Induction Of Endometriosis Research Articles

Ultrastructural Features of Eutopic Endometrium in a Rat Model of Endometriosis

Endometriosis is a chronic recurrent disease that is relatively common. Diagnosis is difficult and often delayed. Current treatments are inadequate with unacceptable side effects and multiple surgeries may be needed. Abnormal eutopic endometrium may play important role in endometriosis-associated infertility. This study aimed to examine the ultrastructural changes in eutopic endometrium in a rat model of surgically induced endometriosis. Endometrial tissue was removed from rats in surgical endometriosis induction group (n=10), sham operated (n=10) and non-operated control (n=10) groups in the diestrus phase of the estrus cycle. They were studied with light, transmission and scanning electron microscope as well as morphometric analysis. Eutopic endometrium in surgically induced endometriosis showed pseudostratified epithelium, vacuolated columnar cells alternated with dark cells. The stroma was edematous exhibiting dilated, congested blood vessels. The mean endometrial mucosal depth and surface epithelial height significantly increased. Ultrastructurally, most luminal epithelial cells showed vacuolation. Mucous secretory granules were surrounded by dilated rough endoplasmic reticulum cisternae. Mitochondria, glycogen deposits and vesicles with electron dense cores were observed. The nuclei were highly euchromatic. Well defined microvilli were noticed with evident apical tight junctions. Scanning electron microscope revealed flattened and structurless surface epithelium with apparent decrease in the number of pinopodes. A different response to sex hormones in different parts of eutopic endometrium was observed. Ultrastructural features of estrogen dominance or progesterone resistance in the eutopic endometrium might account for inappropriate cyclic changes occurring in the disease.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

Exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions.

BackgroundEndometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35 %–50 %, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions.MethodsAs NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2).ResultsOur data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis.ConclusionFor the first time, it was shown that both endogenous as exogenous A-NK cells are able to track, migrate and infiltrate endometriosis lesion. This seems to be a promising result, and if confirmed the efficiency of A-NK cells in killing endometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with endometriosis.

Clarithromycin regresses endometriotic implants in rat endometriosis model

The aim of this study was to investigate the effect of clarithromycin in rat endometriosis and its association with matrix metalloproteinase-9 (MMP-9) expression. After surgical induction of endometriosis, 27 rats were randomised into three groups. Size of endometriotic implants were evalutated and rats in group I (n = 9) were given 100 mg/kg/day of oral clarithromycin, rats in group II (n = 9) were given single 1 mg/kg s.c. injection of leuprolide acetate and rats in group III (n = 9) were not given any medication for 21 days. At the end of 21 days of medication, remaining 23 rats were sacrificed to evaluate morphological and histological features of implants. There was a significant difference between the groups in implant volumes (p = 0.004) before and after medication. Regression of implants were significantly higher in groups I and II than that in control group (p = 0.009 and p = 0.011, respectively). After medication, in group I the implant volume decreased from 62 (12–166) mm3 to 26 (3–87) mm3 (p = 0.012) and in group II the volume decreased from 224 (76–1135) mm3 to 62 (26–101) mm3 (p = 0.028). There was a significant difference between groups in histopathological score (p = 0.024). The epithelial immunohistochemical score of MMP-9 was significantly lower in group II than that in control group (p = 0.014). In conclusion, clarithromycin regresses endometriotic implants in rats, but not via MMP-9.

P-selectin as a potential therapeutic target for endometriosis

To test the hypothesis that P-selectin plays a critical role in the development of endometriosis and that targeting P-selectin has therapeutic potential. Prospective, randomized study. Academic facility. The first experiment used 24 each of female adult P-selectin knockout mice and C57BL/6 wild-type mice, and 96 male green fluorescent protein (GFP) transgenic mice. The second experiment used 16 female adult C57BL/6 mice. In experiment 1, P-selectin knockout and wild-type mice alternately served as donors and recipients for the transplantation of endometrial tissue fragments into the peritoneal cavity to induce endometriosis. Two weeks later all four groups were each randomly divided into two equal-sized subgroups, receiving either green fluorescent protein-expressing platelets or saline infusion. In experiment 2, 2 weeks after the surgical induction of endometriosis, the mice were randomly divided into two groups, one receiving a 2-week treatment with a recombinant P-selectin protein and the other, non-immune IgG, both by intraperitoneal injection. Lesion size, hotplate latency, and immunohistochemistry analysis of platelet aggregation, angiogenesis, transforming growth factor β1, α-smooth muscle actin, collagen I, and the extent of macrophage infiltration in ectopic implants. The extent of fibrosis also was evaluated in experiment 2. P-selectin deficiency significantly retarded the development of endometriosis in a mouse model of endometriosis. In addition, soluble P-selectin treatment markedly reduced the lesion size in mouse through decreased platelet aggregation and angiogenesis, improved general hyperalgesia, and reduced the extent of macrophages infiltration, resulting in reduced fibrotic tissue content. Targeting P-selectin-mediated platelet adhesion onto endometriotic lesions holds promise as a novel therapeutics for treating endometriosis.

Effects of repeated propranolol administration in a rat model of surgically induced endometriosis

ObjectivesTo determine whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model or not. Study designThis was an experimental animal model study. Twenty-four female Wistar albino rats (200–250g) were used to create a model for surgical induction of endometriosis. Two rats died during the surgeries. The rats were randomly divided into treatment (n=11) and control groups (n=10), which were treated with daily intraperitoneal propranolol (10mg/kg) and saline (2mL), respectively. Study duration was 8 weeks. The volumes and histopathological findings of the implants, and immunochemistry for vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-2, and MMP-9 were evaluated. ResultsViable endometriotic implants were created in all animals. In the propranolol-treated group, the mean implant volume significantly decreased after treatment (142.5 vs. 32.1mm3, respectively; p=0.008), while the mean implant volume significantly increased in the control group (141.0 vs. 174.2mm3, respectively; p=0.009). There were also significant reductions in VEGF immunoreactivity scores and both stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores in the propranolol-treated group compared with the control group (p<0.005 for all scores). ConclusionsPropranolol may suppress endometrial tissue by its antiangiogenic activity through inhibitory actions on VEGF, MMP-2, and MMP-9. Therefore, propranolol is a promising candidate drug for effective treatment of patients with endometriosis, which needs to be confirmed with further studies.

ENMD-1068, a protease-activated receptor 2 antagonist, inhibits the development of endometriosis in a mouse model

Protease-activated receptor 2 plays an important role in the pathogenesis of endometriosis. We studied the effect of ENMD-1068, a protease-activated receptor 2 antagonist, on the development of endometriosis in a noninvasive fluorescent mouse model. A red fluorescent protein-expressing xenograft model of human endometriosis was created in nude mice. After endometriosis induction, the mice were injected intraperitoneally with either 25 mg/kg or 50 mg/kg ENMD-1068 or with 200 μL of the vehicle control daily for 5 days. The endometriotic lesions that developed in the mice were then counted, measured, and collected. The lesions were assessed for the production of interleukin 6 and monocyte chemotactic protein-1 by enzyme-linked immunosorbent assays and evaluated for the activation of nuclear factor-κB and the expression of vascular endothelial growth factor by immunohistochemical analyses. Cell proliferation and apoptosis were assessed by immunohistochemistry for Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. ENMD-1068 dose-dependently inhibited the development of endometriotic lesions (P < .05) without apparent toxicity to various organs of the treated mice. Consistently, ENMD-1068 dose-dependently inhibited the expression of interleukin 6 and nuclear factor-κB (P < .05) and cell proliferation (P < .05) in the lesions, as well as increased the percentage of apoptotic cells (P < .05). ENMD-1068 reduced the levels of monocyte chemotactic protein-1 and vascular endothelial growth factor in the lesions (P < .05), but not in a dose-dependent manner. Our study suggests that ENMD-1068 is effective in suppressing the growth of endometriosis, which might be attributed to the drug's antiangiogenic and antiinflammatory activities.

The development of endometriosis in a murine model is dependent on the presence of dendritic cells

Endometriosis is a common condition associated with pelvic pain and infertility. This study group has previously shown that supplementation of dendritic cells led to enhancement of endometriosis lesion growth and angiogenesis. This study determined whether endometriosis is dependent on the presence of endogenous dendritic cells. Surgical induction of endometriosis was performed in CD11c+ DTR/GFP transgenic (Tg) female mice in which dendritic cells were ablated upon injection of diphtheria toxin (DT). Mice were allocated into four groups (n=5 each): group I, wild-type mice treated with vehicle; group II, wild-type mice treated with DT; group III, Tg mice treated with DT; and group IV, Tg mice treated with vehicle. After 10days, mice were killed and endometriosis lesions were analysed by flow cytometry. DT treatment led to ablation of dendritic cells in spleens and endometriosis lesions in Tg mice while no ablation was observed in controls. Corresponding to dentritic cell ablation, endometriosis lesions in group III were ∼5-fold smaller than in the control groups (ANOVA P<0.0001). This study suggests that endometriosis development is dependent on the presence of endogenous dendritic cells. Therapies designed to inhibit dentritic cell infiltration as possible treatments for endometriosis warrant further study.Endometriosis is a common condition associated with pelvic pain and infertility. We have previously shown that supplementation of a specialized group of bone marrow-derived immune cells – dendritic cells – led to enhancement of endometriosis lesion growth and to enhanced blood vessel formation within the lesions. Here we determined whether endometriosis is dependent on the presence of endogenous dendritic cells. Surgical induction of endometriosis was performed in a specialized transgenic mouse model in which dendritic cells could be specifically ablated upon injection of diphtheria toxin. Our results show that, corresponding to dendritic cell ablation, endometriosis lesions in this group were ∼5-fold smaller than in all control groups. Our study indicates that endometriosis development is dependent on the presence of endogenous dendritic cells. Therapies designed to inhibit dendritic cell infiltration as possible treatments for endometriosis warrant further studies.

Effects of tramadol on viscero‐visceral hyperalgesia in a rat model of endometriosis plus ureteral calculosis

The effects of tramadol versus placebo administration on behavioral indicators of ureteral pain, pelvic pain and referred lumbar muscle hyperalgesia were investigated in a rat model of viscero-visceral hyperalgesia from endometriosis plus ureteral calculosis (endo + stone). Fifty female Sprague-Dawley rats underwent surgical induction of endometriosis and, 2 weeks later, were randomly assigned to five groups (10 each), to be treated i.p., twice a day, with tramadol (0.625, 1.25, 2.5, or 5 mg/kg) or saline for 5 days (14-18th day postendometriosis; prestone treatment). On the 21st day, they underwent laparotomy for stone formation in the upper left ureter (dental cement injection). All were video-taped 24 h nonstop for 7 days before and 4 days after stone formation (14-25th day postendometriosis) to record ureteral and pelvic pain behaviors. Lumbar sensitivity (L1) was tested bilaterally, daily over the same period, by verifying presence/absence of vocalization upon muscle pinching at a predefined pressure (calibrated forceps). Additional fifty endo + stone rats underwent the same protocol, except that treatment was performed on 21st-25th day (poststone treatment). Tramadol vs. saline significantly reduced number and duration of ureteral crises, duration of pelvic behavior, and incidence of muscle hyperalgesia (P < 0.0001), with a dose-dependent effect. Prestone treatment was significantly more effective than poststone treatment for the 1.25 dose for all parameters and 2.5 dose for pelvic and muscle parameters (0.003 > P < 0.02). Tramadol, even at low doses, is thus highly protective against pain from 'viscero-visceral hyperalgesia' in endometriosis plus ureteral calculosis; it can represent a valid therapeutic approach in women with these comorbidities.

A physiological approach for treating endometriosis by recombinant pigment epithelium-derived factor (PEDF)

Is pigment epithelium-derived factor (PEDF) expressed in the rodent endometrium and can it be utilized to treat endometriosis without negatively affecting reproductive parameters? PEDF is dynamically expressed in rat endometrium throughout the estrous cycle in a reciprocal manner to vascular endothelial growth factor (VEGF); it possesses potent therapeutic properties for endometriosis that do not compromise the reproductive parameters. Endometriosis pathogenesis depends mainly on neovascularization, with a high local level of VEGF. PEDF, a 50 kDa secreted glycoprotein with a potent anti-angiogenic activity, negates several strong pro-angiogenic factors, such as VEGF. Rat endometrial samples were collected at various days of the estrous cycle (n = 5 rats/day) and mRNA of VEGF and PEDF was determined. Endometriosis was induced by transplanting uterine pieces onto the inner surface of the abdominal wall of recipient rats, resulting in proliferation of the endometrial transplants. Recipient rats were randomly injected intravenously (IV), every third day for the next 3 weeks, with either Tris ('control'; n = 7) or recombinant PEDF (rPEDF; 2 mg/kg/day; 'PEDF prevention'; n = 7), while others were IV injected every third day starting from Day 9 after grafting until the end of 3 weeks, with rPEDF (2 mg/kg/day; 'PEDF treatment'; n = 6). The effect of rPEDF on the duration of the estrous cycle and on the number of ovulated oocytes was evaluated in rats that were randomly divided into four groups and were injected with either Tris or rPEDF every third day for 3 weeks: naive rats (n = 6); rPEDF-treated rats (n = 5); endometriosis-induced rats (n = 5); or endometriosis + rPEDF rats (n = 6). Reproductive parameters: the estrous cycle was evaluated by daily vaginal smears, and the number of ovulated oocytes in the oviductal ampullae of estrus rats was counted. The efficiency of endometriosis induction and treatment was evaluated on the third week after endometrial transplantation, on the day of pro-estrus. Endometrial transplants were isolated and weighted. PEDF and VEGF were monitored by quantitative PCR and immunohistochemistry using confocal microscopy. PEDF mRNA and protein were dynamically expressed in the endometrium all throughout the estrous cycle, reciprocally to VEGF; VEGF was highly expressed during estrus while PEDF expression was low, and vice versa at metestrus II. The weight of the endometrial transplants was significantly reduced after PEDF administration (13% of control for 'PEDF treatment' rats; 7% of control for 'PEDF prevention' rats; P < 0.001). Histology of the transplants' remnants showed a complete loss of their endometrial characteristics. Furthermore, the level of VEGF mRNA in the transplants of PEDF-administered rats was significantly lower (P < 0.05) than in transplants of control rats. Administration of rPEDF had no effect on the estrous cycle or ovulation rate of naive rats, while it had a significantly beneficial effect on the low ovulation rate of endometriosis-induced rats (P < 0.05). The experiments were performed in a rat model. The endometriosis therapeutic potency of PEDF that is exerted reciprocally to VEGF and does not compromise reproductive parameters offers a rational for using PEDF as a treatment for endometriosis with a potential of treating other reproductive angiogenic-related pathologies.

Slit2 Overexpression Results in Increased Microvessel Density and Lesion Size in Mice With Induced Endometriosis

We recently reported that Slit/Roundabout (ROBO) 1 pathway may be a constituent biomarker for recurrence of endometriosis, likely through promoting angiogenesis. In this study, we sought to determine as whether Slit2 overexpression can facilitate angiogenesis, increase lesion size, and induce hyperalgesia in mice with induced endometriosis. We used 30 Slit2 transgenic (S) and 29 wild-type (W) mice and cross-transplanted endometrial fragments from S to W (group SW) and vice versa (group WS), and also within the S and W (groups SS and WW, respectively), into the peritoneal cavity, inducing endometriosis. We also performed a sham surgery within both S and W mice (groups Sm and Wm, respectively). The size of the ectopic implants, microvessel density (MVD) and immunoreactivity to ROBO1, and vascular endothelial cell growth factor (VEGF) in ectopic and eutopic endometrium, along with hotplate and tail-flick tests in all mice, were then evaluated. We found that the induction of endometriosis resulted in generalized hyperalgesia, which was unaffected by Slit2 overexpression. Slit2 overexpression did increase the lesion size significantly and correlated positively with the MVD in ectopic and eutopic endometrium. Slit2 expression levels appear to correlate with the MVD, but not with VEGF immunoreactivity, in ectopic endometrium. Consequently, we conclude that Slit2 may play an important role in angiogenesis in endometriosis. The increased angiogenesis, as measured by MVD, but not VEGF immunoreactivity, likely resulted in increased lesion size in induced endometriosis. Thus, SLIT2/ROBO1 pathway may be a potential therapeutic target for treating endometriosis.

Translational Animal Models to Study Endometriosis-Associated Infertility

Although there is an apparent association between endometriosis and impaired fertility, the pathophysiology of the reduced fecundity in women with endometriosis still remains unclear. Reproduction is a complex and multifactorial process, and possible factors contributing to the reduced fertility of endometriosis patients include defective function of the ovary, gametes, and endometrium as well as developmental disorders of the embryo. Because controlled experiments in humans are limited due to ethical reasons, experimental animal models have been developed mainly in nonhuman primates and laboratory rodents by induction of endometriosis via autologous transplantation of endometrial tissue. Animals with induced endometriosis reveal an impairment of fecundity similar to the situation described for humans and have been used to identify effects of ectopic endometrial tissue on adhesion formation, peritoneal fluid composition, ovarian function, endometrial gene expression, and embryo implantation. These animal models of endometriosis yield a valuable tool to study the mechanisms of endometriosis-associated infertility especially during the onset of the disease that cannot be investigated in women.

Induced Endometriosis in Nonhuman Primates1

Induced Endometriosis in Nonhuman Primates1

Induction of endometriotic nodules in an experimental baboon model mimicking human deep nodular lesions

To establish an experimental model for the study of deep nodular endometriosis. Induction of nodular endometriosis in baboons by grafting different uterine specimens to the peritoneal cavity. Research and university facilities. Ten baboons, to develop a model of induced deep nodular endometriosis. Biopsies of endometrium, and endometrium plus the junctional zone (JZ), full uterine thickness, and myometrium grafted to the peritoneum. Macroscopic descriptions recorded for observed induced lesions; staining with hematoxylin and eosin for histological evaluation and specific antibodies (CK22, CD10) for immunohistochemical studies; and analysis of surface area and volume of lesions, glandular density, and invasion of surrounding organs. The incidence of induced nodular endometriosis was 100%, but the extent depended on the tissue grafted. Lesions induced after grafting specimens containing the JZ were statistically significantly larger than those not containing the JZ. Surrounding organ invasion was reported in more than 40% of lesions after grafting specimens containing the JZ. The first experimental model of nodular endometriosis allows investigation of deeper nodular lesions as well as invasion phenomena associated with nodular lesions.

Natural therapies assessment for the treatment of endometriosis

Can resveratrol and epigallocatechin-3-gallate (EGCG) inhibit the growth and survival of endometriotic-like lesions in vivo in a BALB/c model of endometriosis, and in vitro in primary cultures of human endometrial epithelial cells (EECs)? Resveratrol and EGCG exerted a potent inhibitory effect on the development of endometriosis in a BALB/c murine model and on the survival of EECs. Endometriosis is a common condition associated with infertility and pelvic pain in women of reproductive age. Resveratrol and EGCG are two polyphenols with anticarcinogenic and antioxidant properties that have been proposed as natural therapies to treat endometriosis. Fifty-six 2-month-old female BALB/c mice underwent surgical induction of endometriosis. Treatments with resveratrol or EGCG started 15 days post-surgery and continued for 4 weeks. Human biopsies were taken with a metal Novak curette from the posterior uterine wall from 16 patients with untreated endometriosis and 15 controls who underwent diagnostic laparoscopy for infertility. After the treatments, animals were sacrificed and lesions were counted, measured, excised and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for cell proliferation and vascularization assessment in the lesions. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique was performed for apoptosis evaluation. Peritoneal fluid was collected to analyze vascular endothelial growth factor levels. Human EECs were purified from proliferative-phase endometrial biopsies and cultured. The effect of both polyphenols on cell proliferation was determined by a colorimetric assay using the CellTiter 96®AQueous One Solution Cell Proliferation Assay kit and on apoptosis by the TUNEL technique, using an In Situ Cell Death Detection Kit with Fluorescein. In the mouse model, both treatments significantly reduced the mean number (P < 0.05 versus control) and the volume of established lesions (P < 0.05 versus control). Treatments consistently statistically significantly diminished cell proliferation (resveratrol P < 0.01 and EGCG P < 0.05, versus control), reduced vascular density (resveratrol P < 0.01 and EGCG P < 0.001, versus control) and increased apoptosis within the lesions (resveratrol P < 0.01 and EGCG P < 0.05, versus control). Both compounds induced reduction in human EEC proliferation (P < 0.05 versus basal) and increased apoptosis (P < 0.05 versus basal) in primary cultures. In vitro studies were only carried out in epithelial cells from human eutopic endometrium. The present findings are promising and will assist the development of novel natural treatments for endometriosis. This study was supported by ANPCYT (PICT 6384 BID 1201 OC-AR) and CONICET (PIP 5471), Argentina. None of the authors has any conflict of interest to declare.

Perinatal Exposure to Bisphenol A and Diethylstilbestrol Programs Endometriosis Related Gene Expression in the Mouse Uterus.

Endometriosis is a chronic painful disease that is thought to occur when retrograde menstruation places shed endometrial tissue in the peritoneal cavity where in then implants and invades surrounding tissue. Although retrograde menstruation is thought to occur in up to 90% of women, only 10% of women develop endometriosis. Not only is endometriosis is an estrogen dependent disease in adulthood but the fetal estrogen environment is also associated with the risk of developing endometriosis in women. Conditions during pregnancy associated with high estrogen levels have been correlated with increased risk of endometriosis in adulthood. For example, women whose mothers took the pharmaceutical xenoestrogen DES during their pregnancy have an 80% increased risk of endometriosis. Conversely, maternal smoking, which inhibits placental aromatase expression and results in decreased fetal estrogens, is associated with a four-fold decrease in adult endometriosis risk. In order to better understand the role that developmental xenoestrogen exposure has on programming endometriosis related gene expression, we performed two separate experiments in which we used a mouse model of developmental exposure followed by surgical induction of endometriosis in adulthood. Following perinatal exposure to bisphenol A (BPA) and DES, endometriosis was surgically induced by autotransplantation of endometrial tissue to the arterial cascade of the intestinal mesentery in adulthood. Altered gene expression was assessed with both a global and targeted approach. Developmental exposure to DES and BPA altered the expression of a number of genes as measured by next generation sequencing (RNA-Seq): 22 and 26 genes were upregulated in adulthood and nine and four down regulated by DES and BPA respectively. Interestingly, only 11 genes were altered by both chemicals. In a targeted analysis, the expression of five endometriosis related genes was assessed in the eutopic uterine tissue and endometriotic lesions by real time RT-PCR. Developmental exposure to DES altered the expression of three genes in the eutopic uterus: the expression of Cxcl5 and Ltf was increased and Spp1 tended to be increased in both experiments. Developmental exposure to BPA tended to alter the expression of these genes also. Taken together, these results suggest that developmental exposure to bisphenol A and other xenoestrogens at current environmentally relevant levels may alter the risk for endometriosis in women.

Surgical Induction of Endometriosis in Rhesus Macaques, Macaca mulatta.

Surgical Induction of Endometriosis in Rhesus Macaques, Macaca mulatta.

Stress Exacerbates Endometriosis Manifestations and Inflammatory Parameters in an Animal Model

Women with endometriosis have significant emotional distress; however, the contribution of stress to the pathophysiology of this disease is unclear. We used a rat model of endometriosis to examine the effects of stress on the development of this condition and its influence on inflammatory parameters. Female Sprague-Dawley rats were subjected to swim stress for 10 consecutive days prior to the surgical induction of endometriosis by suturing uterine horn implants next to the intestinal mesentery (endo-stress). Sham-stress animals had sutures only, and an endo-no stress group was not subjected to the stress protocol. At the time of sacrifice on day 60, endometriotic vesicles were measured and colons assessed for macroscopic and microscopic damage. Colonic tissue and peritoneal fluid were collected for inflammatory cell analysis. Endometriosis, regardless of stress, produced a decrease in central corticotropin-releasing factor immunoreactivity, specifically in the CA3 subregion of the hippocampus. Prior exposure to stress increased both the number and severity of vesicles found in animals with endometriosis. Stress also increased colonic inflammation, motility, myeloperoxidase levels, and numbers of mast cells. In summary, prior stress may contribute to the development and severity of endometriosis in this animal model through mechanisms involving cell recruitment (eg, mast cells), release of inflammatory mediators, and deregulation of hypothalamic-pituitary axis responses in the hippocampus.

Towards Endometriosis Diagnosis by Gadofosveset-Trisodium Enhanced Magnetic Resonance Imaging

Endometriosis is defined as the presence of endometrial tissue outside the uterus. It affects 10–15% of women during reproductive age and has a big personal and social impact due to chronic pelvic pain, subfertility, loss of work-hours and medical costs. Such conditions are exacerbated by the fact that the correct diagnosis is made as late as 8–11 years after symptom presentation. This is due to the lack of a reliable non-invasive diagnostic test and the fact that the reference diagnostic standard is laparoscopy (invasive, expensive and not without risks). High-molecular weight gadofosveset-trisodium is used as contrast agent in Magnetic Resonance Imaging (MRI). Since it extravasates from hyperpermeable vessels more easily than from mature blood vessels, this contrast agent detects angiogenesis efficiently. Endometriosis has high angiogenic activity. Therefore, we have tested the possibility to detect endometriosis non-invasively using Dynamic Contrast-Enhanced MRI (DCE-MRI) and gadofosveset-trisodium as a contrast agent in a mouse model. Endometriotic lesions were surgically induced in nine mice by autologous transplantation. Three weeks after lesion induction, mice were scanned by DCE-MRI. Dynamic image analysis showed that the rates of uptake (inwash), persistence and outwash of the contrast agent were different between endometriosis and control tissues (large blood vessels and back muscle). Due to the extensive angiogenesis in induced lesions, the contrast agent persisted longer in endometriotic than control tissues, thus enhancing the MRI signal intensity. DCE-MRI was repeated five weeks after lesion induction, and contrast enhancement was similar to that observed three weeks after endometriosis induction. The endothelial-cell marker CD31 and the pericyte marker α-smooth-muscle-actin (mature vessels) were detected with immunohistochemistry and confirmed that endometriotic lesions had significantly higher prevalence of new vessels (CD31 only positive) than the uterus and control tissues. The diagnostic value of gadofosveset-trisodium to detect endometriosis should be tested in human settings.

Induction of endometriosis alters the peripheral and endometrial regulatory T cell population in the non-human primate

Endometriosis is a gynecological condition that is characterized by extreme abdominal pain and also decreased fertility. Regulatory T cells (Tregs) have immunosuppressive activity critical for embryonic implantation and likewise the acceptance of tissue engraftment. Utilizing the induced non-human primate (Papio anubis) model of endometriosis, we hypothesize that endometriosis decreases the peripheral and endomet rial Treg profile, whereas ectopic lesions have increased Treg localization. Peripheral blood and endometrium were obtained throughout the menstrual cycle prior to and after induction of disease. Animals were randomly assigned to control (n = 7) or diseased (n = 16) treatment groups. Endometriosis was induced by i.p. injection of autologous menstrual tissue for 2 consecutive months during menses. Peripheral blood and endometrial tissue were collected at d9-11PO at 1, 3, 6, 9, 12 and 15 months post-induction of disease for fluorescence-activated cell sorting, quantitative RT-PCR and immunohistochemistry. Ectopic lesions were excised at 1 and 6 months post-inoculation and also harvested at necropsy (15 months) and processed for RNA of IHC. Identification of Tregs through analysis of FOXP3 expression was conducted utlilizing several methodologies. Differences were determined by non-parametric statistical analysis between all treatment groups and time points. In control animals, the proportion of peripheral natural Tregs (nTregs) was reduced (P < 0.05) during the mid- and late secretory stages of the menstrual cycle compared with menses. The induction of disease decreased peripheral Treg expression at early time points (P < 0.05) and this remained low throughout the time course, compared with the pre-inoculatory level of an individual. FOXP3 gene expression and Treg populations were also decreased in the eutopic endometrium (P < 0.05) compared with control animals, whereas these parameters were increased in ectopic lesions (P < 0.05), compared with the eutopic endometrium. Our data suggest that a reduction in peripheral Tregs may be a causative factor for endometriosis-associated infertility, while the increase in ectopic Treg expression may aid lesion development. Furthermore, endometriosis appears to disrupt Treg recruitment in both eutopic and ectopic endometrium.