Osamu Yoshino

Interleukin-1β stimulates the secretion of thymic stromal lymphopoietin (TSLP) from endometrioma stromal cells: possible involvement of TSLP in endometriosis

STUDY QUESTIONnIs thymic stromal lymphopoietin (TSLP) involved in the pathophysiology of endometriosis?nnnSUMMARY ANSWERnTSLP is up-regulated by interleukin (IL)-1β and may be involved in the development of endometriosis.nnnWHAT IS KNOWN ALREADYnEndometriosis is a chronic inflammatory disease in which the Th2 immune response is activated and has been suggested to promote the disease. TSLP is a master cytokine that drive Th2 immune response.nnnSTUDY DESIGN, SIZE, DURATIONnA laboratory study.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnPrimary cultures of endometrioma stromal cells (ESCs) were treated with IL-1β, a typical inflammatory cytokine associated with endometriosis. Gene expression of TSLP in ESCs and secretion of TSLP protein from ESCs were studied using quantitative PCR and a specific ELISA. Interferon γ (IFNγ), a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, were added to the culture to evaluate their effect on the IL-1β-induced secretion of TSLP. Inhibitors of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) were added to the culture to examine intracellular signals involved in IL-1β-induced TSLP secretion. The expression of TSLP in endometrioma tissue was examined by immunohistochemistry. The concentration of TSLP in the serum and peritoneal fluid (PF) of women with or without endometriosis was measured with a specific ELISA.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIL-1β stimulated the expression of TSLP mRNA and secretion of TSLP protein from ESCs. IL-4 enhanced the IL-1β-induced TSLP secretion from ESCs, while IFNγ reduced it. Inhibitors of p42/44 MAPK, p38 MAPK and SAPK/JNK suppressed the IL-1β-induced secretion of TSLP from ESCs. Positive immunostaining of TSLP was observed in the stroma of endometrioma tissue. TSLP concentrations in the serum and PF were both higher in women with endometriosis compared with those without endometriosis.nnnLIMITATIONS, REASONS FOR CAUTIONnThe present study was only in vitro. The samples used for culture were endometrioma tissues, not including other types of endometriosis. Therefore, the present findings should be interpreted with caution.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis study provided new insights in the Th2 immune response-related mechanism in endometriosis.nnnSTUDY FUNDINGnThis study is partly supported by grants from the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology. The authors have no conflicts of interest to declare.

Anti-Mullerian hormone (AMH) is induced by bone morphogenetic protein (BMP) cytokines in human granulosa cells.

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

Bone morphogenetic protein 7 increased vascular endothelial growth factor (VEGF)-a expression in human granulosa cells and VEGF receptor expression in endothelial cells.

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

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinase-1 in Human Uterine Smooth Muscle Cells

The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition.

Interleukin-4 and Prostaglandin E2 Synergistically Up-Regulate 3β-Hydroxysteroid Dehydrogenase Type 2 in Endometrioma Stromal Cells

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

Interleukin-17A is present in neutrophils in endometrioma and stimulates the secretion of growth-regulated oncogene-α (Gro-α) from endometrioma stromal cells.

OBJECTIVEnTo investigate a new role of interleukin (IL)-17A in endometriosis.nnnDESIGNnLaboratory study.nnnSETTINGnUniversity hospital.nnnPATIENT(S)nPatients with ovarian endometrioma undergoing laparoscopy or laparotomy.nnnINTERVENTION(S)nPrimary culture of endometrioma stromal cells (EoSCs) was stimulated with IL-17A. Sections of endometrioma tissue were immunostained with antibodies for IL-17A, growth-regulated oncogene-α (Gro-α), and elastase, a marker of neutrophils. They were also examined with immunofluorescent double staining for IL-17A and myeloperoxidase, another marker of neutrophils.nnnMAIN OUTCOME MEASURE(S)nConcentration of Gro-α was measured using a specific ELISA. Neutrophil chemotaxis was measured with Boyden chamber method. Immunostained sections were examined under microscope.nnnRESULT(S)nInterleukin-17A increased the secretion of Gro-α from EoSCs dose-dependently. The conditioned medium of EoSCs stimulated with IL-17A attracted more neutrophils than that of EoSCs stimulated with vehicle, and the increase was inhibited by the addition of Gro-α-neutralizing antibody. On immunostaining, IL-17A and Gro-α were detected in similar areas of the stroma beneath the epithelium, where Gro-α was detected in cells with a stromal cell appearance whereas IL-17A was detected in neutrophils as determined by detection of elastase. Fluorescent immunostaining corroborated that myeloperoxidase-positive neutrophils were also positive for IL-17A.nnnCONCLUSION(S)nIt is suggested that IL-17A produced by neutrophils stimulates Gro-α secretion from EoSCs, thereby recruiting more neutrophils and inducing perpetuating inflammation in endometriosis.

Thrombin enhances soluble Fms-like tyrosine kinase 1 expression in trophoblasts; possible involvement in the pathogenesis of preeclampsia

OBJECTIVEnTo investigate the possible impact of thrombin on soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in trophoblasts.nnnDESIGNnExperimental.nnnSETTINGnUniversity hospital laboratory.nnnINTERVENTIONS(S)nA trophoblast cell line (HRT-8/SVneo) was treated with thrombin, protease-activated receptor 1 (PAR-1)-specific agonist SFLLERN, and thrombin antagonist PPACK.nnnMAIN OUTCOME MEASURE(S)nmRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts, with the use of real-time polymerase chain reaction; and the secretion of sFlt-1, VEGF, and PlGF protein from trophoblasts, with the use of ELISA.nnnRESULT(S)nAdministration of thrombin (10 U/mL) and PAR-1-specific agonist SFLLRN (300 μmol/L) increased sFlt-1 mRNA expression (4.24 ± 0.74- and 4.21 ± 0.79-fold, respectively) and protein secretion (5.08 ± 0.42- and 1.89 ± 0.16-fold, respectively) in HRT-8/SVneo. The induction of sFlt-1 protein secretion by thrombin was dose dependent. The effect of thrombin was completely reduced by thrombin inhibitor PPACK. Thrombin increased mRNA expression of VEGF but did not change VEGF secretion and PlGF mRNA expression and secretion.nnnCONCLUSION(S)nDuring placental development, thrombin, generated in the local hemorrhage of the uteroplacenta increases trophoblast expression of sFlt-1. Consequently, thrombin may contribute to the pathogenesis of preeclampsia.

The significance of serum anti-Müllerian hormone (AMH) levels in patients over age 40 in first IVF treatment

PurposeAlthough studies of serum anti-Müllerian hormone (AMH) in predicting ovarian reserve are numerous, many studies utilized patients under age 40. However, the assessment of ovarian reserve is especially critical in older infertile women. This study evaluates the significance of AMH level in patients over age 40 at the time of their first in vitro fertilization (IVF) treatment.MethodsForty-nine women over age 40 were studied. Although serum samples were taken prior to their IVF treatments, the data of serum AMH of patients were not taken into consideration to determine the therapy strategy, including follicle induction in which clomiphene citrate and human menopausal gonadotropin were used.Result(s)Twelve out of 49 patients achieved a clinical pregnancy (24.4xa0%). There was a positive correlation between serum AMH levels and the number of oocytes retrieved (Pu2009<u20090.0001). The ROC curve analysis for prediction of poor ovarian response, ≤3 retrieved oocytes, showed that the optimum cut-off level was <u20091.0xa0ng/mL for AMH. The lower AMH group (AMH <u20091.0xa0ng/ml) showed less chance of undergoing embryo transfer than the higher AMH group (AMH ≥1.0xa0ng/ml). There was no difference in pregnancy rate between the two groups. Five out of 12 pregnant women exhibited AMH levels of less than 0.4xa0ng/ml.Conclusion(s)Assessment of serum AMH concentration in older patients is useful for the prediction of oocytes numbers which may be obtained in IVF. A cut-off level of 1.0xa0ng/ml AMH can be used to predict poor ovarian response. This cut-off level of AMH of 1.0xa0ng/ml might be useful to predict whether patients could have an embryo transfer, but had no power to predict achieving pregnancy. On the other hand, our data also showed that patients over age 40 with extreme low levels of AMH still had a chance of pregnancy.

Cyclic stretch augments production of neutrophil chemokines and matrix metalloproteinases-1 (MMP-1) from human decidual cells, and the production was reduced by progesterone.

The purpose of this study was to evaluate the impact of mechanical stretch caused by uterine contraction and progesterone (P4) on decidual cells (DC), neutrophil chemokines, and MMP‐1 expression.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells

OBJECTIVEnTo examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary.nnnDESIGNnMolecular studies.nnnSETTINGnResearch laboratory.nnnPATIENT(S)nEight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for inxa0vitro fertilization.nnnINTERVENTION(S)nLocalizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction.nnnMAIN OUTCOME MEASURE(S)nThe localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR.nnnRESULT(S)nIn the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An inxa0vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC.nnnCONCLUSION(S)nGDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines.