Evaluation of estrogen treatment in an immunodeficient mouse endometriosis model.

BACKGROUND/AIMS: Endometriosis is known to be an estrogen-dependent disease. However, only a few studies have analyzed the effect of estrogen treatment in mice xenotransplanted with human endometrium. The objective of this study was to adapt a previously developed heterologous murine model to the study of estrogens and test the impact of estrone treatment on endometriosis development. METHODS: Human proliferative endometrium was xenotransplanted into the peritoneal cavity of castrated immunodeficient mice. These mice were treated with estrogens by means of subcutaneous estrone-releasing pellets. The effect of estrone on estradiol level, uterine histology and endometriosis development was evaluated after 21 days. RESULTS: Bioactivity of estrone pellets and their metabolization into estradiol were demonstrated. However, there was no impact on endometriosis development (no difference in lesion number, weight, size or fluorescence). This lack of response was not due to absence of estrogen receptor expression, since strong expression was found in all lesions harvested. Surprisingly, castrated nontreated mice presented with lesions showing high proliferative activity, similar to lesions found in treated mice (around 30%). CONCLUSION: The high proliferation observed in lesions recovered from ovariectomized nontreated mice questions the utility of using estrogens in heterologous murine models.

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human endometrial stromal and epithelial cells is regulated by interferon-gamma but not iron.

BACKGROUND/AIMS: Endometrial cells are chronically exposed to iron due to cyclic menstrual bleeding. Iron induces expression of adhesion molecules in endothelial cells. The purpose of this study was to investigate iron incorporation by human endometrial cells and to test whether iron may stimulate expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. METHODS: Endometrial stromal and epithelial cells were cultured in medium alone or supplemented with INF-gamma or transferrin (Tf). Iron incorporation by cells was quantified by densitometry of ferritin immunostaining. ICAM-1 and VCAM-1 expression were evaluated at the transcriptional level by real-time RT-PCR. Membrane-bound and soluble protein levels of ICAM-1 were measured by quantitative immunohistochemistry and ELISA, respectively. RESULTS: Tf induced a significant increase in ferritin immunostaining in both endometrial cell types. Endometrial cells treated with INF-gamma expressed more ICAM-1 and VCAM-1 than untreated cells. By contrast, Tf treatment did not alter ICAM-1 and VCAM-1 expression in cultured endometrial cells. CONCLUSIONS: Endometrial cells are able to incorporate iron from Tf and to metabolize it to ferritin. Iron, unlike interferon-gamma, does not appear to be involved in the regulation of ICAM-1 and VCAM-1 expression in cultured endometrial cells.

Agents blocking the nuclear factor-kappaB pathway are effective inhibitors of endometriosis in an in vivo experimental model.

BACKGROUND: In vitro studies suggest that the transcription factor nuclear factor-kappa B (NF-kappaB) is implicated in the transduction of proinflammatory signals in endometriosis. The aim of this study was to investigate the involvement of NF-kappaB and the processes regulated by NF-kappaB in the initial development of endometriotic lesionsin vivo. METHODS: Endometriosis was induced in nude mice by intraperitoneal injection of fluorescent-labeled menstrual endometrium. Two NF-kappaB inhibitors (BAY 11-7085 and SN-50) were injected intraperitoneally on days 0, 2 and 4 after endometriosis induction, and endometriotic lesions were recovered on day 5. Number, mass, fluorimetry and surface (morphometry) of endometriotic lesions were quantified. NF-kappaB activation, intercellular adhesion molecule (ICAM)-1 expression, cell proliferation and apoptosis were evaluated by immunohistochemical analyses and the TUNEL method. RESULTS: Both NF-kappaB inhibitors induced a significant reduction in lesion development compared to control mice. NF-kappaB activation and ICAM-1 expression of endometriotic lesions were significantly reduced in treated mice, and cell proliferation was significantly reduced in BAY 11-7085-treated mice. Both inhibitors produced a significant increase in apoptosis of endometriotic lesions, as assessed by active caspase-3 immunostaining and the TUNEL method. CONCLUSION: This study demonstrates, for the first time, that the NF-kappaB pathway is implicated in the development of endometriotic lesions in vivo and that NF-kappaB inhibition reduces ICAM-1 expression and cell proliferation, but increases apoptosis of endometriotic lesions, diminishing the initial development of endometriosis in an animal model.

Unsuccessful induction of endometriosis in female rhesus macaques (Macaca mulatta).

BACKGROUND/AIMS: The aim of this study was to induce endometriosis in female rhesus macaques (Macaca mulatta) for research purposes. METHODS: Three female monkeys from 4 to 4.5 years of age underwent three consecutive attempts at endometriosis induction over an 8-month period: (i) the first attempt involved intravaginal sampling of endometrial tissue and transplantation into the intrapelvic cavity; (ii) the second entailed surgical removal of endometrium after hysterotomy and intra-abdominal placement, and (iii) the third used endometrial mucosa obtained by scraping the uterus after hysterectomy, placed in a surgical pouch created in the retrovesical space (Retzius). In each case, the pelvic cavity was closely inspected after 7, 9, and 6 weeks respectively for the presence of endometriotic lesions, and peritoneal biopsies were performed. RESULTS: Neither macroscopic observation nor histological analysis revealed any endometriotic lesions. CONCLUSION: This failure to induce endometriosis in female rhesus macaques suggests that this species is not the most efficient experimental model among primates to investigate endometriosis development or treatment.

Fertility preservation in women with ovarian endometriosis.

Endometriosis is one of the most frequently encountered benign diseases in gynecology. Complete resolution of endometriosis is not yet possible, but therapy has essentially three main objectives: (1) to preserve and improve fertility, (2) to reduce pain, and (3) to delay recurrence for as long as possible. The aim of this paper is to focus on fertility preservation in women with severe endometriosis. In moderate and severe endometriosis, a medico-surgical approach remains the gold standard, but more and more papers are reporting a low ovarian reserve after laparoscopic cystectomy for endometriomas. Indeed, very frequently, normal ovarian tissue is excised together with the endometrioma wall. Ovarian surgery in endometriosis patients should therefore be performed by experienced surgeons in order to both preserve and improve fertility. Preservation of ovarian tissue should be considered in all patients at serious risk of future fertility impairment, particularly before any treatment likely to result in ovarian endometriosis recurrence and/or premature ovarian failure.

Peritoneal endometriosis is an inflammatory disease.

Peritoneal endometriosis is a chronic inflammatory disease characterized by increased numbers of peritoneal macrophages and their secreted products. Inflammation plays a major role in pain and infertility associated with endometriosis, but is also extensively involved in the molecular processes that lead to peritoneal lesion development. Peritoneal oxidative stress is currently thought to be a major constituent of the endometriosis-associated inflammatory response. Excessive production of reactive oxygen species, secondary to peritoneal influx of pro-oxidants such as heme and iron during retrograde menstruation, may induce cellular damage and increased proinflammatory gene expression through nuclear factor-kappa B activation. In particular, prostaglandin biosynthetic enzyme expression is regulated by this transcriptional factor, and increased peritoneal prostaglandin concentrations have been demonstrated in endometriosis. In the light of available data collected from patient biopsies, as well as in vitro and in vivo studies, the respective involvement and potential molecular interactions of iron, nuclear factor-kappa B and prostaglandins in the pathogenesis of endometriosis are explored and discussed. The key role of peritoneal macrophages is emphasized and potential therapeutic targets are examined.

Insights into iron and nuclear factor-kappa B (NF-kappaB) involvement in chronic inflammatory processes in peritoneal endometriosis.

Endometriosis is a chronic pelvic inflammatory process. Local inflammation is known to play a role in pain and infertility associated with the disease, and may be extensively involved in molecular and cellular processes leading to endometriosis development. In this review, we focus on two inflammatory mediators clearly implicated in the pathogenesis of endometriosis, iron and NF-kappaB, and their potential association. Iron is essential for all living organisms, but excess iron results in toxicity and is linked to pathological disorders. In endometriosis patients, iron overload has been demonstrated in the different compartments of the peritoneal cavity (peritoneal fluid, endometriotic lesions, peritoneum and macrophages). This iron overload affects numerous mechanisms involved in endometriosis development. Moreover, iron can generate free radical species able to react with a wide range of cellular constituents, inducing cellular damage. Overproduction of reactive oxygen species also impairs cellular function by altering gene expression via regulation of redox-sensitive transcription factors such as NF-kappaB, which is clearly implicated in endometriosis. Indeed, NF-kappaB is activated in endometriotic lesions and peritoneal macrophages of endometriosis patients, which stimulates synthesis of proinflammatory cytokines, generating a positive feedback loop in the NF-kappaB pathway. NF-kappaB-mediated gene transcription promotes a variety of processes, including endometriotic lesion establishment, maintenance and development. In conclusion, iron and NF-kappaB appear to be linked and both are clearly involved in endometriosis development, making these pathways an attractive target for future treatment and prevention of this disease.

Laparoscopic management of endometriomas using a combined technique of excisional (cystectomy) and ablative surgery.

OBJECTIVE: To describe and evaluate a new technique of laparoscopic treatment of endometriomas that combines excisional and ablative surgery. DESIGN: Descriptive and prospective study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Fifty-two women under 35 years of age presenting for infertility and/or pelvic pain with endometriomas larger than 3 cm were included in the study. None had undergone any surgery for endometriosis. INTERVENTION(S): A large part of the endometrioma wall was first excised according to the cystectomy technique. After this first step, CO(2) laser was used to vaporize the remaining 10%-20% of the endometrioma wall close to the hilus. MAIN OUTCOME MEASURE(S): The feasibility of this new technique was assessed. Ovarian volume and antral follicle count (AFC) were compared between operated ovaries and nonoperated ovaries of patients with endometriosis and controls (women with male factor infertility). RESULT(S): The combined technique was possible in all cases. The volume of the ovary after the combined technique was similar to that of the contralateral normal ovary, as well as to that observed in infertile women without endometriosis presenting for male factor infertility. The AFC on day 2-5 showed the same number of antral follicles in all subgroups. Histopathology of the excised part of the endometrioma revealed the presence of follicles in only one case (2%). The pregnancy rate was 41% at a mean follow-up of 8.3 months. Recurrence of a small endometrioma was observed in only one case (2%). CONCLUSION(S): The combined technique (stripping and ablation) has proved not to be deleterious to the ovary.

Involvement of the nuclear factor-κB pathway in the pathogenesis of endometriosis.

OBJECTIVE: To evaluate the role of nuclear factor-κB (NF-κB) in the pathogenesis of endometriosis. DESIGN: A literature search was conducted in PubMed to identify all relevant citations. RESULT(S): Our findings highlight the important role of NF-κB in the pathophysiology of endometriosis. In vitro and in vivo studies show that NF-κB-mediated gene transcription promotes inflammation, invasion, angiogenesis, and cell proliferation and inhibits apoptosis of endometriotic cells. Constitutive activation of NF-κB has been demonstrated in endometriotic lesions and peritoneal macrophages of endometriosis patients. Agents blocking NF-κB are effective inhibitors of endometriosis development and some drugs with known NF-κB inhibitory properties have proved efficient at reducing endometriosis-associated symptoms in women. Iron overload activates NF-κB in macrophages. NF-κB activation in macrophages and ectopic endometrial cells stimulates synthesis of proinflammatory cytokines, generating a positive feedback loop in the NF-κB pathway and promoting endometriotic lesion establishment, maintenance and development. CONCLUSION(S): NF-κB transcriptional activity modulates key cell processes contributing to the initiation and progression of endometriosis. Because endometriosis is a multifactorial disease, inhibiting NF-κB appears to be a promising strategy for future therapies targeting different cell functions involved in endometriosis development, such as cell adhesion, invasion, angiogenesis, inflammation, proliferation, and apoptosis. Upcoming research will elucidate these hypotheses.

Review: luminescence as a tool to assess pelvic endometriosis development in murine models.

Classic murine endometriosis models may be insufficient to evaluate the effect of therapeutic agents on endometriosis development, because the process of identification and measurement of induced lesions is often impeded, as implants are small and embedded in murine tissue. In this context, as summarized in the current review, luminescence techniques have proved useful for identifying and visualizing or quantifying endometriotic transplants. They are also a valuable tool for endometrial cell tracking in live animals, yielding further information by adding spatial and temporal dimensions to biological processes in vivo. Such approaches involve transplanting luminescently labeled murine or human endometrium into animals. Two main strategies are applied to label endometrium before injection: use of genetically modified tissue or tissue labeled with a fluorescent dye. Each model has its advantages and disadvantages, the choice of model depends on the study objectives/design (long- or short-term studies, homologous or heterologous model).

Iron storage is significantly increased in peritoneal macrophages of endometriosis patients and correlates with iron overload in peritoneal fluid.

OBJECTIVE: To further investigate peritoneal iron disruption in endometriosis by studying iron storage in peritoneal macrophages of patients with endometriosis compared with controls. DESIGN: Cross-sectional study. SETTING: Academic gynecology research unit in a university hospital. PATIENT(S): Fifty patients undergoing laparoscopy. INTERVENTION(S): Collection of peritoneal fluid samples (N = 50) from patients with (n = 27) and without (n = 23) endometriosis undergoing laparoscopy. MAIN OUTCOME MEASURE(S): Quantification of peritoneal macrophage ferritin by immunocytochemical staining and immunodensitometry and measurement of peritoneal iron, transferrin, ferritin, and prohepcidin concentrations. RESULT(S): The optical density of peritoneal macrophage ferritin staining was statistically significantly higher in endometriosis patients than in controls. Higher iron concentrations, transferrin saturations, and ferritin concentrations were also detected in case of endometriosis. A statistically significant positive correlation was found between the optical density of macrophage ferritin staining and peritoneal iron concentrations in endometriosis and control patients. CONCLUSION(S): Iron storage is statistically significantly increased in peritoneal macrophages of patients with endometriosis and correlates with iron overload in peritoneal fluid. The potential implications of iron accumulation in peritoneal macrophages in case of endometriosis are discussed.

Laparoscopic observation of spontaneous human ovulation.

We report laparoscopic observation of spontaneous human ovulation, illustrated by protrusion of the mature follicle and oocyte release into the peritoneal cavity.

Increased activation of nuclear factor-kappa B (NF-kappaB) in isolated peritoneal macrophages of patients with endometriosis.

Endometriosis is characterized by pelvic inflammation that shows an increased number of activated peritoneal macrophages and their secreted products such as cytokines, growth factors, and angiogenic factors. Our results show that activation of nuclear factor-kappa B (NF-kappaB), a pro-inflammatory transcription factor, is statistically significantly increased in peritoneal macrophages from patients with endometriosis when compared with controls.

Nuclear factor-kappa B is constitutively activated in peritoneal endometriosis.

Red (active), black and white endometriotic lesions are characteristic of peritoneal endometriosis. The transcription factor nuclear factor-kappa B (NF-kappaB) activates proinflammatory, proliferative and antiapoptotic genes in many cell types. To determine whether NF-kappaB is activated in peritoneal endometriosis in women, and further ascertain the differential inflammatory status of endometriotic implants, NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression were investigated in peritoneal endometriotic lesions according to their type. Furthermore, p65 and p50 subunits of active NF-kappaB dimers were evaluated in endometriotic lesions to gain some insight into NF-kappaB-implicated pathways. Thirty-six biopsies of peritoneal endometriotic lesions were analyzed. Constitutive NF-kappaB activation, involving p65- and p50-containing dimers, was demonstrated in peritoneal endometriotic lesions by electrophoretic mobility shift assays and supershift analyses, as well as NF-kappaB (p65) DNA-binding activity immunodetection assays. NF-kappaB activation and ICAM-1 expression (evaluated by immunoblotting) were significantly higher in red lesions than black lesions, whereas IkappaBalpha (NF-kappaB inhibitory protein) expression was constant, as shown by western blot analysis. This is the first study to demonstrate constitutive NF-kappaB activation in peritoneal endometriosis in women. NF-kappaB activation and ICAM-1 expression in red lesions confirm the more extensive inflammatory pattern of these lesions compared with black lesions. The involvement of p50/p65 dimers in NF-kappaB activation suggests implication of the classic NF-kappaB activation pathway, making it an attractive therapeutic target in endometriosis.