Gabriela Meresman

Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis

OBJECTIVE To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. DESIGN Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. SETTING Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. PATIENT(S) Women with untreated endometriosis (n = 14) and controls (n = 16). INTERVENTION(S) Collection of endometrial samples during diagnostic or therapeutic laparoscopy. MAIN OUTCOME MEASURE(S) Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. RESULT(S) Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. CONCLUSION(S) Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

Oral contraceptives suppress cell proliferation and enhance apoptosis of eutopic endometrial tissue from patients with endometriosis

OBJECTIVE To evaluate the effects of administering combination oral contraceptives (COCs) to patients with endometriosis on the regulation of cell growth in the eutopic endometrium. DESIGN Prospective study. SETTING Research institute and clinical fertility center. PATIENT(S) Thirteen women with untreated endometriosis and 13 controls. INTERVENTION(S) Biopsy specimens of the eutopic endometrium were obtained from all subjects. Apoptosis, cell proliferation, and Bcl-2 and Bax expression were examined at the epithelial and stromal levels in the eutopic endometrium from patients with endometriosis before and after 30 days of daily exposure to COCs and from controls. MAIN OUTCOME MEASURE(S) Apoptotic cells were detected by using the dUTP nick-end labeling assay; Ki-67, Bcl-2, and Bax expressions were assessed by using immunohistochemical techniques. RESULT(S) After exposure to COCs, apoptosis was significantly increased in the eutopic endometrium compared with before COC administration, both at epithelial and stromal levels. Cell proliferation was significantly lowered by COCs. CONCLUSION(S) COCs showed a positive effect on patients with endometriosis by down-regulating cell proliferation and enhancing apoptosis in the eutopic endometrium.

Effect of Vascular Endothelial Growth Factor Inhibition on Endometrial Implant Development in a Murine Model of Endometriosis

The main factor involved in neovascularization of ectopic endometrial tissue in endometriosis is the vascular endothelial growth factor (VEGF), which is produced both by the endometrial implant and by peritoneal macrophages. On the other hand, bevacizumab is an antiangiogenic agent used in the treatment of different tumors, like colorectal, pulmonary, and recently mammary. We evaluated the effect of the inhibition of VEGF activity with bevacizumab (Avastin) on ectopic endometrial growth in a murine model of endometriosis. Two months old female BALB/c mice had surgery performed to induce endometriotic-like lesions. Treatment with bevacizumab started on post-surgery day 15 and continued during 2 weeks. Then, animals were sacrificed, peritoneal fluid was collected, and endometriotic-like lesions were counted, measured, and removed. Cell proliferation, vascular density, and apoptosis were assessed by immunohistochemistry for proliferating cell nuclear antigen (PCNA), immunohistochemistry for CD34, and Terminal Deoxynucleotidil Transferase-Mediated dUTP Nick End Labeling (TUNEL), respectively. Vascular endothelial growth factor levels were evaluated in the peritoneal fluid by enzyme-linked immunoassay (ELISA). Treatment with bevacizumab significantly inhibited endometriotic lesion development (P < .05). Consistently, bevacizumab significantly inhibited cell proliferation in lesions (P < .01), reduced vascular density (P < .001), as well as increased the apoptotic cell percentage (P < .001). In addition, bevacizumab reduced VEGF levels in peritoneal fluid of endometriosis-induced animals (P < .05). In conclusion, this study suggests a direct effect of bevacizumab on the reduction of endometrial implant growth and supports further research on VEGF inhibition as a novel therapeutic modality in endometriosis.

Gonadotropin-releasing hormone agonist induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis

OBJECTIVE There is growing evidence that suggests a direct action of gonadotropin-releasing hormone agonist (GnRH-a) on endometrial growth. Consequently, our purpose was to evaluate the effect of GnRH-a on in vitro eutopic endometrial cell growth and apoptosis. DESIGN Prospective study. SETTING Research institute and clinical fertility center. PATIENT(S) Sixteen women with untreated endometriosis and 14 controls. INTERVENTION(S) Biopsy specimens of eutopic endometrium were obtained from all subjects. Apoptosis and cell proliferation were examined in epithelial endometrial cell cultures after incubation with leuprolide acetate (LA), antide, and a combination of both. MAIN OUTCOME MEASURE(S) The percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique; cell proliferation was assessed by (3)H-thymidine incorporation. RESULT(S) Leuprolide acetate (LA) (100 ng/mL) enhanced apoptosis in endometrial cultures from patients with endometriosis and controls, and this effect was reversed by antide 10(-7)M. Cell proliferation was down-regulated by LA at 1, 10, and 100 ng/mL in cultures from women without and with endometriosis. The addition of antide 10(-7)M reversed this inhibition. CONCLUSION(S) GnRH-a appears to have a direct effect by enhancing the apoptotic index and decreasing the cell proliferation in endometrial cells.

Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis

BACKGROUND Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs). METHODS AND RESULTS Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 microM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by (3)H-thymidine uptake. Celecoxib at 50, 75 and 100 microM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange-Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 microM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 microM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E(2) (PGE(2)) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE(2) did not reverse celecoxib-induced growth inhibition. CONCLUSIONS This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

The inhibitory effect of celecoxib and rosiglitazone on experimental endometriosis

OBJECTIVE To evaluate the effects of celecoxib and rosiglitazone on the implantation and growth of endometriotic-like lesions in a murine model of endometriosis. DESIGN Prospective experimental study. SETTING Animal research and laboratory facility. ANIMAL(S) Two-month-old female BALB/c mice. INTERVENTION(S) Surgically induced endometriosis in female BALB/C mice; 28 days of treatment with celecoxib, rosiglitazone, or their combination; counting, measuring, excising, and fixing lesions. MAIN OUTCOME MEASURE(S) Immunohistochemical examination for proliferating cell nuclear antigen (PCNA), CD31, and CD34 to assess cell proliferation and vascularization, with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique for apoptosis evaluation. RESULT(S) Celecoxib and the combined treatment (celecoxib and rosiglitazone) statistically significantly reduced the mean number of lesions established per mouse, and all treatments diminished the implant volume. In addition, cell proliferation within the implants was statistically significantly reduced, and apoptosis was statistically significantly enhanced by all treatments. Also, we found that all treatments diminished the vascularized area in the lesion. CONCLUSION(S) These results are promising and reveal that celecoxib and rosiglitazone, combined or separately, have a beneficial effect on overall endometriotic growth.

Effect of aromatase inhibitors on ectopic endometrial growth and peritoneal environment in a mouse model of endometriosis

OBJECTIVE To evaluate the effect of aromatase inhibitors on ectopic endometrial growth and on the release of proangiogenic and proinflammatory factors in peritoneal fluid (PF). DESIGN Prospective experimental study. SETTING Animal research and laboratory facility. ANIMAL(S) Female Balb/c mice 2 months of age. INTERVENTION(S) Mice had surgery performed to induce endometriosis-like lesions. Treatment with anastrozole or letrozole was started on either postoperative day 1 or 28 and continued for 4 weeks. MAIN OUTCOME MEASURE(S) Endometriotic lesions were counted and measured and aromatase expression, cell proliferation, and apoptosis were assessed. Vascular endothelial growth factor (VEGF) and prostaglandin E (PGE) levels were evaluated in the PF. RESULT(S) Endometriosis-like lesions express aromatase P-450. Treatment with either anastrozole or letrozole did not prevent lesion establishment; however, it significantly decreased the size of the endometriotic lesion. When treatment was initiated on postoperative day 1, letrozole and anastrozole decreased cell proliferation and increased apoptosis. When treatment was started on postoperative day 28, both aromatase inhibitors decreased cell proliferation, but only anastrozole augmented apoptosis levels. In addition, letrozole reduced VEGF and PGE levels in PF. Anastrozole diminished VEGF content but did not cause any significant change in PGE levels. CONCLUSION(S) These findings support the further investigation of aromatase inhibition as a treatment option for endometriosis.

Functional and phenotypic alterations in peritoneal macrophages from patients with early and advanced endometriosis

Abstract. The aim of this study was to elucidate whether peritoneal macrophage (pMO) alterations are a generalized feature in all stages of endometriosis and the effect of hormonal treatment on this leukocyte population. For this purpose we quantified the number of pMO, the expression of HLA-DR antigen (pMO DR+), percentages of pMO that reduced nitro-blue tetrazolium (pMO NBT+), and interleukin-1 (IL-1) and prostaglandin E2 (PGE2) production by pMO from patients with early (stages I/II) and advanced (stages III/IV) endometriosis, we also analyzed some of these properties in pMO from patients which had been treated for 6 months with 800 mg/day of Danazol or gonadotropin releasing hormone agonist (GnRHa). We found that there were a significant increase of the pMO number in both types of patients, though the highest values were obtained in early endometriosis (p<0.001). Percentages of pMO DR+ were decreased in all patients (p<0.01) while percentages of pMO NBT+ were significantly increased. Production of IL-1 by early and advanced endometriosis pMO were considerably enhanced. PGE2 release was not altered in early endometriosis pMO but, in advanced endometriosis, pMO PGE2 levels were 100-fold higher than control values. In post-treatment patients, the number of pMO and percentage of pMO NBT+ were similar to early endometriosis patients, though the percentage of pMO DR+ was within the normal range. We conclude that the pMO population, as well as IL-1 and PGE2 production, were altered in all stages of endometriosis, and that these changes could be involved in the pathogenesis of endometriosis and associated infertility. Hormonal treatments do not reverse the pMO changes.

Natural therapies assessment for the treatment of endometriosis

STUDY QUESTION 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)? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. MATERIALS, SETTING, METHODS 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. MAIN RESULTS 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. LIMITATIONS In vitro studies were only carried out in epithelial cells from human eutopic endometrium. WIDER IMPLICATIONS OF THE FINDINGS The present findings are promising and will assist the development of novel natural treatments for endometriosis. STUDY FUNDING 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.

The Role of GnRH Analogues in Endometriosis-Associated Apoptosis and Angiogenesis

It has been postulated that gonadotropin-releasing hormone (GnRH) analogues may act directly on endometrial cells and inhibit their growth and proliferation by regulation of apoptotic and angiogenic mechanisms. Eutopic endometrial cells from patients with endometriosis show an increased proliferation rate and are less susceptible to cell death by apoptosis than those from subjects without the disease. Notably, the GnRH analogue, leuprorelin, inhibits cell proliferation and increases the apoptotic rate in eutopic endometrial cell cultures, an effect that appears to be mediated by an increase in the expression of the pro-apoptotic proteins Bax and FasL and a decrease in the expression of the anti-apoptotic protein Bcl-2. Angiogenesis is an important process in the development of endometrial tissue, and it is regulated by vascular endothelial growth factors (VEGFs) and angiopoietins. VEGF levels are elevated in peritoneal fluid and endometriotic tissue from patients with endometriosis. In addition, it has been demonstrated that the expression of VEGF is potentiated by a variety of cytokines, including IL-1β. Recent studies show that leuprorelin reduces the production of VEGF-A and IL-1β in eutopic endometrial cell cultures, suggesting a mechanism by which it could inhibit the development of endometriosis. Thus, GnRH analogues appear to be effective in reducing the growth of endometrial cells, not only due to their classical pituitary endocrine effects, but also via a direct effect on the endometrial cells themselves.