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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.

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.

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.

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.

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.

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.