Establishment of a novel mouse model of adenomyosis suitable for longitudinal and quantitative analysis and perinatal outcome studies.

The purpose of this study was to establish a novel mouse model of adenomyosis suitable for longitudinal and quantitative analyses and perinatal outcome studies. Using a 30 G needle, the entire uterine wall of one horn was mechanically punctured at a frequency of 100 times/1 cm (adenomyosis horn). The other horn was left unpunctured (control horn). Balb/c mice were sacrificed on day 14 (D14) or day 65 (D65) (n = 3 each). The uterus was fixed, paraffin-embedded, sliced, and stained. Lesions were detected and counted, and their volumes were measured. Cell proliferation and fibrosis were assessed by Ki67 and Masson's Trichrome staining, respectively. Blood vessels were detected using CD31 immunostaining. Some of the mice (n = 4), were mated and the date of delivery, litter size, number of implantations, and number and volume of postpartum lesions were measured. The number of lesions per horn did not differ between D14 and D65. The volume of the entire lesion was significantly greater on D65 than on D14 (p < 0.0001). The volume of the epithelial part of the lesion was significantly greater in D65 (p < 0.0001). The volume of the stromal part of the lesion was also greater on D65 (p < 0.0001). The percentage of Ki67 positive cells in the epithelial part of the lesion was significantly higher on D14 (p < 0.05). In contrast, the percentage of Ki67-positive cells in the stromal part was significantly higher on D65 (p < 0.01). Vascular density in the lesions was higher in on D65 (p < 0.05). The percentage of fibrotic area was significantly higher on D65 (p < 0.01). The date of delivery was slightly earlier than that reported for healthy mice of the same strain. The litter size was smaller than that reported in previous research. The number of implantation sites did not differ between the control and the adenomyosis horn. The number and volume of lesions did not differ between the non-pregnant and postpartum groups. This model can be applied to evaluate the pathogenesis of adenomyosis, validate the efficacy of therapeutic agents, and evaluate the effect of adenomyosis on pregnancy and vice versa.

The roles of polymorphonuclear myeloid-derived suppressor cells in endometriosis.

OBJECTIVES: This study aimed to determine the systemic and local proportions, focal localization, and characteristics of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in endometriosis. STUDY DESIGN: Peripheral blood and peritoneal fluid were obtained from patients with a benign gynecologic condition (controls) or endometriosis. PMN-MDSCs were defined as CD33+HLA-DRlow/-CD14-CD15+ and monocytic (M)-MDSCs were defined as CD33+HLA-DRlow/-CD14+CD15-, and were identified using flowcytometry. Ovarian endometriotic tissues were obtained, and the expression of lectin-type oxidized low density lipoprotein receptor-1 (LOX1) as a marker of PMN-MDSCs, arginine 1 (Arg1), and matrix metalloproteinase 9 (MMP9) were detected using immunohistochemistry. Anti-Ly6G antibody was administered to endometriosis model mice, and the number and weight of the lesions were measured, and cell proliferations and apoptosis in the lesions were analyzed using Ki67 immunohistochemistry and TUNEL assay. RESULTS: In the peripheral blood, the proportion of PMN-MDSCs was significantly higher in endometriosis (3.20 vs 1.63 %, p < 0.05), but the proportion of M-MDSCs did not differ between the groups. In the peritoneal fluid, the proportion of PMN-MDSCs was significantly higher in endometriosis (7.82 × 10-1% vs 6.48 × 10-2%, p < 0.05), whereas the proportion of M-MDSCs did not differ between the groups. PMN-MDSCs were detected in the stromal cell layer of the endometriotic cyst wall. Double staining for LOX1 and Arg1, and LOX1 and MMP9 was confirmed. Administration of Ly6G antibody did not change the number or weight of endometriosis lesions, but significantly decreased Ki67-positive cells and increased TUNEL-positive cells in the lesions. CONCLUSIONS: PMN-MDSCs may contribute to the pathogenesis of endometriosis via Arg1 and MMP9 expression.

Use of selective PGE2 receptor antagonists on human endometriotic stromal cells and peritoneal macrophages.

Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT-PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1ß-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1ß-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.

The effects of tokishakuyakusan, a traditional Japanese medicine (kampo), ferulic acid and paeoniflorin, on human endometriotic stromal cells and peritoneal macrophages.

OBJECTIVES: To evaluate the anti-inflammatory and anti-angiogenetic effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo), and its ingredients, ferulic acid (FA) and paeoniflorin (PA) on endometriotic stromal cells (ESC) and peritoneal macrophages. STUDY DESIGN: Endometriotic tissues were obtained from 16 patients and peritoneal macrophages were obtained from 11 patients that had undergone laparoscopic surgery for ovarian endometriosis. ESC isolated from endometriotic tissues and peritoneal macrophages were cultured, and pre-treated with 300 µg/mL of TSS, 500 µM FA or 50 µM PA. ESC and peritoneal macrophages were then stimulated with IL-1ß. Concentrations of IL-8 and VEGF protein in supernatants were then detected and measured using specific ELISAs. TSS (4 g/kg body weight) was orally administered to female Sprague-Dawley rats. The concentration of FA in plasma and uteri was measured using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS/MS).　 RESULTS: TSS and FA but not PA decreased the secretion of inflammatory cytokine (IL-8) and angiogenic factor (VEGF) in ESC. TSS and FA also suppressed the secretion of inflammatory cytokine (IL-8) from peritoneal macrophages. FA was detected in plasma and in uterine tissues after the oral administration of TSS to rats. CONCLUSIONS: Our study demonstrates that TSS has anti-inflammatory and anti-angiogenic effects on endometriosis related cells by controlling inflammatory cytokine and growth factor secretion from cells, and these effects, at least partially, may be due to the direct effects of the TSS ingredient FA.

Endometriosis Triggers Excessive Activation of Primordial Follicles via PI3K-PTEN-Akt-Foxo3 Pathway.

CONTEXT: The ovarian reserve is reduced in patients with endometriosis. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) Akt-Forkhead box O (Foxo3) pathway is involved in reducing the ovarian reserve. OBJECTIVE: To elucidate the signaling mechanism by which endometriosis decreases ovarian reserve. DESIGN: Studies were conducted by using a mouse model for endometriosis and human ovaries. The endometriosis mouse model was established and ammonium trichloro (dioxoethylene-o,o') tellurate (AS101), an inhibitor of PI3K-PTEN-Akt pathway, was administered to experimental mice. Human ovaries were collected during surgery from patients with endometrioma or from patients with no ovarian pathology (control ovaries). The number of follicles and expression of Foxo3, PTEN, phosphorylated mammalian target of rapamycin and phosphorylated Akt by oocytes in primordial follicles in mouse and human ovaries were detected by immunohistochemical staining and evaluated. RESULTS: In the endometriosis mouse model, the proportion of primordial follicles was diminished, and the proportion of primary, secondary, antral, and growing follicles was increased in comparison with controls. In both mouse and human ovaries, the PI3K-PTEN-Akt-Foxo3 pathway was activated in samples from endometriosis. Administration of AS101 restored the proportion of primordial follicles in endometriotic mice ovaries to control levels. CONCLUSIONS: The current study describes the excessive activation of primordial follicles and the role of the PI3K-PTEN-Akt-Foxo3 pathway in the reduction of ovarian reserve associated with endometriosis. Our results suggest that a PI3K-PTEN-Akt inhibitor should be considered for further investigation as promising medicines for the prevention of the ovarian reserve reduction in patients with endometriosis.

Expression of Nerve Injury-Induced Protein1 (Ninj1) in Endometriosis.

OBJECTIVE: The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs). BACKGROUND: Nerve injury-induced protein 1 (Ninj1) is a molecule originally identified in dorsal root ganglion neurons and Schwann cells after nerve injury and promotes neurite outgrowth. The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs). MATERIALS AND METHODS: Tissues were obtained with consent from patients diagnosed with ovarian endometrioma (n = 15 in total), peritoneal endometriosis (n = 5), adenomyosis (n = 5), and other gynecological disorders (n = 5, control) during surgery. Immunohistochemistry was conducted in order to detect Ninj1 protein expression in the lesion of endometriosis, adenomyosis, and eutopic endometrium. Nerve fibers in the ovarian endometrioma were detected by positive staining of PGP-9.5. To evaluate the effects of IL-1ß on Ninj1 gene expression in endometriosis, ESCs isolated from ovarian endometrioma (n = 5) were treated with IL-1ß (5 ng/mL) for 3 or 6 hours. Messenger RNA (mRNA) expression for Ninj1 was examined using quantitative RT-PCR. RESULTS: The Ninj1 protein was expressed by ovarian endometrioma, peritoneal endometriotic, and adenomyotic tissue. Nerve fibers were found in the areas of positive staining for Ninj1 in ovarian endometrioma. IL-1ß, an indicator of inflammation in endometriosis, significantly increased Ninj1 mRNA expression by ESC. CONCLUSION: Our study demonstrates that Ninj1 is expressed in endometriosis and adenomyosis and is induced by the inflammatory stimuli. Given the neurogenetic property of Ninj1, our results imply that Ninj1, induced by inflammation in endometriosis lesion, may contribute to the pathogenesis of pain symptoms characteristic of endometriosis.

Involvement of immune cells in the pathogenesis of endometriosis.

Endometriosis is characterized by the implantation and growth of endometriotic tissues outside the uterus. It is widely accepted the theory that endometriosis is caused by the implantation of endometrial tissue from retrograde menstruation; however, retrograde menstruation occurs in almost all women and other factors are required for the establishment of endometriosis, such as cell survival, cell invasion, angiogenesis, and cell growth. Immune factors in the local environment may, therefore, contribute to the formation and progression of endometriosis. Current evidence supports the involvement of immune cells in the pathogenesis of endometriosis. Peritoneal neutrophils and macrophages secrete biochemical factors that help endometriotic cell growth and invasion, and angiogenesis. Peritoneal macrophages and NK cells in endometriosis have limited capability of eliminating endometrial cells in the peritoneal cavity. An imbalance of T cell subsets leads to aberrant cytokine secretions and inflammation that results in the growth of endometriosis lesions. It is still uncertain whether these immune cells have a role in the initial cause and/or stimulate actions that enhance disease; however, in either case, modulating the actions of these cells may prevent initiation or disease progression. Further studies are needed to deepen the understanding of the pathology of endometriosis and to develop novel management approaches of benefit to women suffering from this disease.

Drospirenone reduces inflammatory cytokines, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression in human endometriotic stromal cells.

Drospirenone has been used as a progestin in oral contraceptives with ethinyl estradiol (DRSP/EE) and is expected to regulate endometriosis, however, the direct effects of drospirenone on endometriosis have not been clarified. The aim of this study was to evaluate the anti-inflammatory, anti-angiogenic and anti-neurogenic effects of drospirenone on endometriotic stromal cells (ESC). ESC isolated from endometriotic tissues were obtained from patients during laparoscopic surgery for ovarian endometriosis. ESC were exposed to IL-1ß and cultured in the absence or presence of drospirenone. mRNA expression was evaluated using quantitative RT-PCR, and protein was measured using ELISAs. To evaluate the effect of drospirenone on progesterone receptor (PR) and mineralocorticoid receptor (MR), ESC were transfected with siRNA against PR (siPR) and MR (siMR), and cultured in the presence or absence of drospirenone. Drospirenone significantly decreased IL-6, IL-8, VEGF and NGF mRNA expression by ESC. Drospirenone (10-5M) significantly decreased IL-6 secretion and 10-7M drospirenone decreased IL-8 and VEGF secretion. Knockdown of PR, but not MR, negated the effects of drospirenone. In summary, this study demonstrates that drospirenone has anti-inflammatory, anti-angiogenic and anti-neurogenic effects on ESC and these effects are mediated by PR. These drospirenone effects may contribute to the regulatory effects of drospirenone-containing oral contraceptives on endometriosis.

Mannose receptor is highly expressed by peritoneal dendritic cells in endometriosis.

OBJECTIVES: To characterize peritoneal dendritic cells (DCs) in endometriosis and to clarify their role in its etiology. DESIGN: Experimental. SETTING: University hospital. PATIENT(S): Sixty-three women (35 patients with endometriosis and 28 control women) who had undergone laparoscopic surgery. INTERVENTION(S): Peritoneal DCs from endometriosis and control samples were analyzed for the expression of cell surface markers. Monocyte-derived dendritic cells (Mo-DCs) were cultured with dead endometrial stromal cells (dESCs) to investigate changes in phagocytic activity and cytokine expression. MAIN OUTCOME MEASURE(S): Cell surface markers and cytokine expression and identification with the use of flow cytometry or reverse-transcription polymerase chain reaction (RT-PCR). Changes in cytokine expression and phagocytic activity of Mo-DCs cultured with dESCs and d-mannan were measured with the use of flow cytometry and RT-PCR. RESULT(S): The proportion of mannose receptor (MR)-positive myeloid DC type 1 was higher in endometriosis samples than in control samples. The blocking of MR reduced phagocytosis of dESCs by Mo-DCs. Mo-DCs cultured with dESCs expressed higher levels of interleukin (IL) 1ß and IL-6 than control samples. CONCLUSION(S): Peritoneal DCs in endometriosis tissue express high levels of MR, which promotes phagocytosis of dead endometrial cells and thereby contributes to the etiology of endometriosis.

Dienogest reduces proliferation, NGF expression and nerve fiber density in human adenomyosis.

OBJECTIVES: To evaluate the in vivo effect of dienogest on proliferation, apoptosis, aromatase expression, vascular density, nerve growth factor (NGF) expression and nerve fiber density in human adenomyosis tissue. STUDY DESIGN: Twelve women who underwent hysterectomy for adenomyosis were enrolled. Six patients received dienogest treatment prior to hysterectomy (dienogest group), and age-matched six patients who had not received any hormonal treatment for ≥3 months before surgery (control group). Cell proliferation, vascular and nerve fiber density in adenomyosis tissue were evaluated by staining for Ki67, von Willebrand factor and PGP9.5, respectively. Apoptosis was detected using the TUNEL assay. The expression aromatase and NGF were evaluated by staining for corresponding antibodies. RESULTS: The proportion of Ki67 positive epithelial cells was significantly lower in samples from dienogest-treated patients in comparison with controls (p<0.05). The density of blood vessels in adenomyosis was marginally lower in the dienogest group in comparison with controls but statistical significance was not reached (p=0.07). The intensity of NGF expression and the density of nerve fibers were significantly lower in the dienogest group compared with controls (p<0.05 for both). CONCLUSION: This study demonstrates that adenomyosis, taken from patients treated with dienogest, shows remarkable histological features, such as reductions in proliferation, NGF expression and nerve fiber density. These findings indicate the impact of dienogest on local histological events, and explains its therapeutic effect on adenomyosis.

Development of ovarian cancer after excision of endometrioma.

OBJECTIVE: To determine the prevalence rate of subsequent development of ovarian cancer after excision of endometrioma. DESIGN: Retrospective cross-sectional study. SETTING: University hospital. PATIENT(S): A total of 485 women with endometrioma. INTERVENTION(S): Excisions of endometrioma were performed between 1995 and 2004. Data were collected from medical records in 2013. MAIN OUTCOME MEASURE(S): Age, revised American Society for Reproductive Medicine score, cyst diameter, follow-up periods, endometrioma recurrence, and development of ovarian cancer. RESULT(S): Recurrence of endometrioma was recorded in 121 patients (24.9% of the entire cohort), and 4 patients (0.8% of the entire cohort) developed ovarian cancer. All ovarian cancers developed from a recurrent endometrioma (3.3% of patients who experienced recurrence). Recurrence of endometrioma was significantly associated with ovarian cancer development. CONCLUSION(S): Ovarian cancers can develop after excision of endometrioma and are more likely to arise from recurrent endometrioma. Special care such as rigorous follow-up should be practiced to manage patients who experience recurrence of endometrioma.

Effects of 1,25-Dihydroxy Vitamin D3 on Endometriosis.

CONTEXT: Endometriosis is an estrogen-dependent, chronic inflammatory disease. Recent studies have shown that vitamin D (VD) is an effective modulator of the immune system and plays an important role in controlling many inflammatory diseases. OBJECTIVE: The objective of the study was to clarify the in vitro effects of 1,25-dihydroxy vitamin D3 (1,25[OH]2D3) on human endometriotic stromal cells (ESCs) and to determine the serum levels of VD in endometriosis patients. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES: ESCs were isolated from ovarian endometrioma and cultured with 1,25(OH)2D3. Gene expression of IL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, cytosolic prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, matrix metalloproteinase (MMP)-2, and MMP-9 was examined using quantitative RT-PCR. The production of IL-8 and prostaglandin E2 was measured using an ELISA and an enzyme immunoassay. Viable cell number was assessed using a cell-counting assay, and DNA synthesis was assessed using the bromodeoxyuridine incorporation assay. Apoptosis was assessed using flow cytometry. The expression of inhibitory-κBα protein was detected using Western blotting. The serum levels of 25-hydroxyvitamin D3 and 1,25(OH)2D3 were measured by a RIA. RESULTS: In vitro studies showed that 1,25(OH)2D3 significantly reduced IL-1ß- or TNF-α-induced inflammatory responses, such as IL-8 expression and prostaglandin activity. 1,25(OH)2D3 also reduced viable ESC numbers and DNA synthesis but did not affect apoptosis. MMP-2 and MMP-9 expressions were reduced by 1,25(OH)2D3. 1,25(OH)2D3 inhibited nuclear factor-κB activation. The serum 25-hydroxyvitamin D3 levels were significantly lower in women with severe endometriosis than in the controls and women with mild endometriosis. Serum 1,25(OH)2D3 levels were not different between groups. CONCLUSIONS: VD modulates inflammation and proliferation in endometriotic cells, and a lower VD status is associated with endometriosis. Taken together, VD supplementation could be a novel therapeutic strategy for managing endometriosis.

Resveratrol Enhances Apoptosis in Endometriotic Stromal Cells.

PROBLEM: Resistance to apoptosis, together with inflammatory and invasive activity, contributes to the pathogenesis of endometriosis; therefore, approaches that can safely enhance apoptosis in endometriotic tissue are highly sought after as a means of managing the disease. Although resveratrol (RVT) is known to induce apoptosis or increase sensitivity to apoptotic stimuli in various cancer cell types, its effect on human endometriosis has remained uncertain. This study aimed to investigate whether RVT induces or enhances apoptosis in human endometriotic stromal cells (ESCs). METHOD OF STUDY: Endometriotic tissues were collected, during laparoscopies, from women affected by ovarian endometriosis. ESCs were prepared, cultured, and treated with RVT. Apoptosis was assessed by annexin V-PI staining. Survivin mRNA expression in ESCs was examined using RT-PCR. ESCs were pre-treated with or without RVT and then incubated with TNF-α-related-apoptosis-inducing ligand (TRAIL), which is a known pro-apoptotic molecule. RESULTS: RVT alone did not induce apoptosis in ESCs. RVT significantly reduced survivin mRNA expression (P < 0.05). Pre-treatment with RVT significantly enhanced TRAIL-induced apoptosis (8.13 ± 0.83% (control) versus 29.19 ± 7.39% (pre-treated with RVT), P < 0.05). CONCLUSION: This study indicates that RVT suppresses survivin expression and enhances TRAIL-induced apoptosis in ESCs.

Drospirenone induces decidualization in human eutopic endometrial stromal cells and reduces DNA synthesis of human endometriotic stromal cells.

OBJECTIVE: To investigate the in vitro effect of drospirenone on human eutopic endometrial (EuSC) and ectopic endometriotic stromal cells (EcSC). DESIGN: Comparative and laboratory study. The experimental procedures were approved by the Institutional Review Board of the University of Tokyo (registration no. 0324-4). SETTING: University research laboratory. PATIENTS(S): Eight patients undergoing hysterectomy for benign gynecologic disease and 19 patients undergoing cystectomy or adnectomy for endometriosis. INTERVENTION(S): EuSC and EcSC were treated with drospirenone. MAIN OUTCOME MEASURE(S): For the analysis of decidualization of EuSC, cells were observed using microscopy, and the production of PRL was measured using enzyme immunoassay. For the analysis of DNA synthesis of EcSC, 5-bromo-2'-deoxyuridine incorporation was measured by ELISA. Cells in apoptosis were detected and measured by flow cytometry. RESULT(S): Drospirenone induced decidualization in EuSC, and the induction was negated by RU486. Drospirenone reduced DNA synthesis on EcSC, and this reduction was negated by RU486 or P receptor silencing, but not by aldosterone or mineralocorticoid receptor silencing. Drospirenone did not cause EcSC to undergo apoptosis. CONCLUSION(S): Our study demonstrates the direct effects of drospirenone: decidualization of EuSC and reduced DNA synthesis of EcSC, but it does not cause EsSC apoptosis.