Human Endometrial Fragments Research Articles

An efficient protocol for decellularization of the human endometrial fragments for clinical usage.

The present study was aimed tocompare different decellularization protocols for human endometrial fragments. The freeze-thaw cycles in combination with treatment by Triton X-100 and four concentrations of sodium dodecyl sulfate (SDS; 0.1, 0.5, 1, and 1.5%) with two exposure times (24 and 72h) were applied for tissues decellularization. After analysis the morphology and DNA content of tissues the group with better morphology and lower DNA content was selected for further assessments. The nucleus by Acridine orange and extracellular matrix (ECM) using Masson's trichrome, Alcian blue, and periodic acid-Schiff staining were studied. The amount of tissues collagen types I and IV, fibronectin, glycosaminoglycans (GAGs), and elastin was analyzed by Raman spectroscopy. The ultrastructure and porosity of decellularized scaffold were studied by scanning electron microscopy (SEM). The MTT assay was applied for assessments of cytotoxicity of scaffold. The treated group with 1% SDS for 72h showed the morphology similar to native control in having the minimum level of DNA and well preserved ECM. Raman spectroscopy results demonstrated, the amount of collagen types I and IV, GAG, and fibronectin was not significantly different in decellularized scaffold compared with native group but the elastin protein level was significantly decreased (P < 0.001). SEM micrographs also showed a porous and fiber rich ECM in decellularized sample similar to the native control. This combined protocol for decellularization of human endometrial tissue is effective and it could be suitable for recellularization and clinical applications in the future.

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis.

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity. Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Evaluation of the potential therapeutic effects of a double-stranded RNA mimic complexed with polycations in an experimental mouse model of endometriosis

To assess the therapeutic potential of polyinosine-polycytidylic acid, a double-stranded RNA molecule with selective proapoptotic and antiangiogenic activity, complexed with polyethyleneimine (pIC(PEI)) in treating endometriosis. A heterologous mouse model of endometriosis was created by injecting human endometrial fragments into the peritoneum. Endometrial fragments were engineered to express the fluorescent protein mCherry as a reporter to monitor status over the course of the 4-week study. University-affiliated infertility center. Ovariectomized and hormone-replaced nude mice (n = 30) injected with fluorescent-labeled human endometrial fragments at 4-6 weeks of age. Animals (n = 10 per group) were injected with vehicle (control), the anti-VEGF compound CBO-P11 (0.6 mg/kg), or pIC(PEI) (0.6 mg/kg) twice weekly over the course of 4 weeks. Variations in the size of endometriotic implants were estimated by quantifying the expression of mCherry throughout the course of the experiment. Neovascularization, cellular proliferation, and apoptosis were estimated by quantitative immunofluorescence detection of PECAM, α-SMA, Ki67, and TUNEL. pIC(PEI) promoted a significant increase in apoptosis and a decrease in neovascularization in human fragments, but did not reduce the size of endometriotic implants. While pIC(PEI) treatment had significant antiangiogenic and pro-apoptotic effects in this setting, longer periods of exposure than the ones supported by our heterologous model and/or assays in homologous mouse models of endometriosis may be necessary to detect an effect of this compound on lesion size.

Effect of antiangiogenic treatment on peritoneal endometriosis-associated nerve fibers

To investigate the effect of antiangiogenic treatment on experimental endometriotic lesion nerve fibers. Heterologous mouse model of endometriosis. University Institute IVI, University Hospital La Fe. Ovariectomized nude mice (n = 16) receiving human endometrial fragments from oocyte donors (n = 4). Endometrium fragments stuck in the peritoneum of 5-week-old female nude mice treated with vehicle (n = 8) and antiangiogenic agent cabergoline (n = 8; Cb(2,) 0.05 mg/kg/day) for 14 days. Immunofluorescence analysis of von-Willebrand factor (vWF) and vascular smooth muscle cells (αSMA) for evaluating the number of immature blood vessels (IBV) and microvascular density (MVD); immunochemical analysis of protein-gene product 9.5 (PGP 9.5) to assess nerve fibers density (NFD), and blue toluidine staining to confirm presence of mast cells and macrophages in endometriotic lesions. All the results were quantified by morphometric techniques. The IBV, NFD, and number of macrophages and mast cells were statistically significantly decreased in the Cb2-treated group when compared with controls. Antiangiogenic treatment statistically significantly diminishes new blood vessel formation after macrophage, mast cell, and nerve fiber reduction, providing a rationale to test antiangiogenic agents as a novel therapeutic approach to severe pelvic pain associated with human peritoneal endometriosis.

Thrombospondin-1 Mimetic Peptide ABT-898 Affects Neovascularization and Survival of Human Endometriotic Lesions in a Mouse Model

Endometriosis is a common cause of pelvic pain and infertility in women, and a common indication for hysterectomy, yet the disease remains poorly diagnosed and ineffectively treated. Because endometriotic lesions require new blood supply for survival, inhibiting angiogenesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombospondin-1, so we hypothesized that ABT-898 will prevent neovascularization of human endometriotic lesions and that ABT-898 treatment will not affect reproductive outcomes in a mouse model. Endometriosis was induced in BALB/c-Rag2(-/-)Il2rg(-/-) mice by surgical implantation of human endometrial fragments in the peritoneal cavity. Mice received daily injections of ABT-898 for 21 days. Flow cytometry was performed to measure circulating endothelial progenitor cells in peripheral blood. Cytokines were measured in plasma samples. Half of the ABT-898-treated and control mice were euthanized to assess neovascularization of endometriotic lesions, using CD31(+) immunofluorescence. The remaining mice were mated and euthanized at gestation day 12. Endometriotic lesions increased circulating endothelial progenitor cells 13 days after engraftment, relative to baseline. Endometriotic lesions from ABT-898-treated mice exhibited reduced neovascularization, compared with controls, and lesions had fewer CD31(+) microvessels. Chronic treatment with ABT-898 did not lead to any fetal anomalies or affect litter size at gestation day 12, compared with controls. Our results suggest that ABT-898 inhibits neovascularization of human endometriotic lesions without affecting mouse fecundity.

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat and later transferred to the mouse. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system, hormones and environmental factors affect endometriosis as well as the effects of endometriosis on fertility and pain.

The effects of ergot and non-ergot-derived dopamine agonists in an experimental mouse model of endometriosis

Implantation of a retrogradely shed endometrium during menstruation requires an adequate blood supply, which allows the growth of endometriotic lesions. This suggests that the development of endometriosis can be impaired by inhibiting angiogenesis. The growth of endometriotic foci is impaired by commercial oncological antiangiogenic drugs used to block vascular endothelial growth factor (VEGF) signaling. The dopamine agonist cabergoline (Cb2) inhibits the growth of established endometriosis lesions by exerting antiangiogenic effects through VEGFR2 inactivation. However, the use of ergot-derived Cb2 is associated with an increased incidence of cardiac valve regurgitation. To evaluate the potential usage of non-ergot-derived dopamine agonists for the treatment of human endometriosis, we compared the efficacy of quinagolide with that of Cb2 in preventing angiogenesis and vascularization in a heterologous mouse model of endometriosis. Nude mice whose peritoneum had been implanted with eutopic human endometrial fragments were treated with vehicle, 50 μg/kg per day oral Cb2, or 50 or 200 μg/kg per day quinagolide during a 14-day period. At the end of the treatment period, the implants were excised in order to assess lesion size, cell proliferation, degree of vascularization, and angiogenic gene expression. Neoangiogenesis was inhibited and the size of active endometriotic lesions, cellular proliferation index, and angiogenic gene expression were significantly reduced by both dopamine agonists when compared with the placebo. Given that Cb2 and quinagolide were equally effective in inhibiting angiogenesis and reducing lesion size, these experiments provide the rationale for pilot studies to explore the use of non-ergot-derived dopamine agonists for the treatment of endometriosis in humans.

Human endostatin antiangiogenic gene therapy mediated by recombinant adeno-associated virus vector in nude mouse with endometriosis

To study the therapeutic effect of recombinant adeno-associated virus carrying human endostatin gene therapy on endometriosis in mice model. Recombinant adeno-associated virus vector carrying human endostatin gene and enhanced green fluorescent proteins gene (rAAV2-endostatin-EGFP) was constructed. Endometrium was from 12 patients with leiomyoma undergoing hysterectomy in Second Hospital, Tianjin Medical University between November and December 2008. Endometriosis models of nude mice were established by transplanting human endometrial fragments intooperitoneal surface. After 1 week, those 60 mice were divided into 3 groups: treatment group including 20 mice injected with rAAV2-endostatin-EGFP to ectopic lesion, control group including 20 mice injected with rAAV2-EGFP to ectopic lesion and blank control group including 20 mice injected with phosphate buffered saline (PBS) to the ectopic lesion. At 1, 2 and 3 weeks after treatment, those mice underwent laparotomy to observe the location and size of ectopic lesion in abdominal cavity. The expression of endostain protein, number of gland, microvessel density (MVD) and vascular endothelial growth factor (VEGF) were measured in ectopic lesions. The serum level of estradiol and progesterone were detected in nude mice among every groups. (1) All endometriosis of nude mice models were established successfully through peritoneum transplanting. After 1 week's treatment, flat lesion nodes, decreased gland number and narrow and atrophy glandular cavity were observed by light microscope. (2) The endostatin gene was transferred into nude mice successfully and expressed effectively. It was observed that endostatin protein expression was shown with enhanced green fluorescent proteins in ectopic lesion. (3) Glands number of ectopic lesion in rAAV2-endostatin-EGFP group (7.8 ± 1.9, 7.0 ± 1.5 and 5.5 ± 1.7) were significantly less than 10.1 ± 1.7, 10.2 ± 2.0 and 9.8 ± 2.4 in rAAV2-EGFP control group and 10.2 ± 2.2, 10.0 ± 2.0 and 9.7 ± 2.2 in PBS control group at 1, 2 and 3 weeks after treatment (all P < 0.05). Glands number of ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (4) MVD of ectopic lesion in rAAV2-endostatin-EGFP group (12.2 ± 1.5, 11.4 ± 2.1 and 9.0 ± 1.4) was significantly less than those at rAAV2-EGFP control group (16.5 ± 1.7, 16.5 ± 1.9 and 16.9 ± 1.9) and PBS control group (16.2 ± 1.6, 16.0 ± 1.6 and 16.3 ± 1.7) at 1, 2 and 3 weeks after treatment (all P < 0.05). MVD of ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (5) The rate and density of VEGF expression at ectopic lesion in rAAV2-endostatin-EGFP group (35%, 30%, 25% and 1.60 ± 0.43, 1.33 ± 0.30, 1.03 ± 0.36) were significantly less than those at rAAV2-EGFP control group (80%, 75%, 85% and 2.43 ± 0.53, 2.43 ± 0.29, 2.66 ± 0.45) and PBS control group (85%, 90%, 90% and 2.36 ± 0.53, 2.64 ± 0.57, 2.53 ± 0.52) at one 1, 2 and 3 weeks after treatment (all P < 0.05). The expression of VEGF at ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (6) The level of estradial and progesterone in serum of nude mice of rAAV2-endostatin-EGFP group [E(2): (48 ± 7) pmol/L, P: (61 ± 8) nmol/L] did not reach statistical difference when compared with those at rAAV2-EGFP control group [E(2): (50 ± 9) pmol/L, P: (60 ± 10) nmol/L] and PBS control group [E(2): (48 ± 7) pmol/L, P: (58 ± 10) nmol/L, P > 0.05]. The recombinant adeno-associated virus carrying human endostatin gene therapy could inhibit angiogenesis at endometriotic lesions and not influence steroid level. The antiangiogenic gene therapy might become a novel option for endometriosis.

Expression and regulation of estrogen-converting enzymes in ectopic human endometrial tissue

To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. Animal study. Academic medical center. Sixty female nude mice with implanted human endometrial tissue. Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. Transcript levels of steroid hormone receptors, 17beta-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17beta-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

Expression of Glycodelin and Cyclooxygenase‐2 in Human Endometrial Tissue Following Three‐dimensional Culture

Our previous study showed that in vitro culture of human endometrial tissue in a three-dimensional (3D) fibrin matrix could mimic the early stages of endometriosis with invasion, gland and stroma formation and sprouting of new vessels. The objective of the present study was to evaluate the expression of glycodelin (Gd) and cyclooxygenase-2 (COX-2), two angiogenic factors, to further validate the 3D culture model of endometriosis. Human endometrial fragments were obtained from endometrial biopsies and placed in a 3D fibrin matrix culture. Immunohistochemistry with specific antibodies to Gd and COX-2 was used to examine endometrial epithelium and blood vessels, and 4, 6-diamidino-2-phenylindole staining was used for nuclear identification. Three-dimensional culture of human endometrial tissue in the fibrin matrix resulted in the proliferation of endometrial stromal cells, glandular epithelium and angiogenesis. Gd positive glandular epithelium was seen in 85% of wells with developing endometrial glands and COX-2 positive new vessels were seen in 80% of wells with angiogenesis-like structures after 4 weeks of culture. Our findings confirm that angiogenesis occurs following the culture of endometrial tissue in the 3D fibrin matrix, and suggests that Gd and COX-2 might play important roles in promoting neovascularization and cell proliferation in the establishment of endometriosis.

Effect of a statin on an in vitro model of endometriosis

To determine the inhibitory effect of a statin on angiogenesis in a three-dimensional (3-D) culture of human endometrial fragments in vitro. Angiogenesis has been proposed as an important mechanism in the pathogenesis of endometriosis, and statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have been shown to have anti-inflammatory and anti-angiogenic activity. Experimental in vitro study of human endometrial biopsies and 3-D culture in fibrin matrix. Research laboratory at a university-affiliated infertility center. Forty-six normal ovulating women undergoing infertility treatment. Endometrial samples obtained from the fundus of the uterine cavity were minced, and the fragments were placed in a three-dimensional fibrin matrix culture system. Presence or absence of proliferation of stromal cells and invasion of the fibrin matrix, presence or absence of vessel sprouting, and immunohistochemical characterization of cellular components. During the 1st week of culture, invasion of stromal cells into the fibrin matrix occurred in the control group and in some wells outgrowths were observed. After 2 weeks, endometrial glands were observed in the outgrowths at a distance from the main tissue and were growing in conjunction with new vessel formation until the end of culture period. A concentration-dependent effect of lovostatin was seen on cell growth and angiogenesis in the experimental groups. In the presence of 5 and 10 microM of statin, angiogenesis was abolished, and cell proliferation was inhibited. In the presence of 1 microM of lovastatin, angiogenesis was reduced, but cell proliferation was not affected. The statins were shown to be effective in inhibiting the mechanisms of cell proliferation and angiogenesis in an experimental model for the development of endometriosis-like tissue.

Effect of a Statin on an In Vitro Model of Endometriosis

Effect of a Statin on an In Vitro Model of Endometriosis

An in vivo-model to investigate therapeutical approaches for the treatment of peritoneal endometriosis

Endometriosis is a common gynecologic disorder characterized by the presence of endometrial tissue outside the uterus. The disease is associated with dysmenorrhea, pelvic pain and infertility, but the cell biological mechanisms involved in the pathogenesis are yet not understood. It is known that angiogenesis is a prerequisite for the maintenance of endometriotic lesions and it is discussed that an elevated estrogen concentration within the ectopic lesions may support their persistance. To investigate the role of those parameters for the maintenance of ectopic endometrial lesions, we established an in-vivo-model by transplanting human endometrial tissue into the peritoneal cavity of nude mice. At 2 to 28 days after implantation angiogenesis as well as the expression of steroid hormone receptors and estrogen-converting enzymes were investigated in the endometriotic lesions. Neoangiogenesis of mouse vessels into the human endometrial fragments occurred from day 4 after transplantation onwards. This correlated to an increase in transcripts of VEGF and bFGF as well as of their specific receptors. The lesions revealed a well preserved morphology up to 28 days, expression of differentiation markers, of estrogen receptor alpha, progesterone receptors and of different estrogen converting enzymes. We demonstrated that in this in vivo model the expression of steroid hormone receptors as well as of aromatase was regulated in the human ectopic endometrial lesions by systemic application of gestagen, antigestagen (RU486) and the gonadotropin inhibitor danazol. Thus we could show that culturing of human endometrial tissues in the peritoneal cavity of nude mice is an useful experimental model to evaluate cell biological mechanisms responsible for the persistence of ectopic endometrium in the peritoneal cavity.

Quantitative Assessment of Human Endometriotic Tissue Maintenance and Regression in a Noninvasive Mouse Model of Endometriosis

Endometriosis is a prevalent disease characterized by the estrogen-dependent ectopic growth of endometrial tissue. Most of the current medical therapies consist in inducing a hypoestrogenic state in patients, but these treatments are associated with severe side effects and high recurrence rates. The development of convenient and reliable endometriosis animal models would be instrumental to accelerate the emergence of new therapeutic alternatives. Recently, we developed an improved experimental model for endometriosis, relying on the infection of human endometrial fragments by an adenovirus carrying the green fluorescent protein. Following injection of fluorescent fragments into nude mice, the implantation and growth of endometriotic-like lesions could be followed noninvasively. In the present work, we demonstrate that this model can be used to quantify the size of fluorescent endometriotic lesions by in vivo imaging. To this end, we repeatedly measured lesion size over a 4-week period in mice supplemented or not with estradiol. The model was adequate to confirm previous results showing that estrogen is dispensable for the implantation phase of endometrial tissue, whereas it is required for lesion maintenance. As a proof of concept for inducing regression of established lesions, ganciclovir was used to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. A significant decrease in lesion size was observed by in vivo imaging in ganciclovir-treated mice. Together, the data indicate that the noninvasive animal model described here provides a tool for drug testing and/or gene target validation in endometriosis.

Cyr61, a deregulated gene in endometriosis.

Gene expression profiling was performed to identify genes involved in the development of endometriosis. In the secretory phase of the menstrual cycle, several estrogen-regulated genes were up-regulated in endometria of women with endometriosis. The most consistent regulation with one of the highest factors was observed for the Cyr61 gene, which codes for a secreted, cysteine-rich, heparin-binding protein that promotes cell adhesion, migration, and neovascularization. Estrogen responsiveness of endometrial Cyr61 expression was suggested by the higher expression during the proliferative phase and the reduction observed in human endometrial fragments grafted into nude mice subsequently treated with an anti-estrogen. The expression level of Cyr61 was found to be further increased in ectopic endometriotic lesions, as compared to eutopic endometria. In these lesions, an imbalance in expression of the estrogen-converting enzymes 17beta-hydroxysteroid dehydrogenase type 1 and 2 was found, which might explain the elevated Cyr61 level. However, Cyr61 expression was not altered in endometriotic lesions of women treated with a GnRH agonist. These results suggest that Cyr61 may represent a gene characteristic for endometriosis and also play an important role in the development and persistence of endometriotic lesions.

An improved mouse model for endometriosis allows noninvasive assessment of lesion implantation and development

Objective To test whether fragments of human endometrium transduced with the green fluorescent protein (GFP) cDNA and transplanted into nude mice can be noninvasively visualized. Design A murine experimental model for human endometriosis. Setting A biotechnology company. Animal(s) Ovariectomized nude mice. Intervention(s) Whole fragments of human endometrium were transduced in vitro by adenoviral infection with the GFP cDNA before transplantation into nude mice. Animals were noninvasively and repeatedly imaged before lesion collection. Main outcome measure(s) Fluorescence of GFP-expressing human endometrial fragments was evaluated before transplantation into animals. Development of endometriotic lesions was monitored through direct visualization of fluorescent tissue in the living animal or through conventional dissection. Result(s) GFP gene transfer into whole endometrial fragments can be performed, and a high proportion of cells express the reporter gene. Fluorescent endometrial fragments implant in nude mice and form endometriotic-like lesions, which can be directly visualized through the skin of living mice using a simple imaging device. Conclusion(s) This improved mouse model allows noninvasive and dynamic studies of lesion implantation and development to be conducted. In addition to helping better understand the pathophysiology of the disease, this model represents a valuable preclinical tool for testing the efficacy of new drugs targeting endometriosis, which should ultimately accelerate their development phase.

Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Adhesion of human endometrial fragments to peritoneum in vitro

Objective: To evaluate the adhesion of endometrial fragments obtained during the proliferative phase of the menstrual cycle to fresh human peritoneum obtained during abdominal surgery. Design: A prospective, descriptive, morphologic and cell biologic study. Setting: Tertiary care university medical center. Patient(s): Six female volunteers. Intervention(s): After endometrial biopsies performed during diagnostic laparoscopy, endometrial fragments were generated by enzymatic digestion and mechanical separation. Peritoneum was obtained during abdominal operations for benign indications. Main Outcome Measure(s): Adhesion of endometrial fragments was studied by histologic examination and scanning and transmission electron microscopy. Result(s): After incubation, the mesothelium was intact in some areas, whereas in other areas mesothelial cells were damaged or absent. Adhesion of endometrial fragments was observed only at locations where the basement membrane was exposed. In areas largely denuded of mesothelial cells, endometrial fragments spread over the basement membrane to form monolayers. Conclusion(s): Human peritoneum is suitable for studying the adhesion of endometrial fragments. Intact mesothelium prevents the adhesion of endometrial fragments, suggesting that trauma to the mesothelial lining is a prerequisite for endometrial cell adhesion.

Progesterone and antiprogestins, a comparison of their effect on prostaglandin production by human secretory phase endometrium and decidua

The mechanism by which progesterone inhibits PG production is not clear. In systems using isolated human endometrial fragments, progesterone has been shown to inhibit PG production markedly. We have used such a system to test the action of two antiprogesterone steroids RU486 (Roussell-Uclaf) and ZK98734 (Schering) on isolated human endometrial and decidual tissue, with and without added progesterone. Progesterone (200nM) reduced PGF 2α production by the secretory phase endometrium from 10.9ng/mg tissue/24 hr to1.9 ng/mg/24hr on the third day of culture (p<01) and this effect was antagonised by the addition of either 200nM RU486 or 200nM ZK98734 (6.3 and 7.2 ng/mg /24 hr respectively). The antiprogestins on their own showed a slight inhibitory effect on day 3 and RU486 treatment resulted in a significant (p<.05) decrease in PG production from control. PGE and the main 13,14-dihydro-15-keto metabolites of E and F were also significantly decreased by progesterone and restored by the aMiprogestins. The PG production by decidua increased on days 2 and 3 in response to progesterone + antiprogestins but this was not significant. This data shows that the inhibition of PG production shown by progesterone, acting on secretory phase endometrium cultured as tissue fragments, is reversible by the receptor blocking antiprogestins.