Analysis of the risk factors and treatment for repeated implantation failure: OPtimization of Thyroid function, IMmunity, and Uterine Milieu (OPTIMUM) treatment strategy.

PROBLEM: What are the pregnancy outcomes after the OPtimization of Thyroid function, Immunity, and Uterine Milieu (OPTIMUM) treatment strategy in patients with repeated implantation failure (RIF)? METHOD OF STUDY: Infertile women with a history of RIF after more than three embryo transfer (ET) cycles underwent implantation testing, including a hysteroscopy, endometrial biopsy for CD138 immunostaining and bacterial culture, and serum 25-hydroxyvitamin D3 , interferon-γ-producing helper T (Th1) cell, IL-4-producing helper T (Th2) cell, thyroid-stimulating hormone, thyroid peroxidase antibody, and thrombophilia screening between April 2017 and August 2018. We treated chronic endometritis with antibiotics, aberrant high Th1/Th2 cell ratios with vitamin D and/or tacrolimus intake, overt/subclinical hypothyroidism with levothyroxine, and thrombophilia with low-dose aspirin. Of the 116 RIF women, 88 women with 133 ET cycles were recruited from a questionnaire-based survey regarding pregnancy outcomes. Fifty-nine consecutive RIF patients without the OPTIMUM treatment strategy were also recruited as a control. RESULTS: The 116 women with RIF after the OPTIMUM treatment strategy were 38.3 ± 3.8 years old and had an implantation failure history over 5 (3-19) ET cycles. Implantation testing identified impaired intrauterine circumstances in 75 women (64.7%), an aberrant elevated Th1/Th2 cell ratio in 56 women (48.3%), and thyroid abnormalities in 33 women (28.4%). Cumulative ongoing pregnancy rates including spontaneous pregnancy in the patients aged < 40 and ≥ 40 years were 72.7% and 45.5% within two ET cycles, respectively. The pregnancy outcomes in the OPTIMUM group were significantly higher than those in the control. CONCLUSIONS: The OPTIMUM treatment strategy improved pregnancy outcomes in patients with RIF.

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.

The effects of platelet-activating factor on the secretion of interleukin-8 and growth-regulated oncogene alpha in human immortalized granulosa cell line (GC1a).

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.

Granulosa cell tumor associated with secondary amenorrhea and serum luteinizing hormone elevation.

Adult granulosa cell tumors (GCTs) are the most common type of ovarian sex cord tumors. Menstrual irregularity, menorrhagia, or even secondary amenorrhea is frequently observed in premenopausal women bearing GCTs with hormonal activity. We report herein a case of GCT in a patient presenting with secondary amenorrhea and serum luteinizing hormone elevation. A 28-year-old primigravid Japanese woman was admitted complaining of secondary amenorrhea of 2 years' duration. Pelvic examination, transvaginal ultrasonography, and magnetic resonance imaging demonstrated a left ovarian tumor 4 cm in diameter. Serum hormone assays revealed a follicle-stimulating hormone level of 4.8 mIU/ml, luteinizing hormone (LH) of 35.8 mIU/ml, estradiol of 24 pg/ml, progesterone of 1.6 ng/ml, and testosterone of 40 ng/dl. A left salpingo-oophorectomy was performed. The tumor was diagnosed as an adult-type GCT stage IIb (FIGO [International Federation of Obstetricians and Gynecologists], 1988). Spontaneous menstruation occurred soon after the surgery. Serum levels of LH also decreased to normal levels and showed cyclic changes during the menstrual cycle. Subsequently, the patient conceived and delivered a healthy female baby. The tumor recurred in the pelvis 50 months after the initial conservative surgery, with elevated serum LH levels of 36.0 mIU/ml and amenorrhea. The patient was treated by hysterectomy, right salpingo-oophorectomy, omentectomy, paraaortic and pelvic lymphadenectomy, and low anterior resection of the recto-sigmoid colon. Her hormone levels progressed to the postmenopausal state after this surgery. Although LH elevation in patients with GCT is rare and its mechanism is unknown, monitoring of serum LH may provide an additional tumor marker after conservative surgery in such patients.

K252a inhibits proliferation of ovarian cancer cells by upregulating p21WAF1.

The furanosylated indolocarbazole, K252a, belongs to a family of microbial alkaloids that also includes staurosporine, which is known to inhibit proliferation, stimulate apoptosis and induce cell cycle arrest of cancer cells. To elucidate the involvement of K252a in ovarian cancer, we investigated the effects of K252a on the ovarian cancer cell line, SK-OV-3. SK-OV-3 cells were treated with K252a, and its effect on cell growth, cell cycle, and related measurements was assessed. MTT assays showed that the ovarian cancer cell line SK-OV-3 cells were sensitive to the growth inhibitory effect of K252a. Cell cycle analysis indicated that their exposure to K252a decreased the proportion of cells in the S-phase and increased the proportion of cells in the G0/G1 phase of the cell cycle. This occurred in concert with altered expression of p21WAF1 protein related to the G0/G1 phase of the cell cycle. These results raise the possibility that K252a may prove particularly effective in treatment of ovarian cancer.

Expression and regulation of thrombospondin-1 by human endometrial stromal cells.

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.

Endometriotic cells are resistant to interferon-gamma-induced cell growth inhibition and apoptosis: a possible mechanism involved in the pathogenesis of endometriosis.

In order to evaluate the involvement of cell proliferation and apoptosis in the pathogenesis of endometriosis, we investigated the effects of interferon-gamma (IFN-gamma) on cell growth inhibition and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSC), eutopic endometrial stromal cells with endometriosis (ESCwE) and normal endometrial stromal cells (NESC) by modified methylthiazoletetrazolium assay, 5-bromo-2'-deoxyuridine incorporation assay and internucleosomal DNA fragmentation assay. The expression of apoptosis-related molecules and IFN-gamma receptor 1 was also examined in ECSC, ESCwE and NESC using western blot analysis. IFN-gamma significantly inhibited cell proliferation and DNA synthesis of ESCwE and NESC, and induced apoptosis of these cells. In contrast, IFN-gamma did not show apparent effects on the viable cell number, DNA synthesis, or apoptosis of ECSC. An up-regulated expression of Bcl-2 and Bcl-X(L) proteins was observed in ECSC in comparison with ESCwE and NESC, whereas the levels of Bax, Bad, Fas and Fas ligand proteins in ECSC were similar to those in ESCwE and NESC. IFN-gamma receptor 1 expression was detected in ECSC, ESCwE and NESC. Enhanced expression of anti-apoptotic molecules in the ectopic endometrial cells may contribute to the development of endometriosis by conferring resistance to cytokine-induced apoptosis and increasing the chance that these cells will survive and implant outside the uterus. Further investigations on the regulation of cell proliferation in both the endometriotic and the normal endometrium may be important for the elucidation of the pathogenesis of endometriosis.

Cultured human endometrial epithelial cells produce thymus and activation-regulated chemokine with stimulation of interleukin-4 and interleukin-13.

OBJECTIVE: To evaluate the effects of T-helper (Th)1 and Th2 cytokines on the production of thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) by cultured endometrial epithelial cells (EEC) and endometrial stromal cells (ESC). DESIGN: The effects of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon-gamma (IFN-gamma), and tumor necrosis factor-beta (TNF-beta) on the production of TARC and MDC were investigated. SETTING: Research laboratory at a medical school. PATIENT(S): Fifteen endometrial specimens in the mid-late secretory phase were used. INTERVENTION(S): The EEC and ESC were incubated for 24 hours with recombinant human IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-gamma, and TNF-beta. MAIN OUTCOME MEASURE(S): The concentrations of TARC and MDC in the culture media were measured using ELISA. RESULT(S): Small amounts of TARC were detected in the culture medium of nonstimulated EEC. The increase in levels of TARC in the culture media of EEC paralleled the addition of increasing amounts of IL-4 and IL-13. Other cytokines, however, did not affect the production of TARC by EEC. Production of TARC by ESC was not detected under either nonstimulated or cytokine-stimulated conditions. Production of MDC was not detected in the culture media of EEC and ESC. CONCLUSION(S): These results suggest that IL-4 and IL-13 secreted from the embryo during the implantation period may selectively up-regulate the production of TARC by EEC. The controlled production of TARC in the endometrium may contribute to the modulation of the immune reaction by the regulation of Th2 lymphocyte trafficking and functions.

Synergistic effect of interleukin (IL)-1alpha and ceramide analogue on the production of IL-6, IL-8, and macrophage colony-stimulating factor by endometrial stromal cells.

OBJECTIVE: To measure the level of interleukin 6 (IL-6), IL-8, and macrophage colony-stimulating factor (M-CSF) induced by IL-1alpha in endometrial stromal cells (ESC) following treatment with ceramide analogues. DESIGN: The effects of IL-1alpha, 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-1alpha, 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-1alpha, 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-1alpha plus C2-ceramide as compared with IL-1alpha alone. Production of both IL-8 and M-CSF was statistically significantly increased by IL-1alpha plus C6-ceramide as compared with IL-1alpha alone; however, IL-6 production was not increased. CONCLUSION(S): The results suggest that IL-1alpha 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.

Production of macrophage inflammatory protein-3alpha in human follicular fluid and cultured granulosa cells.

OBJECTIVE: To investigate the role of macrophage inflammatory protein (MIP)-3alpha in human ovulation. DESIGN: Study of the levels of MIP-3alpha in serum and follicular fluid. The effects of interleukin (IL)-1alpha, IL-1 receptor antagonist (RA), and tumor necrosis factor (TNF)-alpha on the secretion of MIP-3alpha 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 (i.v.f.-ET). INTERVENTION(S): Follicular fluid was obtained from study participants, and granulosa-lutein cells and GC1a were incubated with IL-1alpha, IL-1RA, or TNF-alpha for 4 to 32 hours. MAIN OUTCOME MEASURE(S): The concentration of MIP-3alpha in human follicular fluid was measured and correlated with oocyte maturation. We also cultured granulosa cells and examined the regulation of MIP-3alpha production. The concentrations of MIP-3alpha in the serum, follicular fluid, and culture medium were measured using enzyme-linked immunoabsorbent assay. RESULT(S): Concentrations of MIP-3alpha were significantly higher in the follicular fluid, but it was not detected in the serum. Concentrations of MIP-3alpha were statistically significantly higher in the follicular fluid containing mature oocytes than in follicular fluid containing immature oocytes. The production of MIP-3alpha was markedly increased over the basal level after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. The stimulatory effect of IL-1alpha was inhibited by IL-1RA. CONCLUSION(S): Our data suggest that MIP-3alpha was present in follicular fluid and correlated with oocyte maturation, and was regulated by IL-1alpha and TNF-alpha. Thus, MIP-3alpha may play an important role in the human preovulatory process.

Down-regulation of interleukin-1 receptor type 1 expression causes the dysregulated expression of CXC chemokines in endometriotic stromal cells: a possible mechanism for the altered immunological functions in endometriosis.

To evaluate the involvement of chemokines in the pathogenesis of endometriosis, we investigated the expression of CXC chemokines in cultured ovarian endometriotic cyst stromal cells (ECSC), endometrial stromal cells with endometriosis (ESCwE), and normal endometrial stromal cells (NESC). Using ELISA, TNF-alpha significantly enhanced the production of IL-8, growth-related oncogene alpha, and epithelial neutrophil-activating peptide-78 in all cases of ECSC (n = 10), ESCwE (n = 6), and, NESC (n = 10). IL-1beta did not affect the production of these chemokines in eight of 10 cases of ECSC. In contrast, IL-1beta significantly enhanced the expression of these chemokines in all cases of ESCwE (n = 6) and NESC (n = 10). Western blot analysis revealed down-regulation of expression of IL-1 receptor type 1 (IL-1-R1) in all cases of ECSC with low response to IL-1beta (n = 8). In contrast, significant IL-1-R1 expression was detected in all cases of NESC. Although IL-1-R1 expression was detected in all cases of ESCwE (n = 6), its expression in ESCwE tended to decrease compared with that in NESC. Moreover, phosphorylation of inhibitor kappaB-alpha was detected in all cases of ESCwE and NESC after stimulation with IL-1beta, but not in ECSC with low response to IL-1beta (n = 8). In contrast, significant IL-1-R2 expression was detected in all cases of ECSC, ESCwE, and NESC. The present findings suggest that the dysregulation of IL-1/IL-1-R system relates to immunological dysfunction in endometriosis. The alteration of the CXC chemokines expression may be important for elucidation of the pathogenesis of endometriosis.

The effect of insulin-like growth factor-I on production of vascular endothelial growth factor by amnion-derived (WISH) cells.

OBJECTIVE: Our objective was to clarify the modulation of vascular endothelial growth factor (VEGF) by amniotic cells. DESIGN: Amnion-derived (WISH) cells were cultured, and the effect of insulin-like growth factor (IGF), mitogen-activated protein (MAP) kinase kinase and/or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126), and phosphatidylinositol (PI) 3-kinase inhibitors (wortmannin) on the production of VEGF was examined. VEGF was assayed using ELISA. The activations of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. RESULTS: In the time course of VEGF production following IGF-I treatment, VEGF production showed significant increases only at 16 and 32 h (p < 0.01). Also, IGF-I increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activities were recognized by treatment with IGF-I and suppressed by U0126 and wortmannin, respectively. When WISH cells were pretreated for 2 h with U0126 and wortmannin and then treated with IGF-I for 16 h, the production of VEGF was significantly decreased in a dose-dependent manner (p < 0.01, p < 0.01, respectively). CONCLUSIONS: WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase interaction with IGF-I receptor, resulting in MAP kinase and PI 3-kinase activation. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and IGF-I may play an important role in the modulation of VEGF production in the placenta.

Differential effects of interferon-alpha and -beta on the secretion of vascular endothelial growth factor by eutopic and ectopic endometrial stromal cells.

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 simultaneously inhibits endostatin production and stimulates vascular endothelial growth factor production by cultured human endometrial stromal cells.

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.

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. 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.

Possible modulators of IL-8 and GRO-alpha production by granulosa cells.

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.

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.

Expression of growth-regulated oncogene beta in an endometrial epithelial cell line, HHUA, and cultured human endometrial cells.

It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. Growth-regulated oncogene (GRO)beta, which belongs to the CXC chemokine family, is a potent chemoattractant for neutrophils. To evaluate the regulation of GRO beta expression in the endometrium, the production of GRO beta by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was examined by using an enzyme-linked immunosorbent assay. Unstimulated HHUA and ESC constitutively secreted GRO beta. Interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma significantly stimulated the expression of GRO beta by HHUA and ESC. Lipopolysaccharide also stimulated the expression of GRO beta by ESC, but not by HHUA. It is suggested that, in the human endometrium, the regulation of GRO beta expression is distinct from that of other CXC chemokines expressed in the endometrium, such as GRO alpha and interleukin-8. The modulation of the GRO beta concentration in the endometrium by inflammatory mediators may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of neutrophils into the endometrium.

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.

Interleukin-13 and tumor necrosis factor-beta 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.