Junichiro Fukuda

Production of vascular endothelial growth factor and angiogenic factor in human follicular fluid

The aim of this study was to quantitate of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and angiogenin in follicular fluid (FF) and to correlate the levels of these substances with oocyte maturation. FF were aspirated from women undergoing in vitro fertilization and embryo transfer. Sera were collected from women with normal menstrual cycles. VEGF in FFs and sera were measured by enzyme linked immunosorbent assay. VEGF, HGF, and angiogenin mRNA expression aspirated folliculars cell was analyzed by reverse transcription and polymerase chain reaction (RT-PCR). The concentrations of VEGF, HGF, and angiogenin in FF were significantly higher than those in serum (P<0.001). VEGF, HGF, and angiogenin mRNA in the aspirated follicles cell was detected by RT-PCR. HGF levels were higher in FF containing mature oocyte. The levels of VEGF in FF containing mature oocytes in women under 39 years of age were significantly lower than those in FF from women more than 40 years old (P<0.01). Our data suggest that VEGF, HGF, and angiogenin may play an important role in follicular growth and development, that VEGF levels in FF appear to be age-dependent; and that VEGF and HGF levels might be valuable biochemical markers of oocyte maturation.

The effect of inflammatory cytokines on secretion of macrophage colony-stimulating factor and monocyte chemoattractant protein-1 in human granulosa cells.

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.

Effects of leptin on the production of cytokines by cultured human endometrial stromal and epithelial cells

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)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-3alpha 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-alpha, MCP-1, and MIP-3alpha were measured using ELISAs. RESULT(S) Unstimulated ESC and HHUA constitutively secreted IL-6, IL-11, LIF, IL-8, GRO-alpha, MCP-1, and MIP-3alpha. The increase in levels of IL-6, IL-8, GRO-alpha, MCP-1, and MIP-3alpha 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.

Possible Modulators of IL‐8 and GRO‐α Production by Granulosa Cells

Problem:  Interleukin‐8 (IL‐8) and growth‐regulated oncogene‐α (GRO‐α) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL‐1α, tumor necrosis factor‐α (TNF‐α), protein kinase C (PKC) activators (TPA), and db‐cyclic adenosine monophosphate (cAMP) on IL‐8 and GRO‐α production by immortalized GC1a and granulosa‐lutein cells.

The production of vascular endothelial growth factor and metalloproteinase via protease-activated receptor in human endometrial stromal cells

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.

Secretion of granulocyte chemotactic protein-2 by cultured human endometrial stromal cells

OBJECTIVE To evaluate the expression of granulocyte chemotactic protein-2 (GCP-2) in human endometrial stromal cells. DESIGN The effects of interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma, and lipopolysaccharide (LPS) on the production of GCP-2 by endometrial stromal cells were investigated. SETTING Research laboratory at a medical university. PATIENT(S) Eight endometrial specimens in the late proliferative phase were used. INTERVENTION(S) Endometrial stromal cells were incubated for 24 hours with IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, IFN-gamma, and LPS.The concentration of GCP-2 in the culture media was measured using an enzyme-linked immunosorbent assay (ELISA). RESULT(S) A small amount of GCP-2 was detected in the culture media of unstimulated endometrial stromal cells. The production of GCP-2 by endometrial stromal cells was stimulated with IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, and LPS in a dose-dependent manner. Interferon-gamma did not affect GCP-2 production by these cells. CONCLUSION(S) These results suggest that GCP-2 is an additional ELR(+)-CXC chemokine expressed in endometrial stromal cells. The modulation of GCP-2 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating the neutrophil trafficking in the endometrium.

Interleukin-13 and tumor necrosis factor-β differentially regulate the production of cytokines by cultured human endometrial stromal cells

OBJECTIVE To evaluate the effects of interleukin (IL)-13, a T-helper (Th)2 cytokine, and tumor necrosis factor (TNF)-beta, a Th1 cytokine, on the production of IL-6 family cytokines and chemokines by endometrial stromal cells (ESC). DESIGN The effects of IL-13 and TNF-beta, on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene alpha (GROalpha), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, T-cell expressed and secreted (RANTES), and eotaxin were investigated. SETTING Research laboratory at a medical university. PATIENT(S) Thirteen endometrial specimens in the late proliferative phase were used. INTERVENTION(S) The ESC were incubated for 24 hours with recombinant human IL-13 and recombinant human TNF-beta. MAIN OUTCOME MEASURE(S) The concentration of IL-6, IL-11, LIF, IL-8, GROalpha, MCP-1, RANTES, and eotaxin in the culture media was measured using ELISA. RESULT(S) The increase in levels of IL-6, IL-8, MCP-1, and eotaxin in the culture media of ESC paralleled the addition of increasing amounts of IL-13 and TNF-beta, whereas the levels of IL-11 and LIF were decreased with increasing amounts of IL-13, but were increased with increasing amounts of TNF-beta. Tumor necrosis factor-beta enhanced the production of GROalpha and RANTES in dose-dependent manner; however, IL-13 did not affect the expression of GROalpha or RANTES. CONCLUSION(S) These results suggest that IL-13 and TNF-beta secreted in the cyclic endometrial tissue and in the decidua may differentially regulate the production of IL-6 family cytokines and chemokines by ESC. The controlled expression of these cytokines in the endometrium may contribute to the modulation of the immune reaction during the menstrual cycle and in early pregnancy by the regulation of leukocyte trafficking and functions.

Synergistic effect of interleukin-1alpha and ceramide analogue on production of prostaglandin E2 and F2alpha by endometrial stromal cells.

PROBLEM: Prostaglandins (PGs) are synthesized in the endometrium. Our objective was to evaluate interleukin (IL)‐1α‐induced production of PGE2 and PGF2α 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‐1α, IL‐1 receptor antagonist (ra), C2‐ceramide and C6‐ceramide. The concentrations of PGE2 and PGF2α 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 PGF2α was significantly increased by IL‐1α and suppressed by IL‐1 ra, in a dose‐dependent manner. PGF2α production was further increased by treatment with the combination of IL‐1α and C2‐ceramide as compared with IL‐1α treatment alone. There was no significant difference in PGE2 production between cells treated with IL‐1α and C2‐ceramide and those treated with IL‐1α alone. Both PGE2 and PGF2α production were significantly increased by treatment with IL‐1α and C6‐ceramide as compared with IL‐1α treatment alone. Treatment of ESC with IL‐1α stimulated PGHS‐2 mRNA. PGHS‐2 mRNA was decreased when IL‐1 ra was added to the IL‐1α‐stimulated cells.
 CONCLUSIONS: These results suggest that IL‐1α stimulates the production of PGE2 and PGF2α by a mechanism that involves the sphingomyelin‐ceramide system, and thus that ceramide may be important in increasing the production of PGE2 and PGF2α in the human endometrium.

The Effects of Platelet-Activating Factor on the Secretion of Interleukin-8 and Growth-Regulated Oncogene α in Human Immortalized Granulosa Cell Line (GC1a)

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) α in cultured human immortalized granulosa cell line (GC1a).

Homologous carcinosarcoma of the uterine corpus associated with tamoxifen therapy

Abstract A very rare case of homologous carcinosarcoma of the endometrium associated with long-term tamoxifen treatment for breast cancer is reported. A 70-year-old Japanese woman was admitted with atypical genital bleeding. Six years earlier, she had undergone total right mastectomy for breast cancer, and administration of tamoxifen had been started postoperatively. On admission, biopsy revealed a homologous carcinosarcoma of the endometrium with metastasis in the endocervix. The patient underwent a Wertheims hysterectomy that revealed stage IIIc disease with pelvic lymph node metastasis. She received six courses of adjuvant chemotherapy. Histological examination showed a homologous carcinosarcoma, composed of poorly differentiated adenocarcinoma and anaplastic sarcoma. No heterologous elements; for example, malignant cartilage or rhabdomyoblasts, were seen. Tamoxifen is a possible etiologic factor in the development of endometrial carcinosarcomas.