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Background To investigate the role of adenosine monophosphate (AMP)-activated protein kinase (AMPK) on the production of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, prostaglandin E2 and F2α induced by IL-1β in endometrial stromal cells (ESCs) following treatment with 5-aminoimidazole-4- carboxamide ribonucleoside (AICAR). Methods Endometrial specimens were obtained and cultured. We examined the effects of IL-1β, IL-1 ra and AICAR on the production of IL-8, MCP-1, PGE2 and PGF2α in human ESCs. The phosphorylations of AMPK, IκB, 4EBP-1, p70S6K and S6 ribosomal protein were analyzed by Western immunoblotting. Results Following stimulation by IL-1β, the production of IL-8, MCP-1, PGE2 and PGF2α showed significant increases, and these increases were suppressed by AICAR. The expression of cyclooxygenase-2 (COX-2) induced by IL-1β and suppressed by AICAR. The phosphorylation of IκB, 4EBP-1, p70S6K and S6 ribosomal protein were inhibited via an AMPK-dependent signal transduction. Conclusions The production of IL-8, MCP-1, PGE2 and PGF2α induced by IL-1β in ESCs were involved in the negative regulatory mechanisms of AMPK. The substances that activate AMPK may be promising agents for the treatment of pathological problems such as dysmenorrhea.

Platelet-activating factor (PAF) is a potent proinflammatory negotiator that shows a distinct spectrum of biological and pharmacological effects and participates in a wide range of pathophysiological conditions. In the reproductive system, PAF has been shown to have an important role in initiating ovulation, progesterone production and chemokine production. The purpose of this article was to review the roles of PAF, a well-known family of messenger phospholipids, in the reproductive process, especially in ovulation. This review highlights the interesting parallels between PAF's mechanism in ovulation and inflammatory process.

Problem Decidual cells are thought to be involved in the maintenance of pregnancy. We conducted this study to evaluate the cellular function of endometrial stromal cells (ESCs) transitioning to decidualization. Methods of study Normal endometrial specimens were obtained from premenopausal patients who had undergone hysterectomies for subserosal leiomyomas. Decidualization of the ESCs (DSCs) was induced by incubating subconfluent cells in media containing medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate. We first analyzed the expression profile of protease-activated receptor-1 (PAR-1) between ESCs and DSCs. To investigate the intracellular signal transduction system in the DSCs, we incubated cells with thrombin receptor activator peptide 6 (TRAP-6). The levels of IL-8, monocyte chemo-attractant protein-1, matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor in the culture medium were measured by enzyme-linked immunosorbent assays. The activation of the MAP kinase signaling pathway was detected by a Western blot analysis. The activation was evaluated for the expression of p21. Results PAR-1 receptor expression is upregulated in DSCs. The productions of chemokine and MMP-1 increased in the DSCs with the addition of TRAP-6. The activity of both the ERK-1 and ERK-2 isoforms was increased by 5-15 minute after TRAP-6 treatment. p70 S6 kinase showed the strongest expression after 1 hour. p21 was strongly observed in ESCs compared to the DSCs. Conclusion Our results suggest that cell function is changed by decidualization in association with increasing PAR-1 expression. The upregulation of PAR-1 may have some influence on pregnancy in the decidua.

Objective To measure the levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) induced by thrombin in endometrial stromal cells (ESC). Design Evaluation of the effects of thrombin, thrombin receptor activator peptide 6 (TRAP-6), and d-phenylalanyl-1-propyl-l arginine chloromethyl ketone (PPACK) on the production of VEGF and MMPs by ESC. Setting Research laboratory at the Oita University Medical School. Patient(s) Eight endometrial specimens in the secretory phase. Intervention(s) ESC were incubated for 24 hours with thrombin, TRAP-6, and PPACK. Main Outcome Measure(s) The levels of VEGF, MMP-1, and active MMP-2 were measured by enzyme-linked immunosorbent assay (ELISA). The presence of protease-activated receptor-1 (PAR-1) and activation of mitogen-activated protein (MAP) kinase were detected by Western blot analysis. Result(s) Following stimulation by thrombin and TRP-6, the production of VEGF, MMP-1, and active MMP-2 statistically significantly increased; U0126 and PPACK statistically significantly suppressed the increases in the production of VEGF, MMP-1, and active MMP-2 induced by thrombin and TRAP-6. Activity by MAP kinase was induced by treatment with thrombin and TRAP-6 and was suppressed by PPACK. Conclusion(s) The results suggest that thrombin stimulates the production of VEGF and MMPs by a mechanism involving the MAP kinase system. The increases in VEGF and MMPs may contribute to neovascularization, which promotes the proliferation of endometrium and placentation.

Problem To investigate the role of platelet-activating factor (PAF) in human ovulation, we studied the regulation of interleukin (IL)-8 and growth-regulated oncogene (GRO) alpha in cultured human immortalized granulosa cell line (GC1a). Method of study GC1a was cultured in serum-free medium, and incubated with carbamyl-PAF (C-PAF) and/or PAF receptor antagonist (WEB 2086). The supernatants were collected, and IL-8 and GRO alpha were measured by enzyme-linked immunosorbent assay. Results After treatment with C-PAF, the levels of IL-8 and GROalpha increased in a time-dependent manner. The levels of IL-8 and GROalpha were significantly increased after treatment with C-PAF in a dose-dependent manner. However, the levels of IL-8 and GROalpha were significantly decreased by treatment with C-PAF and with increasing concentrations of WEB 2086. Conclusion Our data indicated that IL-8 and GROalpha were regulated by C-PAF. The results suggested that PAF may play an important role in human pre-ovulatory processes involving IL-8 and GROalpha production.

Objective To evaluate the involvement of transforming growth factor (TGF)-β isoforms (TGF-β1, TGF-β2, and TGF-β3) on endometrial tissue remodeling during the perimenstrual period. Design The effects of TGF-β isoforms on the cell proliferation, motility, and contractivity of cultured human endometrial stromal cells (ESCs) were investigated. Setting Research laboratory at a medical school. Patient(s) Nine endometrial specimens in the late secretory phase were used. Intervention(s) Endometrial stromal cells were incubated with recombinant human recombinant TGF-β1, TGF-β2, and TGF-β3. Main Outcome Measure(s) The cell proliferation, motility, and contractivity of ESCs were accessed by a modified methylthiazoletetrazolium assay, in vitro wound repair assay, transwell invasion assay, and collagen gel contraction assay. Result(s) All three isoforms of TGF-β significantly inhibited the cell proliferation of ESCs in a dose-dependent manner. In vitro wound repair assay and transwell invasion assay demonstrated that the TGF-β isoforms significantly inhibited the motility of ESCs. However, the TGF-β isoforms were shown to have a clear effect on the collagen gel contractivity of ESCs. Conclusion(s) These results suggest that TGF-β isoforms may promote endometrial tissue repair through the inhibition of the proliferation, expansion, and migration of ESCs, and through the stimulation of the contraction of the collagen gel matrix by these cells. Transforming growth factor-β may be involved in the protection of the endometrium from extensive fibrosis and scarring by regulating ESC function during the perimenstrual period.

Objective To measure the level of interleukin 6 (IL-6), IL-8, and macrophage colony-stimulating factor (M-CSF) induced by IL-1α in endometrial stromal cells (ESC) following treatment with ceramide analogues. Design The effects of IL-1α, IL-1 receptor antagonist (IL-1RA), C2-ceramide, and C6-ceramide on the production of IL-6, IL-8, and M-CSF by ESC. Setting Research laboratory at Oita University Medical School. Patient(s) Eleven premenopausal women who had undergone hysterectomies for subserous myoma provided endometrial specimens in the secretory phase. Intervention(s) The ESC were incubated for 24 hours with IL-1α, IL-1RA, C2-ceramide, and C6-ceramide. Main outcome measure(s) The levels of IL-6, IL-8, and M-CSF in the culture media were measured via enzyme-linked immunoabsorbent assay. Result(s): Following stimulation by IL-1α, the production of IL-6, IL-8, and M-CSF showed a statistically significant increase, and they were suppressed by IL-1RA in a dose-dependent manner. Production of IL-6, IL-8, and M-CSF was not statistically significantly increased by IL-1α plus C2-ceramide as compared with IL-1α alone. Production of both IL-8 and M-CSF was statistically significantly increased by IL-1α plus C6-ceramide as compared with IL-1α alone; however, IL-6 production was not increased. Conclusion(s) The results suggest that IL-1α stimulates the production of IL-8 and M-CSF by a mechanism that involves the sphingomyelin-ceramide system. Ceramide may be important in increasing the production of IL-8 and M-CSF in the human endometrium.

Problem In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein -1 (MCP-1) in human ovulation, we studied the regulation of M-CSF and MCP-1 in cultured human granulosa cells. Method of study Immortalized granulosa cells (GC1a) were cultured in serum-free medium, and incubated with interleukin (IL)-1alpha, IL-1 receptor antagonist (ra) and tumor necrosis factor (TNF)-alpha. The supernatants were collected, and M-CSF and MCP-1 were measured by ELISA. Results The levels of M-CSF and MCP-1 were increased after treatment with IL-1alpha (1 nm) and TNF-alpha (1 nm) in a time-dependent manner. The levels of M-CSF and MCP-1 were significantly increased after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. However, the levels of M-CSF and MCP-1 were significantly decreased by treatment with IL-1alpha (1 nm) and/or increasing concentrations of IL-1 ra. Conclusions Our data indicated that M-CSF and MCP-1 were regulated by IL-1alpha and TNF-alpha. It was suggested that M-CSF and MCP-1 may play an important role in human preovulatory processes.

Platelet activating factor (PAF), a potent phospholipid mediator, has been implicated in a number of reproductive processes through ovulation to parturition. To clarify the regulatory mechanism of PAF metabolism in the decidua, we have investigated the effect of activation of protein kinase C (PKC) on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by human decidual macrophages. Decidual macrophage populations were isolated from human decidua by using enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The cells were treated with a PKC activator (TPA), H-7, dibutyryl cyclic adenosine monophosphate (AMP), Bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetra (acetoxymethyl)-ester (BAPTA/AM) and/or nifedipine. The activity of PAF-AH secreted in the culture medium was assayed. The PKC activator, TPA, inhibited the PAF-AH secretion by decidual cells in a dose-dependent manner. The TPA also decreased the enzyme secretion by flow cytometrically purified macrophages. The inhibitory effect of TPA was blocked by a PKC inhibitor, H-7. Protein kinase A (PKA) activation by dibutyryl cyclic AMP was without effect on the enzyme secretion. Calcium channel blockers, BAPTA/AM and nifedipine had no effect on the PAF-AH secretion. It is suggested that the TPA-induced inhibition of PAF-AH secretion may be mediated, in part, by a PKC-dependent signal transduction, and that activation of PKC may result in the increase in the local concentration of PAF in the decidua because of its inhibitory effect on the PAF-AH secretion by decidual macrophages.

Objective To evaluate the effects of leptin on the production of interleukin (IL)-6 family cytokines and chemokines by human endometrial stromal cells (ESC) and epithelial cells. Design The effects of leptin on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene (GRO)-α, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-3α by ESC and the endometrial epithelial cell line HHUA were investigated. Setting Research laboratory at a medical university. Patient(s) Eight endometrial specimens in the late proliferative phase were used for the isolation of ESC. Intervention(s) ESC and HHUA were incubated for 24 hours with recombinant human leptin. Main outcome measure(s) The concentration of IL-6, IL-11, LIF, IL-8, GRO-α, MCP-1, and MIP-3α were measured using ELISAs. Result(s) Unstimulated ESC and HHUA constitutively secreted IL-6, IL-11, LIF, IL-8, GRO-α, MCP-1, and MIP-3α. The increase in levels of IL-6, IL-8, GRO-α, MCP-1, and MIP-3α in the culture media of ESC and HHUA paralleled the addition of increasing amounts of leptin. In contrast, the levels of IL-11 and LIF were not affected by leptin administration. Conclusion(s) The present findings suggest that leptin may be an additional modulator of IL-6 and chemokine expression in the endometrium. Leptin may contribute to the normal and pathological processes of human reproduction by the regulation of these cytokines in the local environment.

PROBLEM Interleukin-8 (IL-8) and growth-regulated oncogene-alpha (GRO-alpha) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL-1alpha, tumor necrosis factor-alpha (TNF-alpha), protein kinase C (PKC) activators (TPA), and db-cyclic adenosine monophosphate (cAMP) on IL-8 and GRO-alpha production by immortalized GC1a and granulosa-lutein cells. METHOD OF STUDY Confluent granulosa-lutein cells were placed in serum-free medium before incubated for 8 hr with the above-mentioned test agents. Finally, we measured IL-8 and GRO-alpha levels in the culture media using an enzyme-linked immunosorbent assay (ELISA). RESULTS Treatment of granulosa-lutein cells with of IL-1alpha (1 nM), TNF-alpha (1 nM), TPA (1 nM) and db-cAMP (100 microM) produced higher levels of IL-8 than untreated cells by 8 hr (2274.7 +/- 146.3, 1489.8 +/- 190.1, 1452.9 +/- 152.7, 1313.6 +/- 48.4 pg/mL, respectively; control = 457.7 +/- 38.2 pg/mL; P < 0.001). Treatment of granulosa-lutein cells with 1 nM of IL-1alpha, TNFalpha, TPA, and db-cAMP (100 microM) resulted in higher levels of GRO-alpha than untreated cells by 8 hr (993.7 +/- 9.5, 171.4 +/- 6.5, 147.5 +/- 6.7, 472.4 +/- 16.2 pg/mL respectively; control = 73.8 +/- 8.2 pg/mL; P < 0.001). CONCLUSIONS Our data strongly suggests roles for IL-1alpha, TNFalpha, and PKC activators in the inflammation-like mechanism of human ovulation. Furthermore, our study suggests a positive, but still debatable, role for cAMP in the same mechanism.

Objectives : It has been reported that prostaglandin E 2 (PGE 2 ) is synthesized in the amnion and that this synthesis increases during labor. The purpose of this study was to clarify the mechanism for the expression of cyclooxygenase-2 (COX-2) mRNA and the PGE 2 synthesis of amnion-derived (WISH) cells. Study design : Cells were cultured and treated by 12- O -tetradecanoylphorbol-13-acetate (TPA) and dexamethasone (DEX). PGE 2 in the culture medium was measured by ELISA. Total RNA was extracted from the cells, and COX-2 mRNA expression was analyzed by Northern blot analysis. Results : During the time course of PGE 2 production in response to TPA stimulation, the PGE 2 production could not be detected until incubation had continued for 2h, but this production appeared to continue after 4h of incubation. PGE 2 production was significantly increased by TPA and suppressed by treatment with TPA and DEX. During the time course of COX-2 mRNA expression in response to treatment with TPA, the COX-2 mRNA band was detected after 1.5h. The strongest expression of COX-2 mRNA was observed at 2h incubation. After pre-treatment with TPA for 1h, the TPA-induced COX-2 mRNA was suppressed by treatment with DEX for 1 or 2h incubation in a dose-dependent manner. Conclusions : These results suggest that COX-2 mRNA is induced by TPA which activate protein kinase C, and suppressed by DEX in WISH cells.

PROBLEM In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.

Platelet-activating factor (PAF) is involved in such reproductive processes as parturition. We investigated the effect of PAF on the expression of matrix metalloproteinase-1 (MMP-1) and that of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human uterine cervical fibroblasts. Uterine cervical tissue was obtained from patients who underwent cesarean section at term. Collagenase-dispersed fibroblasts were cultured and used in the experiments. PAF receptor was identified in the uterine cervical fibroblasts by use of reverse transcription-polymerase chain reaction and Southern blot analysis. Northern blot analysis showed that PAF increased the expression of MMP-1 mRNA in a time-dependent manner, whereas expression of TIMP-1 mRNA was not affected by PAF. Concentration of MMP-1 protein in the PAF-treated culture media significantly exceeded that in control cultures. The PAF-induced production of MMP-1 protein was abolished by treatment with WEB 2170, a specific PAF receptor antagonist. Results suggest that PAF may accelerate collagenolysis in the human uterine cervix by inducing an imbalance in the activity between MMP-1 and TIMP-1, thus contributing to the cervical ripening during parturition.

PROBLEM: The aim of this study was to clarify the physiological effects of interferon (IFN)-γ on secretion of vascular endothelial growth factor (VEGF) by endometrial stromal (ES) cells. METHOD OF STUDY: ES cells were obtained from human uterine endometrium by enzymic digestion and filtration. The effects of IFN-γ on production of VEGF by ES cells were examined by analyzing VEGF mRNA expression with Northern blotting analysis and by assaying VEGF protein. RESULTS: IFN-γ inhibited VEGF mRNA and protein expression by ES cells in a dose-dependent manner. In ES cells treated with IFN-γ, VEGF production was not significant until 6 hr of incubation and was significantly affected after 6 hr of incubation, but decreased significantly after 1 to 48 hr. IFN-γ also suppressed VEGF mRNA expression by ES cells. CONCLUSIONS: ES cells produce VEGF, which may contribute to endometrial neovascularization and proliferation. IFN-γ may play an important role in regulating VEGF production by ES cells.

To examine the production of hepatocyte growth factor (HGF) by human endometrial stromal cells (ESC) in vitro, concentrations of HGF in the culture media of ESC were measured after the addition of various amounts of 12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin, lipopolysaccharide (LPS), interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNFalpha), interferon-gamma (IFNgamma), or ethynylestradiol-17alpha using an ELISA. The expression of HGF mRNA was also assayed by a reverse transcription-polymerase chain reaction. The concentration of HGF in the culture media of unstimulated ESC was below the detection level of the assay. TPA stimulated the secretion of HGF by ESC in a dose-dependent manner. TPA also induced the transcription of HGF mRNA by ESC. Forskolin, LPS, IL-1beta, IL-6, IL-8, TNFalpha, IFNgamma, or ethynylestradiol-17alpha did not alter HGF mRNA or protein levels. TPA-stimulated production of HGF was partially inhibited by the addition of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or sphingosine. These results suggest that a protein kinase C-dependent pathway may play an important role in the regulation of HGF production by ESC. HGF secreted by ESC may be involved in the regeneration of the endometrium during the normal menstrual cycle.

Summary PAF, a potent lipid mediator, has been implicated in a number of reproductive processes, including parturition. PAF has been shown to stimulate prostaglandin E 2 production in fetal membranes and to cause myometrial contraction. PAF metabolism in the decidua and its modulation by endotoxin or cytokines has been investigated. Decidual macrophages secrete PAF-AH activity of the plasma type that inactivates PAF. LPS, TNF-α, and IL-1β inhibited the PAF-AH secretion by decidual macrophages. The LPS-inhibition was partially reversed by IL-1ra or by neutralizing antibodies against TNF-α and IL-1β. The effect of IL-1β on the secretion was abolished by IL-1ra. IFN-γ, IL-6, and IL-8 also inhibited the PAF-AH secretion, while M-CSF increased the enzyme secretion. These observations lend additional support to the concept that PAF is pathophysiologically involved in parturition or preterm labor caused by chorioamnionitis, forming a PAF-mediated cytokine network at the fetal-maternal decidual interface.

Tumor necrosis factor-alpha stimulates the secretion of vascular endothelial growth factor by cultured human oviductal epithelial cells and stromal fibroblasts. Tumor necrosis factor-alpha-stimulated vascular endothelial growth factor secretion by these cells may control oviductal fluid secretion by regulating vascular permeability.

In order to investigate the role of insulin-like growth factors (IGFs) in human ovulation, we evaluated the concentrations of IGF-binding proteins (IGFBPs) in human follicular fluid (FF). The concentrations of IGFBP-1 in the FFs of 15 women undergoing in vitro fertilization and embryo transfer were measured and related to those of 17beta-estradiol (E2), progesterone and androstenedione in the FFs. IGFBP-1 levels in the FFs were positively correlated with those of E2 and progesterone. No correlation was found between the IGFBP-1 and androstenedione levels in FFs. The concentrations of IGFBP-1 were significantly increased in the FFs which contained mature oocytes compared with those of immature oocytes, whereas IGFBP-3 in FFs tended to decrease as oocytes matured. It is suggested that IGFs may play important roles in human preovulatory processes, and that IGFBP-1 may be a valuable biochemical marker in the evaluation of oocytes.

In order to investigate the roles of epidermal growth factor (EGF) and transforming growth factor (TGF)-α in ovulation, we studied the production of interleukin (IL)-8 and growth-regulated oncogene (GRO)-α in cultured human granulosa-lutein cells.Granulosa-lutein cells obtained from the follicular fluids of in vitro fertilization and embryo transfer patients were cultured and treated with EGF, TGF-α, tumor necrosis factor (TNF)-α or 12-O-tetradecanoylphorbol 13-acetate (TPA). An immortalized granulosa cell line (GC1a) was also cultured and treated with EGF, TGF-α or mitogen-activated protein kinase kinase inhibitor. The supernatants were collected, and IL-8 and GRO-α were measured by ELISA.The levels of IL-8 and GRO-α were significantly increased after treatment with EGF, TGF-α, TNF-α and TPA by primary cultured granulosa-lutein cells. The levels of IL-8 and GRO-α were also significantly increased after treatment with EGF or TGF-α in a dose-dependent manner by GC1a. When GC1a was treated with EGF, TGF-α or U0126, the levels of IL-8 and GRO-α were significantly decreased.Our data indicate that the production of IL-8 and GRO-α is upregulated by EGF and TGF-α. It is suggested that EGF and TGF-α may play an important role in luteinization processes involving IL-8 and GRO-α production.

BACKGROUND: In order to investigate the regulation of chemokines [interleukin-8 (IL-8), growth-regulated oncogene (GRO)α, monocyte chemoattractant protein-I (MCP-I)) induced by thrombin in endometrial stromal cells (ESCs), the effects of thrombin, a protease activated receptor (PAR)-I antagonist (PPACK), mitogen-activated protein kinase kinase inhibitor (U0126), phospholipase C inhibitor (U-73122), an antagonist of the intracellular InsP3 receptor (2-aminoethoxy-diphenylborate (2-APB)] and a protein kinase C inhibitor (GF-109203X) on the production of chemokines by ESCs were evaluated. METHODS: ESCs from eight endometrial specimens in the secretory phase were cultured and incubated for 24 h with thrombin and PPACK, U0126, U-73122, 2-APB or GF-109203X. The levels of IL-8, GROα and MCP-I in the culture medium were measured by means of ELISA. The activation of MAP kinase was detected by western blot analysis using anti-phosphorylated MAP kinase (ERK I /2) antibody. RESULTS: Following stimulation by thrombin, the production of IL-8, GROα and MCP-I increased significantly in a dose-dependent manner. PPACK, U0126, U-73 122, 2-APB or GF-109203X suppressed the increases in production of IL-8, GROα and MCP-I induced by thrombin (P < 0.001, P < 0.001 and P < 0.001, respectively). MAP kinase activities were induced by treatment with thrombin, and were suppressed by PPACK, U0126, U-73122, 2-APB or GF-109203X. CONCLUSIONS: Our results suggest that thrombin stimulates the production of IL-8, GROα and MCP-I via PAR-I by a mechanism involving the MAP kinase system. The increases in IL-8, GROα and MCP-I may contribute to the maintenance of implantation involving leukocyte chemotaxis.

Background: Metallothionein (MT) is known to bind to metals with high affinity. The potential for MT-1 mRNA expression in endometrial stromal cells (ESC) and amniotic cells in response to cytokines and cadmium chloride (CdCl2) was evaluated. Methods: Human ESC were cultured and treated with interleukin-1α, 12-O-tetradecanoylphobol 13-acetate (TPA), forskolin, transforming growth factor-β, and CdCl2. Amnion-derived (WISH) cells were also cultured and treated with the same reagents. The levels of MT mRNA were evaluated by Northern blot analysis in ESC and WISH cells. Results: In response to treatment with CdCl2 (0.01–10 µM), the expression of MT mRNA markedly increased in ESC and WISH cells in a dose-dependent manner. On the other hand, the expression of MT mRNA did not increase after treatment with interleukin-1α (1 nM), TPA (10 nM), forskolin (1 µM) or transforming growth factor-β (1 nM) in these cells. Conclusion: These findings indicate that MT expression in ESC and WISH cells is sensitive to the CdCl2 concentration, which is known to be evaluated in cigarette smokers. The present results suggest that increased levels of MT may affect metal metabolism at the feto-maternal interface.

Background:Wepreviously demonstrated a remarkably higher association of pelvic endometriosis in women with ovarian endometrioma. However, information regarding occurrence of pelvic endometriosis in women with demoid cyst is unknown even thesewomenexperience similar retrogrademenstruation like womenwithovarianendometriosis.During theprocessof endometrial cell attachment, an event of immunedefect or tolerance occurs that finally decide the survival or clearance of autologous cells in pelvic environment. Here, we retrospectively evaluated recorded files of laparoscopy from 1982 to 2008 at our university and examined the incidence of pelvic endometriosis in women with benign ovarian cysts. Material and methods: We performed a retrospective study conducted in 2988 cases that had undergone laparoscopic surgery between September 1982 and April 2008 for the indication of ectopic pregnancy, tubal infertility and other benign gynecologic diseases. We analyzed occurrence of pelvic endometriosis in women with ovarian endometrioma and dermoid cyst during 25 years observation period.With informed consent, cystwall from20 womenwithdermoidcysts and20womenwithovarianendometrioma and corresponding eutopic endometria and peritoneal tissues were prospectively collected during laparoscopy. Tissue expression of FOXP3+ Treg cells was examined by immunohistochemistry and peritoneal fluid (PF) levels of TGF and IL-6 were measured by ELISA. Results: Among 2988 laparoscopy cases, 350 cases (11.7%)were found to have ovarian endometrioma, 414 (13.8%) cases had dermoid cyst and 398 (13.3%) cases were treated for fertility screening or uterine myoma with bilateral pathology free ovary. A predominance of both ovarian endometrioma and dermoid cysts was found inwomen in their 20th and30thyears of age.Womenwithdermoid cysts had a significantly decreased occurrence of pelvic endometriosis (74 cases, 18.6%) when compared with that of women with ovarian endometrioma (267 cases, 76.3%) and with women with bilateral normal ovary (188 cases, 47.2%). A significantly higher expressionof FOXP3, a transcriptional factorofTregcells,was found in cyst wall and peritoneum of women with dermoid cysts having peritoneal lesions comparing to similar tissues derived from dermoid cysts without any peritoneal lesions. A relationship between FOXP3 expression and TGF /interleukin-6 levels in peritoneal fluid (PF) will be discussed. Conclusions: Higher FOXP3+ Treg cells in the peritoneum may be associated with decreased occurrence of peritoneal lesions in women with benign ovarian cyst. A possible immunosurveillance secondary to different pelvic environmentmight be involved in this protective effect against endometriosis.

Objective The aim of this study is to evaluate the effects of interleukin (IL)-13, a Th2 cytokine, on the production of vascular endothelial growth factor (VEGF) and soluble fms -like tyrosine kinase-1 (sFlt-1) in human oviductal cells in vitro . Study design Human oviductal epithelial cells (OECs) were isolated from five premenopausal patients. The secretion of VEGF 165 and sFlt-1 by cultured OECs in response to IL-13 was measured using an enzyme-linked immunosorbent assay. Results The secretion of VEGF 165 and sFlt-1 was detected in cultured OECs under untreated conditions. IL-13 enhanced the secretion of VEGF 165 and sFlt-1 by OECs in a dose-dependent manner. Conclusion The present findings suggest that IL-13 is a regulatory factor of VEGF and sFlt-1 production in the human fallopian tubes. IL-13 in the local environment may stimulate oviductal vascular permeability by inducing the production of VEGF by oviductal cells. The modulation of VEGF secretion by IL-13 secreted by the peri-implantation embryo may contribute to the normal and pathological processes of human reproduction during the peri-implantation period.

Objective To evaluate the effect of hypoxia on the production of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) in the human fallopian tube. Design The secretion of VEGF and sFlt-1 by cultured oviductal epithelial cells (OECs) and oviductal stromal fibroblasts (OSFs) in response to hypoxia was investigated. Setting Research laboratory at a medical school. Patient(s) Normal oviducts obtained from seven premenopausal patients were used. Intervention(s) Oviductal epithelial cells and OSFs were incubated under normoxic (20% O 2 ) or hypoxic (2% O 2 ) conditions. Main Outcome Measure(s) The concentrations of VEGF and sFlt-1 in the culture media of OECs and OSFs were measured by enzyme-linked immunosorbent assays. Result(s) The secretion of both VEGF and sFlt-1 was detected in cultured OECs and OSFs and was found to have been stimulated under hypoxic conditions in these cells. Conclusion(s) The present findings suggest that hypoxia in the local environment may stimulate oviductal vascular permeability by inducing the production of VEGF by oviductal cells. Simultaneous up-regulation of sFlt-1 secretion by these cells under hypoxic conditions may prevent excessive up-regulation of vascular permeability. The modulation of the bias of VEGF and sFlt-1 in the fallopian tubes may contribute to the normal and pathological processes of oviductal fluid secretion by regulating oviductal vascular permeability during the menstrual cycle.

Objective To investigate the role of macrophageinflammatory protein (MIP)-3α in human ovulation. Design Study of the levels of MIP-3α in serum and follicular fluid. The effects of interleukin (IL)-1α, IL-1 receptor antagonist (RA), and tumor necrosis factor (TNF)-α on the secretion of MIP-3α by primary cultured granulosa-lutein cells and an immortalized granulosa cell line (GC1a) were investigated. Setting Research laboratory at a university medical school. Patient(s) Forty-six patients with sterility undergoing in vitro fertilization and embryo transfer (IVF-ET). Intervention(s) Follicular fluid was obtained from study participants, and granulosa-lutein cells and GC1a were incubated with IL-1α, IL-1RA, or TNF-α for 4 to 32 hours. Main outcome measure(s) The concentration of MIP-3α in human follicular fluid was measured and correlated with oocyte maturation. We also cultured granulosa cells and examined the regulation of MIP-3α production. The concentrations of MIP-3α in the serum, follicular fluid, and culture medium were measured using enzyme-linked immunoabsorbent assay. Result(s) Concentrations of MIP-3α were significantly higher in the follicular fluid, but it was not detected in the serum. Concentrations of MIP-3α were statistically significantly higher in the follicular fluid containing mature oocytes than in follicular fluid containing immature oocytes. The production of MIP-3α was markedly increased over the basal level after treatment with IL-1α and TNF-α in a dose-dependent manner. The stimulatory effect of IL-1α was inhibited by IL-1RA. Conclusion(s) Our data suggest that MIP-3α was present in follicular fluid and correlated with oocyte maturation, and was regulated by IL-1α and TNF-α. Thus, MIP-3α may play an important role in the human preovulatory process.

Thrombospondin-1 (TSP-1) production was modulated by EGF, IFN-gamma, and in vitro decidualization. It is suggested that TSP-1 may contribute to the regulation of neovascularization in the endometrium and gestational tissues.

Interferon (IFN)-beta, not IFN-alpha, suppresses vascular endothelial growth factor secretion in endometriotic cyst stromal cells. It was suggested that IFN-beta may be more suitable than IFN-alpha as an anti-endometriotic agent.

Hypoxia downregulated the concentration of endostatin in the culture media of human endometrial stromal cells but did not affect the messenger (m)RNA expression of collagen XVIII. Both mRNA and protein expression of vascular endothelial growth factor were upregulated in a hypoxic condition.

PROBLEM Prostaglandins (PGs) are synthesized in the endometrium. Our objective was to evaluate interleukin (IL)-1alpha-induced production of PGE2 and PGF2alpha in endometrial stromal cells (ESC) following treatment with ceramide analogues. METHODS OF STUDY ESC were obtained from human uterine endometrium by enzymic digestion and filtration. ESC were treated with IL-1alpha, IL-1 receptor antagonist (ra), C2-ceramide and C6-ceramide. The concentrations of PGE2 and PGF2alpha in media were determined using ELISA. The induction of prostaglandin H synthase (PGHS)-2 mRNA was also ascertained by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS The production of PGE2 and PGF2alpha was significantly increased by IL-lalpha and suppressed by IL-1 ra, in a dose-dependent manner. PGF2alpha production was further increased by treatment with the combination of IL-1alpha and C2-ceramide as compared with IL-1alpha treatment alone. There was no significant difference in PGE2 production between cells treated with IL-1alpha and C2-ceramide and those treated with IL-1alpha alone. Both PGE2 and PGF2alpha production were significantly increased by treatment with IL-1alpha and C6-ceramide as compared with IL-1alpha treatment alone. Treatment of ESC with IL-1alpha stimulated PGHS-2 mRNA. PGHS-2 mRNA was decreased when IL-1 ra was added to the IL-1alpha-stimulated cells. CONCLUSIONS These results suggest that IL-1alpha stimulates the production of PGE2 and PGF2alpha by a mechanism that involves the sphingomyelin-ceramide system, and thus that ceramide may be important in increasing the production of PGE2 and PGF2alpha in the human endometrium.

Although preimplantation embryo and decidual cells secrete significant amounts of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF); its precise function in early pregnancy has yet to be established. To investigate the effect of PAF on cytokine synthesis, we measured the cytokine concentration in the culture media of two human cell lines: normal endometrial stromal cells (ESC) and endometrial stromal sarcoma cells (MaMi), following stimulation with a non-metabolized PAF analogue, carbamyl-PAF (C-PAF). Enzyme-linked immunosorbent assays were used to measure five cytokines: interleukin (IL)-6, IL-8, macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein-1alpha (MIP-1alpha) and tumour necrosis factor-alpha (TNF-alpha). We also evaluated the mRNA expression for IL-6 and IL-8 in ESC after C-PAF stimulation using Northern blot analysis. Non-stimulated ESC and MaMi cells both secreted IL-6, IL-8, and M-CSF, but not MIP-1alpha or TNF-alpha. The concentrations of IL-6, IL-8, M-CSF, MIP-1alpha, and TNF-alpha in the culture media of both cell lines increased in parallel with increasing amounts of C-PAF. C-PAF stimulated IL-6 and IL-8 transcription in ESC. These results suggest that PAF secretion by decidual tissues and developing embryos may induce cytokine synthesis by the ESC, as part of the cytokine network in the feto-maternal unit. An increase in the local cytokine concentration may be an important factor in the maintenance of early stages of gestation.

A case of mucin-producing adenocarcinoma arising from a mature cystic teratoma of the right ovary is reported in a 67-year-old multiparous Japanese woman. The patient underwent total hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and adjuvant chemotherapy containing cisplatin, Adriamycin, and cyclophosphamide with the diagnosis of ovarian cancer, probable stage Ic. Treatment was successful even though the tumor extended through the cyst wall.

Objective To examine the role of platelet-activating factor (PAF) metabolism in the periovulatory processes. Design The PAF-acetylhydrolase activity in the follicular fluid (FF) obtained in conjunction with IVF-ET procedure was assayed and its activity was related to oocyte maturation. The PAF-acetylhydrolase activity also was related to the concentration of various ovarian hormones. Setting All patients were managed and treated at Oita Medical University Hospital, Oita, Japan. Patients The study concerned 30 women between 28 and 36 years of age with tubal infertility. Main Outcome Measure The activity of PAF-acetylhydrolase in FF was assayed as well as E 2 and P. Oocyte maturation also was evaluated. Results Platelet-activating factor-acetylhydrolase activity was decreased significantly in the FFs of patients with a successful outcome of their pregnancies compared with the nonsuccessful group. Estradiol levels were negatively correlated with PAF-acetylhydrolase activities in the FFs. No correlation was found between the PAF-acetylhydrolase activity and P concentration in the FF. Significantly more mature and less immature oocytes were recovered in the group who subsequently became pregnant compared with the nonpregnant group. Conclusions It is suggested that the decrease in PAF-acetylhydrolase activity may result in an increase of PAF in the FFs, which in turn may contribute to a successful pregnancy. The determination of PAF-acetylhydrolase activity in FF may serve as a prognostic marker for the evaluation of oocytes that are utilized in IVF-ET procedure.

We evaluated the effect of transferrin on the regulation of granulosa cell function in humans by evaluating the production of progesterone (P) in the preovulatory phase in vivo, and in cultured porcine granulosa cells in vitro. Twenty-five women treated for in vitro fertilization and embryo transfer had their serum levels of 17 beta-estradiol (E2) and P determined on the day of administration of human chorionic gonadotropin. Transferrin concentrations were also determined in ovarian follicular fluid. In an in vitro study, porcine granulosa cells were cultured in the presence of follicle-stimulating hormone (FSH) and transferrin. Serum levels of P showed a significant negative correlation with those of transferrin (r = -0.53, p0.01), whereas serum levels of E2 did not (r = 0.14). When the subjects were divided into two groups by serum P concentration (low P1 ng/ml, high Por = 1 ng/ml) serum concentrations of transferrin were significantly increased in the group with the low versus the high level of p (p0.01). Production of P by porcine granulosa cells was suppressed by transferrin in the presence of various concentrations of FSH. Increasing the dose of transferrin significantly suppressed the production of P by those cells in a dose-dependent fashion. The production of P during the preovulatory phase may be suppressed by transferrin in the granulosa cells.

A transverse vaginal septum is the most common congenital anomaly of the vagina. Vaginal atresia is seen less frequently. There have been some variations in cases of transverse vaginal septum and vaginal atresia. This is the first report of a patient with retrohymenal vaginal atresia and perforate transverse vaginal septum.

Background Women with polycystic ovary syndrome (PCOS) are generally insulin- resistant and are consequently often treated with metformin. We investigated the effect of metformin and AICAR on the AMP-activated protein kinase (AMPK) pathway. Methods We evaluated the effects of 5-amino-imidazole-4-carboxyamide-1- beta-D-ribofuranoside (AICAR) and metformin on tumor necrosis factor (TNF)-alpha- stimulated chemokine production in human granulosa cells. The phosphorylations of AMPK, I-kappaB, 4E-BP-1, p70S6K were analyzed by western immunoblotting. Results AICAR and metformin markedly reduced the IL-8 and GROalpha production induced by TNF-alpha. AICAR and metformin also reduced the TNF-alpha-induced phosphorylation of I-kappaB. The phosphorylations of I-kappaB, 4EBP-1, p70S6K were inhibited via an AMPK-dependent signal transduction. Conclusions These results suggest that metformin promotes granulosa cell function by reducing a TNF-alpha- and chemokine-mediated inflammatory reaction through an AMPK-dependent pathway. These finding may have implications for metformin’s actions during the treatment of PCOS with metformin.