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Bone morphogenetic protein (BMP) cytokine is known to regulate ovulation, as BMP-6 null mice exhibit a decrease in the number of ovulatory follicles without effect on either the morphology or the dynamics of follicular development. In the present study, the role of BMP-6 in ovulatory process was investigated using human granulosa-lutein cells (GCs). Granulosa-lutein cells, obtained from in vitro fertilization patients, were cultured with BMP-6 followed by RNA extraction. The neutrophil-chemotactic activity of the supernatant of cultured GC was investigated. Bone morphogenetic protein 6 significantly increased growth-regulated oncogene α (GRO-α) messenger RNA (mRNA) and protein expression in GC. In the neutrophil-chemotaxis assay, the GC supernatant cultured with BMP-6 attracted more neutrophils than control samples, which was negated with anti-GRO-α neutralizing antibody. Bone morphogenetic protein 6 also suppressed the relative expression of the protease inhibitors, secretory leukocyte peptidase inhibitor, and whey acid protein 14 mRNA in GC. Bone morphogenetic protein 6 might play a role in ovulation by increasing the accumulation of neutrophils in the ovulatory follicle and suppressing the effect of protease inhibitors.

The formation of an individual capillary network in the theca cell layer is required for ovarian folliculogenesis. Although vascular endothelial growth factor (VEGF) is critical for this process, the regulation of VEGF has been unclear. In the present study, the relationship between VEGF and intraovarian cytokine, bone morphogenetic protein 7 (BMP-7) was investigated. Granulosa cells (GC), obtained from in vitro fertilization patients, were cultured with BMP-7 followed by RNA extraction. Human umbilical vein endothelial cells (HUVECs) were also cultured with BMP-7 followed by RNA extraction, tube formation assay, or cell count analysis. The BMP-7 stimulated VEGF messenger RNA (mRNA) and protein expression in GC significantly. In HUVEC, BMP-7 increased an approximately 1.8-fold in the cell number and induced the tube formation significantly compared to control. The BMP-7 also induced a 2-fold increase in VEGF receptor mRNA transcript relative abundance in HUVEC. The BMP-7, a theca cell-derived factor, may stimulate endothelial cell to form vasculature in the follicle via 2 distinct mechanisms, induction of VEGF expression in GC and increased sensitivity of endothelial cells to VEGF.

Purpose: Although studies of serum anti-Müllerian hormone (AMH) in predicting ovarian reserve are numerous, many studies utilized patients under age 40. However, the assessment of ovarian reserve is especially critical in older infertile women. This study evaluates the significance of AMH level in patients over age 40 at the time of their first in vitro fertilization (IVF) treatment., Methods: Forty-nine women over age 40 were studied. Although serum samples were taken prior to their IVF treatments, the data of serum AMH of patients were not taken into consideration to determine the therapy strategy, including follicle induction in which clomiphene citrate and human menopausal gonadotropin were used., Result(s): Twelve out of 49 patients achieved a clinical pregnancy (24.4 %). There was a positive correlation between serum AMH levels and the number of oocytes retrieved (P < 0.0001). The ROC curve analysis for prediction of poor ovarian response, ≤3 retrieved oocytes, showed that the optimum cut-off level was < 1.0 ng/mL for AMH. The lower AMH group (AMH < 1.0 ng/ml) showed less chance of undergoing embryo transfer than the higher AMH group (AMH ≥1.0 ng/ml). There was no difference in pregnancy rate between the two groups. Five out of 12 pregnant women exhibited AMH levels of less than 0.4 ng/ml., Conclusion(s): Assessment of serum AMH concentration in older patients is useful for the prediction of oocytes numbers which may be obtained in IVF. A cut-off level of 1.0 ng/ml AMH can be used to predict poor ovarian response. This cut-off level of AMH of 1.0 ng/ml might be useful to predict whether patients could have an embryo transfer, but had no power to predict achieving pregnancy. On the other hand, our data also showed that patients over age 40 with extreme low levels of AMH still had a chance of pregnancy.

The aim of this study is to examine the regulation of follistatin, an activin-binding protein, in endometriosis. Endometrioma stromal cells (EoSCs) were obtained from 9 patients undergoing laparoscopy of the ovarian endometrioma. In cultured EoSCs, interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), which could induce activin-A, also induced follistatin messenger RNA (mRNA) and protein. The cystic fluid of endometrioma from 8 patients was obtained to measure the concentration of activin-A and follistatin by enzyme-linked immunosorbent assay (ELISA). Also, activin activity in the fluid was examined by erythroid differentiation assay using mouse erythroleukemia F5-5.fl cells. In the cystic fluid of endometrioma, the mean values of activin-A and follistatin concentration were 36.8 ng/mL and 4.0 ng/mL, respectively. In a bioassay, all 8 samples exhibited activin activity, which was equivalent to recombinant activin-A activity of 12.8 ± 1.4 ng/mL. Although follistatin was present in the cystic fluid of endometrioma, the activity of activin, which is an exacerbation factor of endometriosis, was predominant in vivo.

Problem: The purpose of this study was to evaluate the impact of mechanical stretch caused by uterine contraction and progesterone (P₄) on decidual cells (DC), neutrophil chemokines, and MMP-1 expression., Method of Study: DC were cultured, and cyclic stretch was applied using a computer-operated cell stretch system. Interleukin (IL)-8, growth-regulated oncogene (GRO) α, matrix metalloproteinase (MMP)-1, and mRNA and/or protein expression/activity was measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. The effect of P₄ was also studied., Results: Cyclic mechanical stretch increased IL-8, GROα, mRNA and protein, and MMP-1 production and activity level. Supernatant from stretched cells induced neutrophil chemotactic activity significantly. P₄ suppressed the effect of stretch., Conclusion: The current study demonstrates that cyclic mechanical stretch stimulates the production of neutrophil chemokines and MMP-1 from human decidual cells, and the production was reduced by progesterone. These findings suggest that decidual cells are responding to mechanical and endocrine signals and induce biochemical factors and thereby contribute to the regulation of human labor., (© 2013 John Wiley & Sons A/S.)

Context: Endometriosis is a chronic inflammatory disease in which immune response and production of estrogen in endometriotic tissues are involved in the development of the disease. Prostaglandin E2 (PGE2) stimulates aromatase (P450arom) expression in endometrioma stromal cells (ESCs) and increases the production of estrogens. On the other hand, an accumulating amount of evidence suggests that IL-4, a typical Th2 cytokine, plays important roles in the disease., Objective: The objective of the investigation was to study the effect of IL-4 on the expression of 3β-hydroxysteroid dehydrogenase (HSD3B2), a pivotal enzyme for estrogen production, in ESCs., Design, Patients, and Main Outcome Measures: ESCs were isolated from ovarian endometrioma tissues and cultured with IL-4 and PGE2. CP-690550, a Janus protein tyrosine kinase 3 inhibitor, and HSD3B2 small interfering RNA were added to the culture. Gene expression of HSD3B2 and P450arom was examined by quantitative RT-PCR. Dehydroepiandrosterone (DHEA) was added to the culture, and then the combined enzyme activity of HSD3B2, which converts DHEA to androstenedione, and P450arom, which converts androstenedione to estrone, was examined by measuring estrone concentration in the supernatants with a specific enzyme immunoassay., Results: IL-4 increased the expression of HSD3B2 mRNA in a dose-dependent manner. CP-650550 inhibited the IL-4-induced increase in HSD3B2 mRNA expression. PGE2 also increased the expression of HSD3B2 mRNA, and the combination of IL-4 and PGE2 synergistically increased the expression of HSD3B2 mRNA. IL-4 had no effect on the expression of P450arom mRNA, whereas PGE2 increased the expression of P450arom mRNA. Although PGE2 alone increased the production of estrone from DHEA, the combination of IL-4 and PGE2 significantly augmented the production of estrone from DHEA. The enhanced production of estrone by the combination of IL-4 and PGE2 was inhibited by CP-690550 and HSD3B2 small interfering RNA., Conclusions: IL-4 in combination with PGE2 may enhance estrogen production in endometriotic tissues, implying an elaborate mechanism that Th2 immune response augments inflammation-dependent progression of the disease.

Problem: The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition., Method of Study: Uterine smooth muscle cells (USMC) were isolated from uterine myometrial tissues and cultured. The effects of cyclic stretch on mRNA and/or protein expression of IL-8, Groα, and pro-MMP-1 by USMC were measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. To evaluate the effect of progesterone (P(4) ), USMC were pretreated with P(4) for 24 hr., Results: Cyclic stretch increased IL-8 and Groα mRNA and protein and pro-MMP-1 production significantly. Supernatants from stretched cells induced neutrophil chemotactic activity significantly; these effects were abrogated by anti-IL-8 or Groα neutralizing antibodies. Stretch effects were reduced by P(4) ., Conclusion: These results suggest that uterine contraction may induce neutrophil infiltration and MMP-1 production, which may contribute to cervical ripening and rupture of membrane. The inhibitory effects of P(4) may explain the mechanism by which progestin prevents preterm labor., (© 2012 John Wiley & Sons A/S.)

Problem: The aim of this study is to evaluate the expression and regulation of proprotein convertase subtilisin/kexin (PCSK) 6, which is known to be an important factor in the production of bone morphogenetic protein (BMP) cytokines in human ovary., Method of Study: The localization of PCSK 6 protein in normal human ovaries was examined by immunohistochemistry. Human granulosa cells (GC), obtained from 34 patients undergoing ovarian stimulation for in vitro fertilization, were cultured with BMP-2, BMP-6, BMP-7, BMP-15, growth differentiation factor (GDF)-9, and activin-A with or without FSH. PCSK 6 mRNA expression level was evaluated by quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR)., Results: An immunohistochemistry study revealed that GC expressed PCSK 6 throughout follicular development, beginning in the primary follicle stage, while oocytes expressed PCSK 6 from the primordial follicle stage onwards. An in vitro study demonstrated that BMP-2, BMP-6, BMP-7, and BMP-15, not activin-A and GDF-9, decreased PCSK 6 gene expression in human GC. FSH induced PCSK 6 mRNA in the presence of activin-A or GDF-9. GDF-3, which is an inhibitor of BMP cytokines, also induced PCSK 6 mRNA expression., Conclusions: PCSK 6, which is a critical factor to produce BMP cytokines, was suppressed with BMP stimulation in human GC, suggesting the presence of a negative feedback system in the follicular development process., (© 2012 John Wiley & Sons A/S.)

Objectives: Serum concentration of anti-Mullerian hormone (AMH) is used as a biomarker in clinical practice. Therefore, it is important to elucidate the mechanism by which AMH is regulated in granulosa cells (GC). An important first step in understanding AMH regulation is to determine which factors up-regulate AMH expression., Study Design: Human GC, obtained from 28 women undergoing oocyte retrieval for in vitro fertilization, were stimulated with various intraovarian cytokines including bone morphogenetic protein (BMP)-2, -6, -7 -15, activin-A and growth differentiation factor (GDF)-9 (100 ng/ml). The expression of AMH mRNA was evaluated with reverse transcription and quantitative real-time polymerase chain reaction (PCR), and AMH protein in cultured supernatant was measured with EIA kit., Results: BMP-2, -6, -7 and -15, but not activin-A and GDF-9, significantly induced AMH expression in GC at mRNA and protein level, while all stimuli increased FSH receptor mRNA and decreased steroidogenic acute regulatory protein (StAR) mRNA level., Conclusions: Among the transforming growth factor (TGF)-β superfamily, BMP-2, -6, -7 and -15 significantly induced AMH expression in human GC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR1 and PAR2 are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR1, thrombin, and a typical activator of PAR2, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR1 activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR2 activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR1 activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR2 activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR1 and PAR2 in the endometrium and endometriosis, and suggest PAR1 and PAR2 as possible therapeutic targets for endometriosis.

Background: The relationship between fibroids and infertility remains a critical and unresolved question. During the implantation phase, it is known that uterine peristalsis is dramatically reduced, which is thought to facilitate implantation of the embryo to the endometrium. In the previous study, using a cine MRI mode, we found that less than half of the patients with intramural fibroids exhibited abnormal uterine peristalsis during the mid-luteal phase. In the present study, we further investigated whether myomectomy for patients in the high peristalsis group is a constructive method to normalize uterine peristalsis., Methods: The frequency of junctional zone movement was evaluated using a cine MRI mode during the mid-luteal phase. Fifteen infertility patients, who had intramural myomas and exhibited abnormal uterine peristalsis (≥2 times/3 min) in their first MRI, underwent myomectomy and a second MRI. After receiving the second MRI, patients underwent infertility treatment for at least 8 months, and pregnancy rate was evaluated prospectively., Results: Among 15 patients, the frequency of uterine peristalsis was normalized (0 or 1 time/3 min) in 14 patients. Following myomectomy and second MRI test, 6 of the 15 patients achieved pregnancy (n = 15, pregnancy rate: 40%)., Conclusions: The presence of uterine fibroids might induce abnormal uterine peristalsis in some patients, leading to infertility, and myomectomy may improve fertility in these patients., (Copyright © 2012 AAGL. Published by Elsevier Inc. All rights reserved.)

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades an essential amino acid, tryptophan, and plays a role in inhibiting the proliferation of T cells and intracellular pathogens. Inhibiting IDO in mice leads to fetal rejection, suggesting its significance in establishing pregnancy. Toll-like receptor 3 (TLR-3) is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I interferon. Using a human trophoblast cell culture system, we studied the effect of TLR-3 ligation on IDO expression and function by treating trophoblasts with polyinosinic-polycytidylic acid [poly(I:C)] (a synthetic double stranded RNA, which mimics viral RNA). Real-time PCR and Western blot analysis revealed that IDO mRNA and protein expression was significantly induced by poly(I:C). The activity of IDO was also increased by poly(I:C) given that the L-kynurenine concentrations were elevated in conditioned media. Conditioned media from poly(I:C)-treated trophoblasts were found to inhibit the proliferation of human T cells significantly. Poly(I:C) was also shown to induce interferon (IFN)-β mRNA expression in trophoblasts. Recombinant human IFN-β increased IDO mRNA expression in trophoblasts more rapidly than poly(I:C). Pretreating with neutralizing antibody against IFN-β significantly suppressed IDO induction by poly(I:C). Collectively we have demonstrated that ligation of TLR-3 by poly(I:C) induces IDO expression in human first-trimester trophoblasts via an IFN-β-dependent pathway. These findings suggest that upon viral infection, trophoblasts induce IDO and in turn contribute to antimicrobial activity and maintenance of fetomaternal tolerance.

Extragenital endometriosis severely impairs the quality of life for affected women but its standard management has not yet been well established because of its relatively low incidence. As extragenital organs, intestine, followed by urinary tract, is the most common place affected by endometriosis, for which surgical treatment is sometimes difficult and accompanied by severe complications. Recently, dienogest, a novel progestin, has emerged as a new alternative for endometriosis, especially for endometriosis-associated pain. In this report, we presented four cases with rectosigmoidal and one with bladder endometriosis, treated with oral 2 mg/day dienogest for over 6 months. For all cases, the measurable extragenital lesions exhibited the reduction in their size after 10 to 11 months of use, accompanied with immediate relief of subjective symptoms related with extragenital lesions. This report suggests that dienogest can be a novel conservative alternative for extragenital endometriosis.

Problem: Preeclampsia is a pregnancy disorder characterized by systemic inflammation. High mobility group box 1 (HMGB1) is a molecule known to act as a 'danger signal' by participating in various inflammatory processes, but data in regard to preeclampsia are sparse. The aim of this study was to analyze placental and serum HMGB1 levels in normal pregnancy and preeclampsia., Method of Study: Sera were collected from women with preeclampsia soon after the manifestation of the disease and before commencing any medication. Placental samples were collected immediately after delivery. Expressed isoforms of HMGB1 (28- and 30-kDa) in the placenta were evaluated by Western blot analysis. Serum HMGB1 concentrations were measured using enzyme-linked immunosorbent assays (ELISA)., Results: Two isoforms of HMGB1 are expressed by the human placenta. The 28- and 30-kDa HMGB1 isoforms were expressed highly in preeclamptic placental tissue; however, compared with normotensive control tissue, differences in detected expression levels did not reach statistical significance. No significant difference was observed in serum HMGB1 levels between control and preeclampsia., Conclusion: Inflammation provoked by HMGB1 is likely to be involved in the proinflammatory process in preeclamptic placenta. Further studies are needed to elucidate the precise role of HMGB1 in preeclampsia., (© 2011 John Wiley & Sons A/S.)

Objective: To examine the effects of interleukin (IL)-17F on the secretion of IL-8 and the gene expression of cyclooxygenase 2 (COX2) in endometriotic stromal cells., Design: In vitro experimental study using human samples., Setting: University hospital., Patient(s): Endometriotic tissues were obtained from women with ovarian endometriomas undergoing laparoscopic surgery., Intervention(s): Endometriotic stromal cells (ESCs) were cultured with IL-17F., Main Outcome Measure(s): Concentrations of IL-8 were measured by a specific ELISA, and messenger RNA levels of IL-8 and COX2 were measured by real-time reverse transcription-polymerase chain reaction (PCR)., Result(s): IL-17F increased the secretion of IL-8 from ESCs, and the effect was inhibited by antibodies for IL-17 receptor A and IL-17 receptor C. Tumor necrosis factor α (TNF-α) synergistically enhanced IL-17F-induced increase in IL-8 secretion from ESCs. The IL-17F increased the gene expression of IL-8 and COX2 in ESCs., Conclusion(s): These findings suggest that IL-17F may stimulate the development of endometriosis by up-regulation of IL-8 and COX2., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by several serine proteases. PAR2 activation in endometriotic stromal cells (ESCs) has been implicated in the development of endometriosis but the regulatory mechanism of PAR2 expression in ESC is unknown. Our objective was to study the mechanism by which PAR2 expression may be regulated in endometriotic lesions., Methods: Primary cultures of ESCs were treated with transforming growth factor-β (TGF-β) 1, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and the expression of PAR2 was examined by real-time quantitative PCR. ESCs pretreated with or without TGF-β1 were treated with PAR2 agonist peptide (PAR2AP) and the secretion of the pro-endometriotic cytokine, IL-6, was measured using a specific enzyme-linked immunosorbent assay. Effects of TGF-β type 1 inhibitor, SB431542, and PAR2 small interfering RNA (siRNA) on the TGF-β1 stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion were also evaluated. To study intracellular signaling, effects of inhibitors of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K) and of Smad4 siRNA on the TGF-β1-induced PAR2 gene expression were studied., Results: Only TGF-β1, but neither TNF-α nor IL-1β, increased gene expression of PAR2. Activation of PAR2 with PAR2AP increased the secretion of IL-6 from ESCs. As expected, TGF-β1 pretreatment dose-dependently enhanced the PAR2AP-induced increase in IL-6 secretion from ESCs. Treatment of ESCs with the TGF-β type 1 inhibitor, SB431542, inhibited both TGF-β1-stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion. Transfection of ESCs with PAR2 siRNA produced a similar inhibition of IL-6 secretion. The TGF-β1-induced increase in PAR2 gene expression was repressed by inhibition of p38 MAPK, p42/44 MAPK or PI3K, but not by knockdown of Smad4 expression., Conclusions: In view of significant roles of PAR2 and IL-6 in endometriosis, the TGF-β1-induced increase in PAR2 expression may be an elaborate mechanism that augments the progression of the disease.

Objective: To examine the regulation and the function of activin-A in stromal cells derived from endometrioma., Design: Molecular studies., Setting: University research laboratory., Patient(s): Endometrioma stromal cells (EoSC) were obtained from 28 patients with ovarian endometrioma undergoing laparoscopy., Intervention(s): EoSC were cultured with inflammatory stimuli or recombinant activin-A, followed by RNA extraction., Main Outcome Measure(s): Activin mRNA expression was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR), and activin-A concentration of supernatant of cultured EoSC was evaluated by ELISA. Also, the effect of activin-A on EoSC was evaluated with real-time RT-PCR and cell proliferation assay., Result(s): Inflammatory stimuli, interleukin (IL) -1β, and tumor necrosis factor (TNF) -α induced inhibin/activin-βA subunit mRNA and activin-A protein expression in EoSC. Additionally, activin-A enhanced EoSC proliferation and increased the expression of IL-6 and protease-activated receptor (PAR)-2 mRNA., Conclusion(s): An in vitro study revealed that activin-A, which is induced by IL-1β or TNF-α, might promote endometriosis by stimulating IL-6 and PAR-2 mRNA expression and increasing the proliferation of EoSC., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Magnetic resonance imaging was used to measure the endometrial T2 relaxation times of patients with infertility with fibroma. Although the location of fibromas did not influence the T2 relaxation times, we did observe a significant decrease in endometrial T2 relaxation times after myomectomy., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Problem: A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP-2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP-2 in the human ovary., Method of Study: BMP-2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP-2 or human chorionic gonadotrophin (HCG), followed by RNA extraction., Results: BMP-2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP-2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP-2 and increased BMP and activin membrane-bound inhibitor (BAMBI), an antagonist of BMP-2., Conclusion: Expression and disappearance of BMP-2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP-2 function by controlling BMP-2 and BAMBI expression., (© 2010 John Wiley & Sons A/S.)

The gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are of particular importance in ovarian physiology. However, FSH receptors and LH receptors are not expressed until the secondary follicle stage, indicating that initiation of follicular growth is independent of the gonadotropins. Among many intra-ovarian growth factors, many studies have shown that bone morphogenetic proteins (BMPs) play pivotal roles in regulating the early phases of follicular growth. The BMP system induces the gonadotropin system by modulating gonadotropin receptors in early-stage follicles. Interestingly, the BMP system also prevents precocious maturation of the follicle by suppressing luteinization. Signals provoked by the preovulatory LH surge eliminate BMPs, enabling luteinization to progress. Thus, the BMP system and the gonadotropin system seem to cooperate in regulating follicular development, maturation, and luteinization.

Endometriosis is a disease characterized by the presence of endometriotic tissue outside the uterine cavity. Although its pathogenesis remains to be elucidated, immune status is suggested to play an important role in the initiation and the progression of the disease. In particular, immune cells in lymphoid lineage that comprised T and B lymphocytes and natural killer cells play essential roles in determining either accept or reject survival, implantation, and proliferation of endometrial and endometriotic cells. Numerous studies have shown aberrant functions of these immune cells in women with endometriosis. The abnormalities include reduced activity of cytotoxic T cells and NK cells, cytokine secretion by helper T cells, and autoantibody production by B lymphocytes. These alterations are suggested to be induced by various manners and promote the disease. Understanding of these immune aspects in endometriosis is thus expected to benefit the treatment of the disease.

In a novel paradigm of T cell differentiation, type 17 T helper (Th17) cells may play a significant role in endometriosis, a chronic inflammatory disease. However, the mechanism regulating the accumulation of Th17 cells in endometriotic tissues remains unknown. We hypothesized that Th17 cells migrate to endometriotic tissues through an interaction of the chemokine CC chemokine ligand (CCL)20 and its receptor CCR6. Using endometriotic tissues from women with endometriosis, we demonstrated, by flow cytometry, that Th17 cells in endometriotic tissues express CC chemokine receptor (CCR)6. Immunohistochemistry also revealed that CCL20 was expressed in the epithelial cells and stromal cells beneath the epithelium of endometriotic tissues. CCR6+ cells were small and round and scattered in the stroma in which abundant CCL20+ cells were detected. CCL20 caused selective migration of Th17 cells in the peripheral blood in a migration assay. IL-1β, TNF-α, and IL-17A increased the secretion of CCL20 in cultured endometriotic stromal cells. Inhibitors of p38- and p42/44-MAPKs, and stress-activated protein kinase/c-Jun kinase suppressed the secretion of CCL20 increased by IL-1β, TNF-α, and IL-17A. This suggests that the CCL20/CCR6 system is involved in the migration of Th17 cells to endometriotic tissues and that proinflammatory cytokines contribute to the development of endometriosis via up-regulation of CCL20 secretion from endometriotic stromal cells.

Objective: To study the relationship between eotaxin and interleukin-4 (IL-4) in the pathophysiology of endometriosis., Design: Comparative and laboratory study., Setting: University teaching hospital reproductive endocrinology and infertility practice., Patient(s): Ectopic endometrial tissues were collected from women with endometriosis., Intervention(s): Ectopic endometrial stromal cells (ESCs) were isolated and cultured with IL-4. Ectopic endometriotic tissues were immunostained for eotaxin and IL-4., Main Outcome Measure(s): Gene expression of eotaxin was determined by standard and real-time reverse-transcriptase polymerase chain reaction. Secretion of eotaxin from ESC was measured using specific ELISA. The immunostained sections were examined., Result(s): Interleukin-4 (IL-4) increased mRNA expression and protein secretion of eotaxin from ESC in a dose-dependent manner. Immunohistochemical analysis showed that eotaxin-positive cells colocalized with IL-4-positive cells and accumulated around the blood vessels in the stroma of endometriotic tissue., Conclusion(s): IL-4 induces eotaxin in ESCs, which might promote angiogenesis and the subsequent development of endometriosis., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In an animal endometriosis model, the administration of hyaluronic acid (HA) reagent significantly suppressed the formation of endometriotic lesions in both number and weight. This effect was found when HA treatment was conducted at the time of endometrial fragment inoculation., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To examine the effect of bone morphogenetic protein 7 (BMP-7) on FSH receptor (FSHR) expression in human granulosa cells., Design: Laboratory study using human samples., Setting: University hospital., Patient(s): Human granulosa cells were obtained from 60 women undergoing oocyte retrieval for IVF., Intervention(s): Human granulosa cells (GCs) were cultured with recombinant BMP-7, followed by RNA extraction., Main Outcome Measure(s): mRNA levels of GCs were measured by real-time reverse-transcription polymerase chain reaction., Result(s): Bone morphogenetic protein 7 increased FSHR gene expression in human luteinized granulosa cells, whereas it decreased LH receptor gene expression. Bone morphogenetic protein 7 also increased FSH-induced cyclic adenosine monophosphate production in GCs, indicating up-regulation of the cellular response to FSH. Although BMP-7 increased gene expression of activin-betaA and -betaB in GCs, inhibition of activin function did not affect the BMP-7-induced FSHR gene expression., Conclusion(s): These findings provide new insight into the biologic function of BMP-7 in the human ovary and demonstrate its unique mechanism of regulating FSHR action., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Immunohistochemical staining using human normal ovaries showed that bone morphogenetic protein-6 (BMP-6) was abundantly present in the granulosa cells (GC) of healthy tertiary follicles but not in atretic follicles. An in vitro study showed that BMP-6 induced gene expression of FSH receptor, inhibin/activin beta subunits, and anti-Müllerian hormone (AMH) in human GCs, suggesting that BMP-6 is an important mediator to support healthy follicle growth in the human ovary.

Postinjury (primary) abdominal compartment syndrome (ACS) was described more than 15 years ago as severe abdominal distension with high peak airway pressures, CO(2) retention, and oliguria, which led to unplanned re-exploration after damage-control laparotomy. Later, a more elusive type of ACS was recognized, which develops without abdominal injuries (secondary ACS). Both syndromes were recently characterized, their independent predictors were identified, and preventive strategies were developed to reduce their incidence. Once viewed as a syndrome with almost uniform mortality, systematic preventative strategies and therapeutic efforts have reduced the prevalence, morbidity, and mortality of the syndrome. This review was designed to summarize the recent advances in the management of ACS, to classify the currently available evidence, and to identify future directions of research and clinical care.

IL-1, secreted by human embryos and trophoblast cells, is important for successful implantation and pregnancy. We previously reported that IL-1beta induced IL-8 production in human endometrial stromal cells (ESCs) and that induction was regulated by substances implicated in implantation. In the present study using human primary cells in culture, we measured IL-1beta-induced production of IL-8 from endometrial epithelial cells (EECs) and ESCs and examined effects of the endometrium-derived IL-8 on migration and number of first-trimester villous cytotrophoblast cells (vCTs). Both basal and IL-1beta-induced IL-8 levels of cell supernatants were much higher in EECs than ESCs. Addition of IL-1beta to EECs increased the chemotactic activity of the supernatants to vCTs, and this effect was suppressed by immunoneutralization with anti-IL-8 antibody. Supernatants of IL-1beta-stimulated EECs yielded significantly higher number of vCTs compared with those of untreated EECs, and the effect was inhibited by IL-8 antibody. These findings suggest that IL-1 promotes implantation by stimulating EECs to produce IL-8, which subsequently induces migration of vCTs and contributes to survival of vCTs.

Several lines of evidence indicate that the Th2 immune response is associated with endometriosis. Although an increased concentration of interleukin (IL)-4, a typical Th2 cytokine, has been reported in endometriotic tissues, the implication of this for endometriosis has not been determined. To investigate a possible role of IL-4 in the development of endometriosis, we examined the presence of IL-4-producing cells in endometriotic tissues and the effect of IL-4 on proliferation of endometriotic stromal cells. Endometriotic stromal cells were isolated from endometriotic tissues obtained from women undergoing surgery for endometrioma. Immunohistochemistry of endometriotic tissues revealed that IL-4-positive cells were abundant in the stroma. The effect of IL-4 on proliferation of endometriotic stromal cells was studied using cell counting and BrdU incorporation assays. IL-4 (0.1 to 10 ng/ml) significantly increased cell number and BrdU incorporation in a dose-dependent manner, and the proliferative effect of IL-4 was inhibited by anti-IL-4 receptor antibody. IL-4-induced activation of mitogen-activated protein kinases in endometriotic stromal cells was examined by Western blotting. IL-4 induced phosphorylation of p38 mitogen-activated protein kinase, stress-activated protein kinase/c-Jun kinase, and p42/44 mitogen-activated protein kinase and inhibitors of these kinases suppressed IL-4-induced proliferation of endometriotic stromal cells. These findings suggest that proliferation of endometriotic stromal cells induced by locally produced IL-4 is involved in the development of endometriosis.

The concentration of the soluble form of CD44, a competitive inhibitor for CD44 and hyaluronic acid binding, is higher in the peritoneal fluid of patients with endometriosis than in the peritoneal fluid of patients without endometriosis.

The microRNA (miRNA) processing enzyme Dicer1 is required for zygotic and embryonic development, but the early embryonic lethality of Dicer1 null alleles in mice has limited our ability to address the role of Dicer1 in normal mouse growth and development. To address this question, we used a mouse mutant with a hypomorphic Dicer1 allele (Dicer(d/d)) and found that Dicer1 deficiency resulted in female infertility. This defect in female Dicer(d/d) mice was caused by corpus luteum (CL) insufficiency and resulted, at least in part, from the impaired growth of new capillary vessels in the ovary. We found that the impaired CL angiogenesis in Dicer(d/d) mice was associated with a lack of miR17-5p and let7b, 2 miRNAs that participate in angiogenesis by regulating the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1. Furthermore, injection of miR17-5p and let7b into the ovaries of Dicer(d/d) mice partially normalized tissue inhibitor of metalloproteinase 1 expression and CL angiogenesis. Our data indicate that the development and function of the ovarian CL is a physiological process that appears to be regulated by miRNAs and requires Dicer1 function.

IL-17A is secreted from Th17 cells, a discovery leading to revision of the mechanism underlying the role of Th1/Th2 in the immune response. Strong evidence suggests that immune responses associated with inflammation are involved in the pathogenesis of endometriosis. In the present study, we first demonstrated that the presence of Th17 cells in peritoneal fluid of endometriotic women by flow cytometric analysis and IL-17A-positive cells in endometriotic tissues by immunohistochemistry. To investigate the role of IL-17A in the development of endometriosis, we then studied the effect of IL-17A on IL-8 production, cyclooxygensase-2 expression, and cell proliferation of cultured endometriotic stromal cells (ESCs). IL-17A enhanced IL-8 secretion from ESCs in a dose-dependent manner. The IL-17A-induced secretion of IL-8 from ESCs was suppressed by anti-IL-17 receptor A antibodies or inhibitors of p38 MAPK, p42/44 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase. Addition of TNFalpha synergistically increased IL-17A-induced IL-8 secretion from ESCs. IL-17A also enhanced the expression of cyclooxygensase-2 mRNA and proliferation of ESCs. IL-17A may play a role in the development of endometriosis by stimulating inflammatory responses and proliferation of ESCs.

Problem: Ovarian follicles undergo a dynamic change to provide a mature ovum, and the process involves angiogenesis, follicular cell proliferation and leukocyte recruitment. Midkine (MK) is a heparin-binding growth factor that has angiogenic, mitogenic, and chemotactic activities. In the present study, we investigated the presence of MK and its possible role in human ovarian follicles., Method of Study: Follicular fluid (FF) and luteinized granulosa cells (LGC) were collected from women undergoing in vitro fertilization and embryo transfer. Expression of MK protein in FF was examined by Western blotting. Concentrations of MK, estradiol and oxygen in FF were measured. 5-bromo-2'-deoxyuridine (BrdU) incorporation assay was performed in LGC. Normal ovarian tissues were obtained surgically and used in in-situ hybridization of MK mRNA., Results: The presence of MK protein was verified in FF. MK mRNA was expressed in both granulosa cells and theca cells of large follicles. There is a significant negative correlation between the concentrations of MK and oxygen in FF, and a significant positive correlation between the concentrations of MK and estradiol. MK promoted BrdU uptake in LGC., Conclusion: The present findings imply that hypoxic condition, a characteristic of growing follicles, associates with the production of MK. Given that MK is involved in granulosa cell proliferation and estradiol production in developing follicles, MK may play a role as a local regulator in the human ovary.

Context: Metformin, a widely used treatment for diabetes that improves insulin sensitivity, also has both antiinflammatory properties and a modulatory effect on ovarian steroid production, two actions that have been suggested to be efficacious in therapy for endometriosis., Objective: To determine whether metformin may be effective for the treatment of endometriosis, we evaluated the effects of this agent on inflammatory response, estradiol production, and proliferation of endometriotic stromal cells (ESCs)., Design: ESCs derived from ovarian endometriomas were cultured with various concentrations of metformin., Main Outcome Measures: IL-8 production, mRNA expression and aromatase activity, and 5-bromo-2'-deoxyuridine incorporation in ESCs were measured., Results: Metformin dose-dependently suppressed IL-1beta-induced IL-8 production, cAMP-induced mRNA expression and aromatase activity, and 5-bromo-2'-deoxyuridine incorporation in ESCs., Conclusion: These results suggest that further investigation into the unique therapeutic potential of metformin as an antiendometriotic drug is warranted.

Innate immunity in the endometrium has fundamental significance for reproduction. Although toll-like receptors (TLRs) play central roles in innate immune responses, their expression in the human endometrium remains to be fully elucidated. We have examined the gene expression of TLR2, TLR3, TLR4, and TLR9 in endometrial tissues by real-time quantitative PCR and in situ hybridization. The expression levels of the four genes in endometrial tissues varied in a similar pattern during the menstrual cycle; the levels were high in the perimenstrual period and low in the periovulatory period. Expression of the four genes was detected in both epithelial cells and stromal cells throughout the menstrual cycle. Expression levels were higher in epithelial cells for TLR3 and in stromal cells for TLR4, while they were comparable in epithelial cells and stromal cells for TLR2 and TLR9. These findings imply that differential spatio-temporal expression patterns of TLRs subserve proper innate immunity of the endometrium.

IFN-gamma secreted by a human embryo and trophoblast cells during implantation is suggested to play an important role in implantation and pregnancy. In the present study, we explored expression and possible functions of CXCL11, a CXC chemokine strongly induced by IFN-gamma, and its receptor CXCR3 in the human endometrium. Secreted CXCL11 protein was not detected in cultured endometrial stromal cells (ESC) but was detected in cultured endometrial epithelial cells (EEC). IFN-gamma stimulated the protein levels of CXCL11 in a dose-dependent manner in EEC and ESC. CXCL11 secreted from EEC with 100 ng/ml IFN-gamma was 220-fold of the control, and 100-fold as compared with that secreted from ESC with the same dose of IFN-gamma. CXCR3 was expressed in EEC, ESC, and trophoblast cells. Addition of IFN-gamma to EEC increased the chemotactic activity of its culture medium to trophoblast cells and T cells, and the effect was suppressed by immunoneutralization with Abs of three CXCR3 ligands, including anti-CXCL11 Ab. CXCL11 significantly increased BrdU incorporation of ESC, which was inhibited by a p42/44 MAPK pathway inhibitor PD98059. In contrast, CXCL11 significantly decreased BrdU incorporation and increased the release of lactate dehydrogenase and the positive staining of annexin V in EEC. These findings suggest that IFN-gamma promotes implantation by stimulating EEC to produce CXCL11, which induces migration of trophoblast cells and T cells, proliferation of ESC, and apoptosis of EEC.

In various cells including endometriotic cells, p38 mitogen-activated protein kinase (MAPK) plays essential roles for inflammation, an etiological factor for endometriosis. We evaluated the effect of FR 167653, a p38 MAPK inhibitor, on the development of endometriosis using a murine model. As an endometriosis model, estradiol-treated ovariectomized BALB/c mice were injected intraperitoneally with endometrial fragments of the syngenic donor mice. The animals were injected with either 30mg/kg FR 167653 or only vehicle (control) s.c. twice a day, starting 2 days before endometrial injection. Three weeks later, the peritoneal fluids and the developed endometriotic lesions were collected. Both the weight of all the endometriotic lesions per mouse and the concentrations of interleukin-6 and monocyte chemoattractant protein-1 in the peritoneal fluid were significantly lower in the FR 167653-treated mice than in the control mice. These findings suggest that FR 167653 may inhibit the development of endometriosis possibly by suppressing peritoneal inflammatory status.

Mutations in the bone morphogenetic protein 15 (BMP-15) gene cause female infertility in the monoovulatory human and sheep; however, in the polyovulatory mouse, loss-of-function of BMP-15 results only in reduced ovulation rate. To elucidate the cause of these species-specific differences, we investigated the functional role of BMP-15 in the mouse ovary. Here, we found that the functional mature form of BMP-15 is barely detectable in the mouse oocytes until just before ovulation, when it is markedly increased. Further, we found that BMP-15 induces cumulus expansion in mouse cumulus-oocyte complexes. The oocyte culture medium from immature mice primed with pregnant mare serum gonadotropin followed by human chorionic gonadotropin also stimulated cumulus expansion, and this activity was attenuated by BMP-15 antibody. Interestingly, the oocyte culture medium from mice treated with pregnant mare serum gonadotropin alone had no effect. Moreover, BMP-15 stimulated the expression of EGF-like growth factors in cumulus cells as well as a series of molecules downstream of EGF-like growth factor signaling, including cyclooxygenase 2, hyaluronan synthase 2, tumor necrosis factor-stimulated gene 6, and pentraxin 3, all of which are necessary for normal cumulus expansion. An antagonist of the EGF receptor completely abolished the effect of BMP-15 in inducing cumulus expansion. These results are consistent with the phenotype of BMP-15-null mice, which exhibit normal folliculogenesis but have defects in the ovulation process. The species-specific differences in the phenotypes caused by BMP-15 mutations may thus be attributed to the temporal variations in the production of the mature form of BMP-15.

Adiponectin, a pleiotropic cytokine, exerts its effects via the specific receptors AdipoR1 and AdipoR2. Whereas circulating adiponectin concentrations decrease in women with endometriosis and endometrial cancer, possible effects of adiponectin and the presence of the receptors in the endometrium have not been determined. In this study, we examined the expression of adiponectin receptors AdipoR1 and AdipoR2 in the human endometrium and assessed effects of adiponectin in endometrial cells. Expression of AdipoR1 and AdipoR2 in endometrial tissues was evaluated by real-time quantitative PCR, in situ hybridization, and Western blotting. The effects of adiponectin on phosphorylation of AMP-activated protein kinase, a regulator of energy homeostasis, in cultured endometrial stromal cells (ESCs) and epithelial cells (EECs) were studied by Western blotting. The effects of adiponectin on IL-1beta-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from cultured ESCs were determined using specific ELISAs. The expression of AdipoR1 and AdipoR2 was detected in the endometrium. The expression of both genes was increased in the midluteal phase, the period of embryo implantation. In situ hybridization revealed that both AdipoR1 and AdipoR2 appeared to be equally expressed in the epithelial cells and in the stromal cells. Adiponectin increased phosphorylation of AMP-activated protein kinase in ESCs and EECs. Adiponectin decreased IL-1beta-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from ESCs. These findings suggest that adiponectin exerts energy-homeostatic and antiinflammatory effects in the endometrium, and these effects might be relevant to pathological and physiological endometrium-related events such as implantation and endometriosis.

Decidualization is an essential process of endometrial differentiation for embryo implantation and maintenance of pregnancy. Recently, uterine movement-induced mechanical stress was noticed to have possible effects on endometrial functions. In this study, we addressed the possible effect of mechanical stress on the process of decidualization of endometrial stromal cells (ESC). ESC were cultured on flexible-bottomed culture plates. After decidualization was achieved with estradiol and progesterone for 12 days, cultures were continued for 24 h with or without cyclic stretch (25% elongation) in serum-free conditions at a rate of 2 cycles/min using a computer-operated cell tension system. Concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker of decidualization, in the conditioned medium were measured by specific ELISA, and IGFBP-1 mRNA expression in the ESC was measured by RT-PCR. Cyclic stretch remarkably increased IGFBP-1 secretion from decidualized ESC. It also increased IGFBP-1 mRNA in decidualized ESC. The increase in IGFBP-1 secretion was inhibited by actinomycin D but not by indomethacin, PD-98059, or H-89. Conditioned medium of decidualized ESC cultured with cyclic stretch increased IGFBP-1 secretion from decidualized ESC cultured under stationary conditions. These findings imply that uterine movement modulates decidualization of the endometrium and has a regulatory effect on reproduction.

Background: Inflammation has been proposed to play essential roles in the pathophysiology of endometriosis, in which neutrophils and mast cells have been suggested to be involved. We studied whether the protease-activated receptor 2 (PAR2), which is activated by enzymes from neutrophils and mast cells, in endometriotic stromal cells (ESC) has any implication in the development of the disease., Methods: Cultured ESC were stimulated with various concentrations of a specific PAR2 agonist peptide. Proliferating activity of the cells was determined using immunostaining of proliferating cell nuclear antigen (a cell proliferation marker), 5-bromo-2'-deoxyuridine incorporation into DNA and cell count. The concentrations of interleukin (IL)-6 and IL-8 were measured using specific enzyme-linked immunosorbent assay kits. The phosphorylation of three mitogen-activated protein kinases (MAPK), i.e. p38 MAPK, p42/44 MAPK and stress-activated protein Kinase/c-jun N terminal Kinase, in ESC was examined with Western blot analysis., Results: Activation of PAR2 stimulated the proliferation of ESC and the secretion of IL-6 and IL-8 from ESC in a dose-dependent manner. Activation of PAR2 stimulated the phosphorylation of all three MAPK, and inhibitors of each MAPK suppressed the PAR2 activation-induced proliferation of ESC., Conclusions: The activation of PAR2 in ESC may be involved in the pathophysiology of endometriosis by inducing the growth and inflammation of endometriotic lesions.

Background: Adiponectin is a pleiotropic cytokine originally discovered as an adipocyte-specific gene product. Serum adiponectin concentrations have been reported to be low in women with endometrial cancer, breast cancer and uterine leiomyoma, suggesting possible involvement of adiponectin in these estrogen-related diseases. We thus addressed the relevance of adiponectin to endometriosis, an estrogen-dependent disease, in the present study., Methods: Women with (n = 48) and without (n = 30) endometriosis undergoing laparoscopy were recruited in this study. Blood samples were collected, and serum adiponectin concentrations were measured using a specific enzyme-linked immunosorbent assay. The relationship between laparoscopic findings and serum adiponectin concentrations was analysed., Results: The adiponectin concentrations in the serum of the women with endometriosis (median, 13.1 microg/ml; interquartile range, 10.2 - 16.7) were significantly lower than those of the women without endometriosis (15.9 microg/ml, 13.5 - 19.5; P = 0.008). A significant negative correlation was found between serum adiponectin concentrations and both endometriosis scores (R = - 0.307, P = 0.006) and adhesion scores (R = - 0.254, P = 0.026) of the revised American Society for Reproductive Medicine classification of endometriosis., Conclusions: The present findings suggest that adiponectin is implicated in the pathophysiology of endometriosis.

Background: GnRH II is the second form of GnRH and is widely distributed in peripheral tissues of the female reproductive tract as well as in the central nervous system. In the present study, we studied the possible implication of GnRH II in endometriosis., Methods: Effects of GnRH II on 5-bromo-2'-deoxyuridine (BrdU) uptake by cultured endometriotic stromal cells were examined. Effects of GnRH II on interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8 were also studied. mRNA levels of GnRH I, GnRH II, type I GnRH receptor and type II GnRH receptor were determined by real-time quantitative RT-PCR in endometrial tissues of women with or without endometriosis and in endometriotic tissues., Results: GnRH II dose-dependently suppressed BrdU uptake by endometrial stromal cells. Treatment with IL-1beta markedly increased mRNA levels of COX-2 and IL-8 in endometrial stromal cells and IL-8 protein secretion by these cells, while these increments were significantly suppressed by supplementation with GnRH II. The mRNA levels of GnRH II were lower in endometrial and endometriotic tissues of women with endometriosis than in endometrial tissues of women without endometriosis, both in the proliferative phase and the secretory phase. In addition, as for GnRH I, type I GnRH receptor and type II GnRH receptor, the mRNA levels were lower in endometrial tissues of women with endometriosis than in those without endometriosis in the secretory phase., Conclusions: In the light of the demonstrated antiproliferative and anti-inflammatory effects of GnRH II on endometrial stromal cells, the lower expression of GnRH II in eutopic and ectopic endometrium of women with endometriosis suggests that endogenous GnRH II-mediated cytostatic regulation may be impaired in the development of endometriosis.

Problem: Adiponectin is a novel adipocytokine of extreme importance for the metabolism. Recent studies have indicated that adiponectin suppresses inflammation, angiogenesis, and fibrosis, which are central pathogenic factors for endometriosis. We addressed the possibility that adiponectin is implicated in endometriosis., Method of Study: We measured concentrations of adiponectin in peritoneal fluid (PF) of women with (n = 54) or without (n = 26) endometriosis using specific enzyme-linked immunosorbent assay., Results: Adiponectin concentrations in PF of women with endometriosis were significantly lower than those of women without endometriosis. With respect to the stages of the disease, the concentrations of adiponectin in women with stage III/IV endometriosis were significantly lower compared to those in women without endometriosis and with stage I/II endometriosis. Body mass index were comparable among the groups., Conclusions: These findings imply that adiponectin may be an anti-endometriotic factor, possibly due to its anti-inflammatory, anti-angiogenic, and anti-fibrotic properties.

Background: Aiming at improving an animal model of endometriosis, we developed a homologous mouse model using 'green mice' that ubiquitously express green fluorescent protein., Methods: Endometrial fragment obtained from estradiol (E2)-supplemented ovariectomized 'green mice' was minced and injected into the peritoneal cavity of ovariectomized wild-type mice. The recipient wild mice were raised with or without E2 supplementation for 2 weeks, and then were euthanized. Endometriotic lesions that developed in the abdomen were examined both macroscopically and microscopically under fluorescence, and weight of the lesions was measured., Results: The endometriotic lesions were more clearly detected under fluorescence imaging than by conventional macroscopic examination. Histologically, endometriotic lesions deriving from 'green mice' were sharply distinguished from surrounding host tissues under fluorescence microscopy. More lesions developed in E2-supplemented than control recipient mice. The measured fluorescence intensity of endometriotic lesions showed significant positive correlation with their weight (R=0.844, P<0.0001), and was significantly higher in E2-supplemented mice than in vehicle-supplemented mice (P=0.0062)., Conclusion: The present endometriosis model using 'green mice' would be useful for expeditious identification and quantitative evaluation of endometriotic lesions.

Endometriosis is known to be associated with local inflammatory reactions. Given the emerging concept of thrombin and its specific receptor, protease-activated receptor 1 (PAR1), as important players in inflammation and cell proliferation, we investigated whether thrombin and PAR1 might be involved in the pathophysiology of the disease, using a primary cell culture system of endometriotic tissues. PAR1 mRNA was expressed in primary endometriotic stromal cells (ESCs). Thrombin and SFLLRN (Ser-Phe-Leu-Leu-Arg-Asp), a PAR1 agonist peptide, increased the mRNA expression of IL-8, monocyte chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2) and the protein secretion of IL-8 nd MCP-1 in ESCs. The addition of thrombin inhibitor d-phenylalanyl-l-prolyl-l arginine chloromethyl ketone (PPACK) together with thrombin inhibited the thrombin-induced secretion of IL-8 and MCP-1. Thrombin, but not SFLLRN, activated matrix metalloproteinase-2 in ESCs, and the effect was inhibited by PPACK. Thrombin and SFLLRN increased proliferating cell nuclear antigen-positive ratio of ESCs, indicating their cell proliferation-stimulating effects. The thrombin-induced increase in proliferating cell nuclear antigen-positive ratio was diminished by PPACK. These findings imply that the thrombin system might be involved in the pathophysiology of endometriosis, stimulating inflammatory responses of endometriotic cells and their mitogenic activity.

Objective: To investigate the role of interleukin (IL)-16 in peritoneal fluid in the pathogenesis of endometriosis., Design: Comparative and laboratory study., Setting: University of Tokyo Hospital., Patient(s): Peritoneal fluids (PFs) were collected from women without endometriosis (n = 34) and with endometriosis (stages I/II, n = 30; stages III/IV, n = 58). Peritoneal fluid mononuclear cells (PFMCs) were collected from six women., Intervention(s): The PFs were collected; PFMCs were isolated and cultured with or without recombinant human (rh) IL-16., Main Outcome Measure(s): Concentrations of IL-16 in PFs were measured by enzyme-linked immunosorbent assay (ELISA). Concentrations of IL-6, tumor necrosis factor-alpha (TNF-alpha), and IL-1beta in culture media of PFMCs were determined by ELISA., Result(s): The IL-16 concentrations in the PF of women with advanced endometriosis (stages III/IV) (330 pg/mL, 231-501; median, interquartile range) were significantly higher (P=.0016) than those without endometriosis (229 pg/mL, 174-311). The PFMCs cultured with rhIL-16 released an increased amount of IL-6, TNF-alpha and IL-1beta, which was a 1.17-, 1.33-, and 1.54-fold increase, respectively, over that in the control culture., Conclusion(s): The present study indicates that IL-16 in PF may play a role in the pathogenesis of endometriosis by initiating or sustaining inflammatory responses in the peritoneal cavity.

Background: The present study was conducted to assess whether midkine (MK), a multifunctional molecule known to stimulate tumor growth, may be involved in the development of endometriosis., Methods: The mitogenic activity of MK on cultured endometriotic stromal cells was examined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation. Concentrations of MK in the peritoneal fluid (PF) of women without or with endometriosis and those under GnRH agonist treatment were measured using a specific enzyme immunoassay. The expression of MK mRNA in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues was evaluated by RT-PCR., Results: MK significantly increased BrdU incorporation into the DNA of cultured endometriotic stromal cells. The MK concentrations in the PF of the women with advanced endometriosis (stages II, III and IV) Median: 1.21 ng/ml; interquartile range 0.80-2.27 were significantly higher than those of the women without endometriosis and with stage I endometriosis (0.06 ng/ml, 0.67-1.26, P < 0.05). As for the menstrual phase, the MK concentration in PF in the inteal phase (1.32 ng/ml. 0.72-2.21) were significantly higher than those in the follicular phase (0.95 ng/ml, 0.68-1.24, P < 0.05). In addition, women with adnexal adhesions had higher concentrations of MK in PF than those without adhesions (P < 0.05). The MK concentrations of the women under GnRH agonist treatment were significantly lower than those of the other groups (P < 0.001). The expression of MK mRNA was detected in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues., Conclusions: The present findings suggest that MK may play roles, such as stimulation of endometriotic cell proliferation, in the development of endometriosis.

Protease-activated receptor 2 (PAR2) is activated by various proteases released from the leukocytes, such as neutrophils and mast cells. Because these leukocytes reside in the endometrium, we speculated that PAR2 might be activated there. In this study, we investigated the presence and possible roles of PAR2 in the endometrium. During the menstrual cycle, the expression of PAR2 mRNA in human endometrial tissues is increased from the late secretory phase to the menstrual phase and in early pregnancy. In vitro, PAR2 agonist peptide (PAR2AP) stimulated IL-8 production in both endometrial epithelial cells (EECs) and stromal cells (ESCs). PAR2AP also stimulated the mRNA expression of stem cell factor, a known activator for mast cells, in ESCs, and activated matrix metalloproteinase-7, an epithelial cell-specific matrix metalloproteinase, in EECs. In addition, PAR2AP significantly increased the 5-bromo-2'-deoxyuridine incorporation in ESCs. PAR2AP induced the phosphorylation of three MAPKs, i.e. p38 MAPK, p42/44 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase, in ESCs. Inhibitors of all three MAPKs inhibited PAR2AP-induced secretion of IL-8 in both EECs and ESCs. This is the first report demonstrating the presence of PAR2 in the human endometrium. The increased expression of PAR2 around the menstrual period, its up-regulation of molecules important for endometrial remodeling, and its mitogenic effect on endometrial cells raise the expectation of the possible involvement of PAR2 in menstruation and other architectural changes of the endometrium occurring during the menstrual cycle. MAPKs may mediate PAR2 functions in these processes.