated from menstrual blood, cultured in Dulbecco's modified Eagle's medium (DMEM), identified by immunocytochemistry and flow cytometry, differentiated into endometrial cells in vitro, and finally transplanted into the axillary subcutaneous tissue of non­obese diabetic/severe combined immunodeficiency (NOD­SCID) mice to create endometrial tissue. Additionally, the cloning efficiency and POU domain class 5 transcription factor 1 (OCT­4) positivity of MenSCs from patients with severe IUA were compared with those from healthy women. Immunocytochemistry and flow cytometry results showed that 95.1±0.8% cells were OCT­4­positive, 0.9±0.4% were cluster of differentiation (CD)45­positive, 1.8±0.9% were STRO­1­positive and 1.0±0.4% were human leukocyte antigen­antigen D related­positive. Following differentiation in vitro, the results of immunocytochemistry, reverse transcription­polymerase chain reaction and western blot analysis showed that the expression of cytokeratin (CK) and vimentin (VIM) was increased in MenSCs compared with that in control subjects. Subsequent to transplantation in mice administered with sequential 17ß­estradiol and progesterone, CK, VIM, estrogen receptor and progesterone receptor were expressed in the transplantation regions, suggesting that MenSCs could differentiate into endometrial tissues in vivo. The cloning efficiency and OCT­4 positivity of MenSCs from patients with severe IUA was significantly decreased. In conclusion, to the best of our knowledge, this is the first study in which MenSCs could differentiate into endometrial cells in vitro and create endometrial tissue in NOD­SCID mice in vivo, with impaired cloning efficiency and OCT­4 expression of MenSCs from patients with IUA. This study will provide a theoretical basis for the treatment of IUA with MenSCs.

A Novel Experimental Model of Colorectal Endometriosis.

PURPOSE: Endometriosis is a disease that affects 6-10% of the female population, mainly women of reproductive age, and causes a variety of cyclic symptoms. Deep infiltrating endometriosis and in particular bowel involvement presents a challenge for modern surgery. To date, there are no experimental animal models in this field, demonstrating experimental induction of endometriosis directly attached to surface of the colon imitating human colorectal endometriosis; hence, the implementation of novel pharmaceutical and surgical strategies for the management of colorectal endometriosis is mainly limited to clinical studies. AIM OF THE STUDY: To investigate whether induction of colorectal endometriotic lesions in is feasible in rats. MATERIALS AND METHODS: Twenty, female, adult, non-pregnant Sprague Dawley rats sustained uterine horn resection, which was then placed around the rectum of the rat with the endometrial surface in direct contact with the bowel serosa and approximated in the serosal surface of the colon with two sutures. RESULTS: Two weeks following, surgery rats were euthanized and the bowel was surgically explored. The presence of a cystic lump at the site of the surgical intervention was evaluated macroscopically and microscopically. Histopathology documented the presence of cystic endometriosis. The endometriotic focus was adherent to the bowel wall by large fibrous nodules with concomitant replacement of part of the outer longitudinal muscle layer. CONCLUSIONS: The findings of our study support that the proposed experimental model of colorectal endometriosis is feasible, easily reproducible and may be implemented in future research in this field.

Therapeutically Targeting the Inflammasome Product in a Chimeric Model of Endometriosis-Related Surgical Adhesions.

Development of adhesions commonly occurs in association with surgery for endometriosis. Even in the absence of surgery, women with endometriosis appear to be at an enhanced risk of developing adhesions. In the current study, we utilized a chimeric mouse model of experimental endometriosis in order to examine the role of inflammasome activation in the development of postsurgical adhesions. Mice were randomized to receive peritoneal injections of human endometrial tissue fragments or endometrial tissue conditioned media (CM) from women with or without endometriosis 16 hours after ovariectomy and placement of an estradiol-releasing silastic capsule. A subset of mice receiving CM was also treated with interleukin (IL) 1 receptor antagonist (IL-1ra). Our studies demonstrate that peritoneal injection of endometrial tissue fragments near the time of surgery resulted in extensive adhesive disease regardless of tissue origin. However, adhesion scores were significantly higher in mice receiving CM from tissues acquired from patients with endometriosis compared to control tissue CM ( P = .0001). Cytokine bead array analysis of endometrial CM revealed enhanced expression of IL-1ß from patients with endometriosis compared to controls ( P < .01). Finally, the ability of human tissue CM to promote adhesive disease was dramatically reduced in mice cotreated with IL-1ra ( P < .0001). Our data implicate enhanced expression of IL-1ß in women with endometriosis as a potential causal factor in their increased susceptibility of developing postsurgical adhesions. Thus, targeting inflammasome activation may be an effective strategy for the prevention of surgical adhesions in patients with endometriosis.

Leucine-rich repeat-containing G-protein-coupled receptor 5-positive cells in the endometrial stem cell niche.

OBJECTIVE: To study, isolate and characterize leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5)-positive cells from human endometrium to determine their functional relevance. DESIGN: Prospective experimental animal study. SETTING: University research laboratories. ANIMAL(S): Nonobese diabetic mice (NOD-SCID) (strain code 394; NOD.CB17-Prkdcscid/NcrCrl). INTERVENTION(S): Human LGR5+ cells were labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and injected under the kidney capsule in immunocompromised mice. MAIN OUTCOME MEASURE(S): Epithelial and stromal LGR5+ cells were isolated from human endometrium by means of fluorescence-activated cell sorting, and phenotypic characterization was performed by means of flow cytometry with the use of hematopoietic and mesenchymal markers. Engrafted SPIO-labeled LGR5+ cells were localized with the use of Prussian blue staining and immunohistochemistry against CD9 and Vimentin. Deep transcriptomic profiling of LGR5+ cells was performed with the use of microarrays and RNA sequencing. RESULT(S): The percentage of LGR5+ cells in human endometrium represented 1.08 ± 0.73% and 0.82 ± 0.76% of total cells in the epithelial and stromal compartments, respectively. LGR5+ cells were phenotypically characterized by abundant expression of CD45 hematopoietic marker and no expression of surface markers CD31, CD34, CD133, CD73, and CD90. Coexpression with the macrophage marker CD163 was detected. Xenotransplantation of labeled LGR5+ cells into the kidney capsules of immunocompromised mice resulted in a weak endometrial reconstitution from this cell of origin. Transcriptomic profiling revealed new attributes for LGR5+ cells related to their putative hematopoietic origin. CONCLUSION(S): These data suggest that endometrial LGR5 is not an endogenous stem cell marker. Instead, LGR5+ cells appear to be recruited from blood to be part of the stem cell niche at the perivascular microenvironment to activate the endogenous niche.

[Effect of hypobaric hypoxic preconditioning on surgically induced endometriosis by allotransplant of uterine tissue in rats].

OBJECTIVE: To determine the effects of hypobaric hypoxia pretreatment on surgically induced endometriosis in rats. METHODS: Six rats were randomized into 2 groups and exposed to hypoxia (8% O2) and normoxia (21% O2) for 8 h. The uterine endometrium was intraperitoneally implanted into estrogen-treated ovariectomized Lewis rat, and the growth and quality of the implants were measured. The changes in apoptosis, protein and gene expressions in the serum, abdominis effusion fluids and implants were tested by ELISA, immunohistochemical staining, TUNNEL assay, Western blotting and RT-PCR. RESULTS: The volume of the implants in the hypoxic pretreatment group was significantly increased compared with the normoxia group. High expressions of Ki67, CD31, VEGF, and HIF-1α and lowered cell apoptosis were found in the hypoxia-pretreated implants compared with the normoxic group. VEGF level in the serum and peritoneal fluid were increased in hypoxia-pretreated group, but TNFα level was comparable between the 2 groups. CONCLUSION: Hypoxia play an important role in the occurrence and progression of endometriosis by increasing cell proliferation and angiogenesis and decreasing cell apoptosis in the implants in the rat model.

Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice.

BACKGROUND: The endometrial regenerative cell (ERC) is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4)-induced acute liver injury (ALI). METHODS: An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse) into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction. RESULTS: ERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G) was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA) was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs) was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1ß, IL-6, and TNF-α but higher level of IL-10 in both serum and liver. CONCLUSIONS: Human ERCs protect the liver from acute injury in mice through hepatocyte proliferation promotion, as well as through anti-inflammatory and immunoregulatory effects.

Viburnum opulus L.: A remedy for the treatment of endometriosis demonstrated by rat model of surgically-induced endometriosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Endometriosis is a gynecological disorder characterized by the presence of endometrial tissue outside the uterine cavity. The fruits of Viburnum opulus L. have been used to treat gynecological disorders including primary and secondary dysmenorrhea and ovarian cysts. MATERIALS AND METHODS: Air-dried and powdered fruits of V. opulus were extracted sequentially with n-hexane, ethyl acetate (EtOAc), and methanol (MeOH), respectively for four days. Endometriosis was induced by suturing 15mm piece of endometrium into abdominal wall of Sprague Dawley rats. In second laparotomy, the dimensions of endometrial implants were measured and intra-abdominal adhesions were scored. The abdomen was closed. Extracts were daily administered to the rats. At the end of the experiment, rats were sacrified and endometriotic foci areas and intra-abdominal adhesions were re-evaluated. The tissues were also histopathologically investigated. Furthermore, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels of peritoneal fluid were measured. HPLC analyses were conducted on the most potent EtOAc and MeOH extracts to determine the amount of the major compound, chlorogenic acid. RESULTS: The endometriotic volumes were found to be reduced significantly in the EtOAc extract-administered group to 30.1mm3 and in the MeOH extract-administered group to 34.7mm3 as compared to the control group. No adhesion was observed in the reference and EtOAc groups. Histopathological data also supported the results. Both EtOAc and MeOH extract-administered groups displayed significant remission in the levels of TNF-α, VEGF and IL-6. When the active extracts were subjected to HPLC analysis, chlorogenic acid was found to be the major compound and the amount of this compound was calculated as 0.5112±0.0012mg and 1.7072±0.0277mg/100mg extract, repectively. CONCLUSIONS: The results of the present study indicate that the effectiveness of the fruit extract of V. opulus could be partially attributed chlorogenic acid. Other phenolic compounds could potentiate the activity due to their amount.