Assessment of the Ferroptosis Regulators: Glutathione Peroxidase 4, Acyl-Coenzyme A Synthetase Long-Chain Family Member 4, and Transferrin Receptor 1 in Patient-Derived Endometriosis Tissue.

Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case-control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = -2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2-13.5) g/dL versus 12.5 (12.2-13.4) g/dL in the control group; t (54) = -0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations.

A physiological concentration of anandamide promotes the migration of human endometrial fibroblast and the interaction with endothelial cells invitro.

INTRODUCTION: The mechanisms that govern fibroblast behavior during the vascular adaptations of the uterus at early pregnancy remain unknown. Anandamide, an endocannabinoid, binds to cannabinoid receptors (CBs), and regulates gestation and angiogenesis. Its tone is regulated by fatty acid amide hydrolase (FAAH) within the uterus. We investigated the role of anandamide in endometrial fibroblasts migration and whether anandamide modulates fibroblasts-endothelial crosstalk. METHODS: T-hESC and EA.hy926 cell lines were used as models of endometrial stromal and endothelial cells, respectively. T-hESC were incubated with anandamide plus different agents. Migration was tested (wound healing assay and phalloidin staining). Protein expression and localization were studied by Western blot and immunofluorescence. To test fibroblast-endothelial crosstalk, EA.hy926 cells were incubated with fibroblast conditioned media obtained after T-hESC migration. RESULTS: Anandamide 1 nM increased T-hESC migration via CB1 and CB2. Cyclooxygenase-2 participated in anandamide-stimulated fibroblast migration. Prostaglandin F2alpha, and not prostaglandin E2, increased fibroblast wound closure. CB1, CB2, cyclooxygenase-2 and FAAH were expressed in T-hESC. Anandamide did not alter cyclooxygenase-2 localization but induced its cytoplasmic and nuclear expression through CB1 and CB2. URB-597, a FAAH selective inhibitor, also increased T-hESC migration via both CBs, and augmented cyclooxygenase-2 expression. Conditioned media from anandamide-induced T-hESC wound healing closure stimulated endothelial migration and did not alter their proliferation. Soluble factors from cyclooxygenase-2 were secreted by T-hESC and participated in T-hESC-induced EA.hy926 migration. Although anandamide-conditioned media augmented in EA.hy926 the expression of γH2AX, a marker of DNA damage, cyclooxygenase-2 was not involved in this effect. DISCUSSION: Our results provide novel evidence about an active role of anandamide on endometrial fibroblast behavior as a mechanism regulating uterine vascular adaptations in early gestation.

Effects of pharmacological inhibition of hyaluronic acid synthesis on experimental endometriosis.

BACKGROUND: Dysregulated hyaluronic acid (HA) metabolism has been shown to be implicated in several pathologies including endometriosis. 4-Methylumbelliferone (4MU) is an HA synthesis inhibitor with proven antitumour activity. In this study, we aim to evaluate the effect of 4MU on endometriosis development both in vivo and in vitro. METHODS: Endometriosis was surgically induced by uterine tissue auto-transplantation in 32 two-month-old BALB/c mice. Animals were designated into the early or late starting treatment group, which initiated on day 2 or day 15 after surgery, respectively. Within each group, 4MU 200 mg/kg/day or vehicle (Control) were administered by oesophageal gavage for 28 days. After sacrifice, the percentage of developed lesions, lesion size, cell proliferation, vascularization and HA deposition within the endometriotic-like lesions were evaluated. Cell viability was assessed in endometrial epithelial cells (ECC-1) and in endometrial stromal cells (t-HESC); and migration was evaluated in t-HESC. RESULTS: There was a significant reduction in the percentage of developed lesions in mice that started the 4MU treatment on day 2 compared with its respective control group, and compared with those that started treatment on day 15. However, no significant changes were found when analysing endometriotic-like lesion's cell proliferation, vascularization and HA deposition. In vitro, both cell viability and migration were inhibited by 4MU treatment. CONCLUSIONS: The inhibition of HA synthesis could be a beneficial and alternative option to treat endometriosis at the early stage of the disease. Further research is necessary to elucidate 4MU's mechanism of action and better strategies for delivering this promising drug.

Resveratrol impairs cellular mechanisms associated with the pathogenesis of endometriosis.

RESEARCH QUESTION: Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes? DESIGN: In-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR. RESULTS: Resveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells. CONCLUSION: Resveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

Endocrine disruptor hexachlorobenzene induces cell migration and invasion, and enhances aromatase expression levels in human endometrial stromal cells.

Endometriosis is the presence and growth of endometrial tissue outside of the uterus. Previous studies have suggested that endocrine disrupting chemicals such as organochlorine pesticides could be a risk factor for endometriosis. Hexachlorobenzene (HCB) is a weak ligand of the aryl hydrocarbon receptor (AhR) and promotes metalloproteinase and cyclooxygenase-2 (COX-2) expression, as well as, c-Src activation in human endometrial stromal cells (T-HESC) and in rat endometriosis model. Our aim was to evaluate the effect of HCB exposure on oestrogen receptor (ER) ɑ and ß, progesterone receptor (PR) and aromatase expression, as well as, on cell migration and invasion in T-HESC and primary cultures of endometrial stromal cells from eutopic endometria of control subjects (ESC). Results show that HCB increases ERɑ and aromatase protein levels and reduces PR content in both T-HESC and ESC. However, the pesticide only increases ERß expression in ESC, without changes in T-HESC. Moreover, cell migration and invasion are promoted by pesticide exposure involving the AhR, c-Src, COX-2 and ER pathways in T-HESC. HCB also triggers ERɑ activation via phosphorylation in Y537 through AhR/c-Src pathway. Our results provide experimental evidence that HCB induces alterations associated with endometriosis, suggesting that these mechanisms could contribute to pesticide exposure-induced endometriosis development.

Plants as source of new therapies for endometriosis: a review of preclinical and clinical studies.

BACKGROUND: Given the disadvantages and limitations of current endometriosis therapy, there is a progressive increase in studies focusing on plant-derived agents as a natural treatment option with the intention of achieving high efficiency, avoiding adverse effects and preserving the chance for successful pregnancy. The heterogeneity of these studies in terms of evaluated agents, applied approaches and outcomes illustrates the need for an up-to-date summary and critical view on this rapidly growing field in endometriosis research. OBJECTIVE AND RATIONALE: This review provides a comprehensive overview of plant-derived agents and natural treatment strategies that are under preclinical or clinical investigation and critically evaluates their potential for future endometriosis therapy. SEARCH METHODS: An English language PubMed literature search was performed using variations of the terms 'endometriosis', 'natural therapy', 'herb/herbal', 'plant', 'flavonoid', 'polyphenol', 'phytochemical', 'bioactive', 'Kampo' and 'Chinese medicine'. It included both animal and human studies. Moreover, the Clinicaltrials.gov database was searched with the term 'endometriosis' for clinical trials on plant-derived agents. No restriction was set for the publication date. OUTCOMES: Natural therapies can be assigned to three categories: (i) herbal extracts, (ii) specific plant-derived bioactive compounds and (iii) Chinese herbal medicine (CHM). Agents of the first category have been shown to exert anti-proliferative, anti-inflammatory, anti-angiogenic and anti-oxidant effects on endometrial cells and endometriotic lesions. However, the existing evidence supporting their use in endometriosis therapy is quite limited. The most studied specific plant-derived bioactive compounds are resveratrol, epigallocatechin-3-gallate, curcumin, puerarin, ginsenosides, xanthohumol, 4-hydroxybenzyl alcohol, quercetin, apigenin, carnosic acid, rosmarinic acid, wogonin, baicalein, parthenolide, andrographolide and cannabinoids, with solid evidence about their inhibitory activity in experimental endometriosis models. Their mechanisms of action include pleiotropic effects on known signalling effectors: oestrogen receptor-α, cyclooxygenase-2, interleukin-1 and -6, tumour necrosis factor-α, intercellular adhesion molecule-1, vascular endothelial growth factor, nuclear factor-kappa B, matrix metalloproteinases as well as reactive oxygen species (ROS) and apoptosis-related proteins. Numerous studies suggest that treatment with CHM is a good choice for endometriosis management. Even under clinical conditions, this approach has already been shown to decrease the size of endometriotic lesions, alleviate chronic pelvic pain and reduce postoperative recurrence rates. WIDER IMPLICATIONS: The necessity to manage endometriosis as a chronic disease highlights the importance of identifying novel and affordable long-term safety therapeutics. For this purpose, natural plant-derived agents represent promising candidates. Many of these agents exhibit a pleiotropic action profile, which simultaneously inhibits fundamental processes in the pathogenesis of endometriosis, such as proliferation, inflammation, ROS formation and angiogenesis. Hence, their inclusion into multimodal treatment concepts may essentially contribute to increase the therapeutic efficiency and reduce the side effects of future endometriosis therapy.

PI3K/AKT pathway is altered in the endometriosis patient's endometrium and presents differences according to severity stage.

Based on the inflammatory nature and hormone-dependency of endometriosis, PI3K/AKT signaling appears to influence its progression. Could the endometriosis stages be linked to differential changes in PI3K/AKT pathway regulation? The objective is to evaluate the expression of PI3K, PTEN, AKT and p-AKT in endometrial human biopsies, according to the presence or absence of the disease, and to assess the underlying differences regarding the endometriosis stages. Biopsy specimens of the ectopic and eutopic endometrium were obtained from twenty women with untreated peritoneal endometriosis as well as endometrium biopsies from nine controls. Our study revealed an increased expression of PI3K in eutopic and ectopic endometrium from patients with endometriosis, and a reduced expression of PTEN and increased levels of AKT phosphorylation, compared to control endometrium. Both eutopic and ectopic endometrium from patients with minimal-mild endometriosis expressed a significant reduced PTEN level compared to the respective endometrium from patients with moderate-severe endometriosis. The ratio p-AKT/total AKT showed higher levels of AKT phosphorylation in endometriotic tissue from patients with minimal-mild endometriosis. This study has firmly confirmed the alteration in PI3K/AKT pathway regulation and demonstrated clear differences between the stages of endometriosis, emphasizing the importance of this pathway in the first stage of the disease.

Active compounds present inRosmarinus officinalis leaves andScutellaria baicalensis root evaluated as new therapeutic agents for endometriosis.

RESEARCH QUESTION: Can carnosic acid, (CA) rosmarinic acid (RA) and wogonin (WG) inhibit the growth of cultured human endometrial stromal cells and endometriotic-like lesions induced in a BALB/c model of endometriosis? DESIGN: Primary stromal cell cultures were established from endometrial biopsies from women with endometriosis and controls. The human endometrial stromal cell line T-HESC was also used for in-vitro experiments. Endometriosis was surgically induced in BALB/c mice, which were randomly assigned to CA 2 mg/kg/day (n = 11); CA 20 mg/kg/day (n = 10); RA 1 mg/kg/day (n = 11); RA 3 mg/kg/day (n = 10); WG 20 mg/kg/day (n = 12); intraperitoneal vehicle control (n = 8) or oral vehicle control (n = 11). After surgery, CA and RA were administered intraperitoneally on days 14-28. WG was administered orally by intragastric gavage on days 14-26. RESULTS: CA, RA and WG significantly inhibited in-vitro cell proliferation in primary and T-HESC cell cultures (P < 0.05). CA and WG induced cell cycle arrest of T-HESC at the G2/M phase (P < 0.01). RA reduced intracellular ROS accumulation (P < 0.001), whereas WG increased it (P < 0.05). WG significantly inhibited oestrogen receptor alpha expression in T-HESC (P < 0.01). In-vivo, CA, RA and WG significantly reduced lesions size (P < 0.05). All compounds significantly decreased the percentage of cells in proliferation (P < 0.05) whereas RA and WG further increased the percentage of apoptotic cells (P < 0.05) in endometriotic-like lesions. CONCLUSIONS: The results are promising; further investigation of these compounds as new therapeutics is needed.

TNFRp55 deficiency promotes the development of ectopic endometriotic-like lesions in mice.

Endometriosis is an inflammatory disease depending on estradiol, with TNF-α being one of the most representative cytokines involved in its pathogenesis. TNF-α acts through its bond to the TNFRp55 and TNFRp75 membrane receptors. The aim of this study was to analyze the effect of the TNFRp55 deficiency on the development of ectopic endometriotic-like lesions. Endometriosis was induced surgically in mice of the C57BL/6 strain, wild type (WT) and TNFRp55-/- (KO). After four weeks, the peritoneal fluid was collected and the lesions were counted, measured with a caliper, removed, weighed, fixed or kept at -80°C. We evaluated the cell proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry and apoptosis by TUNEL technique in the ectopic lesions. MMP-2 and MMP-9 activities (factors involved in invasiveness) were measured by zymography in the peritoneal fluid; estradiol and progesterone levels were measured by radioimmunoassay in the lesions and in the peritoneal fluid. We found that in KO animals the mean number of lesions established per mouse, the lesion volume, weight and cell proliferation increased and apoptosis decreased. In addition, the activity of MMP-2 and the estradiol level increased, whereas the progesterone level was not significantly modified. In conclusion, the deficiency of TNFRp55 promoted the establishment and development of endometriosis through an increase in the lesion size and high levels of estradiol which correlate with an increase in the MMP-2 activity. This is evidence of the possible association of the deregulation of the TNFRp55 expression and the survival of the endometriotic tissue in ectopic sites.

Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells.

Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells.

Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions.

BACKGROUND: The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis. OBJECTIVE: To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis. MATERIALS AND METHODS: The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20 mg/kg 4-MU (n = 8) or 80 mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry. MAIN RESULTS: Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions. CONCLUSIONS: The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.

Interplay between Endometriosis and Pregnancy in a Mouse Model.

OBJECTIVES: To evaluate the effect of endometriosis on fertility and the levels of the IL-2 and IFN-γ in the peritoneal fluid in a mouse model; to evaluate the effect of pregnancy on endometriotic lesion growth, apoptosis and cell proliferation. STUDY DESIGN: Two month old C57BL/6 female mice underwent either a surgical procedure to induce endometriosis or a sham surgery. Four weeks after surgery mice were mated and sacrificed at day 18 of pregnancy. Number of implantation sites, fetuses and fetal weight were recorded. Endometriotic lesions were counted, measured, excised and fixed. Apoptosis and cell proliferation were evaluated in lesions by TUNEL and immunohistochemistry for PCNA respectively. Levels of IL-2 and IFN-γ were assessed by ELISA in the peritoneal fluid. RESULTS: Pregnancy rate (i.e. pregnant mice/N) decreased in mice with endometriosis. However there were no significant differences in resorption rate, litter size and pup weight between groups. IFN-γ augmented in endometriosis mice independently of pregnancy outcome. Additionally IFN-γ increased in pregnant endometriosis mice compared to pregnant sham animals. While IFN-γ increased in non pregnant versus pregnant mice in the sham group, IL-2 was increased in non pregnant mice in the endometriosis group. The size of endometriotic lesions increased in pregnant mice while apoptosis increased in the stroma and cell proliferation decreased in the epithelium of these lesions. Additionally, leukocyte infiltration, necrosis and decidualization were increased in the same lesions. CONCLUSIONS: Pregnancy rate is reduced in this mouse model of endometriosis. Levels of IL-2 are increased in the peritoneal fluid of mice with endometriosis suggesting a role of this cytokine in infertility related to this disease. The size of endometriotic lesions is increased in pregnant mice; however pregnancy has a beneficial effect on lesions by decreasing cell proliferation and by increasing apoptosis, decidualization and necrosis.

Local VEGF inhibition prevents ovarian alterations associated with ovarian hyperstimulation syndrome.

The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases its severity in rats.

Targeting galectin-1-induced angiogenesis mitigates the severity of endometriosis.

Endometriosis is characterized by the presence of endometrial tissue outside the uterus that causes severe pelvic pain and infertility in women of reproductive age. Although not completely understood, the pathophysiology of the disease involves chronic dysregulation of inflammatory and vascular signalling. In the quest for novel therapeutic targets, we investigated the involvement of galectin-1 (Gal-1), an endogenous glycan-binding protein endowed with both immunosuppressive and pro-angiogenic activities, in the pathophysiology of endometriotic lesions. Here we show that Gal-1 is selectively expressed in stromal and endothelial cells of human endometriotic lesions. Using an experimental endometriosis model induced in wild-type and Gal-1-deficient (Lgals1(-/-) ) mice, we showed that this lectin orchestrates the formation of vascular networks in endometriotic lesions in vivo, facilitating their ectopic growth independently of vascular endothelial growth factor (VEGF) and the keratinocyte-derived CXC-motif (CXC-KC) chemokine. Targeting Gal-1 using a specific neutralizing mAb reduced the size and vascularized area of endometriotic lesions within the peritoneal compartment. These results underline the essential role of Gal-1 during endometriosis and validate this lectin as a possible target for the treatment of disease.

Anastrozole and celecoxib for endometriosis treatment, good to keep them apart?

Endometriosis is a benign gynecological disease. Cyclooxygenase-2 (COX-2) and aromatase proteins have been shown to be overexpressed in eutopic endometrium from women suffering from this disease compared to disease-free women. Furthermore, inhibition of these molecules individually was demonstrated to have antiproliferative and proapoptotic effects both in vitro and in vivo in several models. In this study, the effect of combining celecoxib, a selective COX-2 inhibitor, and anastrozole, an aromatase inhibitor, on the implantation and growth of endometriotic like lesions in a murine model of endometriosis was evaluated. Endometriosis was surgically induced in female BALB/c mice. After 28 days of treatment with celecoxib, anastrozole, or their combination, animals were killed and lesions were counted, measured, excised, and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for assessment of cell proliferation and vascularization. TUNEL technique was performed for apoptosis evaluation. Celecoxib was the only treatment to significantly reduce the number of lesions established per mouse, their size and vascularized area. In addition, cell proliferation was significantly diminished and apoptosis was significantly enhanced by both individual treatments. When the therapies were combined, they reversed their effects. These results confirm that celecoxib and anastrozole separately decrease endometriotic growth, but when combined they might have antagonizing effects.

[Relevance of apoptosis in the female reproductive system]. / Relevancia de la apoptosis en la reproducción femenina.

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.

Relevancia de la apoptosis en la reproducción femenina / Relevance of apoptosis in the female reproductive system

La apoptosis es un proceso genéticamente controlado mediante el cual las células inducen su propia muerte. Mensualmente y en forma cíclica, el ovario, el endometrio y la glándula mamaria atraviesan por ciclos de proliferación celular y apoptosis respondiendo a los cambios en la secreción hormonal. Durante el desarrollo embrionario, la apoptosis está implicada en procesos relacionados con la escultura de los diferentes órganos, a través de la eliminación de estructuras innecesarias y con el control de las células defectuosas. Asimismo, la apoptosis juega un papel fundamental en la función ovárica. La reserva folicular se establece durante la vida fetal y luego se va eliminando gradualmente. La apoptosis está involucrada tanto en la muerte celular durante el proceso de reclutamiento del folículo dominante, como en la luteólisis. Durante la pubertad la apoptosis contribuye a la formación del espacio luminal de los ductos terminales de la mama. A su vez, el proceso de involución mamaria luego de la lactancia se caracteriza por una apoptosis masiva de las células epiteliales secretoras. Así como la apoptosis está involucrada en los cambios fisiológicos que ocurren a nivel endometrial, también se ha asociado a la muerte celular programada con procesos patológicos, especialmente en aquellos caracterizados por el incremento en el crecimiento celular como es el caso de la endometriosis. El delicado balance entre la apoptosis y la proliferación celular es fundamental ya que permite que los tejidos puedan responder en forma cíclica a los cambios hormonales fisiológicos y prevenir procesos de transformación neoplásica.

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.

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 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.

Apoptosis is increased and cell proliferation is decreased in out-of-phase endometria from infertile and recurrent abortion patients.

BACKGROUND: Various endometrial abnormalities have been associated with luteal phase deficiency: a significant dyssynchrony in the maturation of the glandular epithelium and the stroma and a prevalence of out-of-phase endometrial biopsy specimens. Out-of phase endometrium is a controversial disorder related to failed implantation, infertility and early pregnancy loss. Given that the regulation of the apoptotic process in endometrium of luteal phase deficiency is still unknown, the aim of this study was to evaluate cell proliferation, apoptosis and the levels of the main effector caspase, caspase-3 in the luteal in-phase and out-of-phase endometrium. METHODS: Thirty-seven endometrial samples from sterile or recurrent abortion patients were included in this study: 21 in-phase samples (controls) and 16 samples with out-of-phase endometrium. Biopsy specimens of eutopic endometrium were obtained from all subjects during days 21-25 of the menstrual cycle. The endometrium with endometrial maturity of cycle day 25 or less at the time of menstruation was considered out-of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhibiting DNA fragmentation, by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxygenin nick-end labeling (TUNEL) technique. Expressions of Proliferating Cell Nuclear Antigen (PCNA) as a marker of cell proliferation, and of cleaved caspase-3 as a marker of apoptosis, were assessed by immunohistochemistry in the luteal in-phase and out-of-phase endometrium from infertile and recurrent abortion patients. RESULTS: Luteal out-of-phase endometrium had increased apoptosis levels compared to in-phase endometrium (p < 0.05). Caspase-3 evaluation confirmed these results: the luteal out-of-phase endometrium showed augmented cleaved caspase-3 expression (p < 0.005). As well, our data demonstrated that the luteal out-of-phase endometrium expresses decreased PCNA levels (p < 0.05), showing that cell proliferation is diminished in this tissue. CONCLUSIONS: this study represents the first report describing variations at the cell proliferation and cell death levels in the out-of-phase endometrium in comparison with in-phase endometrium from infertile and recurrent abortion patients. Further studies are needed to elucidate a potential role of these alterations in the physiopathology of luteal phase deficiency.