Hiroharu Kobayashi

Met Is the Most Frequently Amplified Gene in Endometriosis-Associated Ovarian Clear Cell Adenocarcinoma and Correlates with Worsened Prognosis

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.

Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway

OBJECTIVE To investigate the protective effect of sphingosine-1-phosphate (S1P) against H(2)O(2)-induced apoptosis in human granulosa cell cultures with freshly harvested granulosa cells. DESIGN Experimental study. SETTING Academic medical center for reproductive medicine. PATIENT(S) Cultures of primary granulosa cells isolated from women undergoing in vitro fertilization (IVF). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Cell apoptosis and Western blot analysis of signaling pathway proteins. RESULT(S) We found that S1P (1 and 10 mM) statistically significantly decreased granulosa cell apoptosis after H(2)O(2) treatment. The decreased cell apoptosis induced by S1P was abolished after treatment with VPC23019, an inhibitor of S1P1 and S1P3 receptors, W146, an inhibitor of S1P1 receptors, and CAY10444, an inhibitor of S1P3 receptors. A Western blot analysis revealed that the level of phospho-Akt increased and peaked at 10 minutes after 10 mM S1P exposure. CONCLUSION(S) Treatment with S1P can inhibit the apoptosis of granulosa cells in response to oxidative stress induced by H(2)O(2). The protective effect of S1P is mediated by activating the PI3K/Akt pathway, and the antiapoptotic effect of S1P is mainly mediated through the S1P1 and S1P3 receptor.

Chemical anchoring of lauryl methacrylate-based reversed phase monolith to 1/16″ o.d. polyetheretherketone tubing

In this paper, we describe a method for the preparation of easy-to-use reversed-phase monolithic microbore columns. Polyetheretherketone (PEEK) tubing with an outer diameter of 1/16″ and an inner diameter of 1.0 mm was used as a column housing (empty column), and in it lauryl methacrylate (LMA) was copolymerized with ethylene dimethacrylate (EDMA). In order to chemically anchor the polymer monolith to the tube wall, the inner wall surface was pretreated by the following two-step procedure. (1) 50% sulfuric acid was filled into the PEEK tubing and left to stand for 6 h to generate sulfonate groups on the surface. (2) After washing with Milli-Q water, the sulfonated PEEK surface was brought into contact with 1 M glycidyl methacrylate in dichloromethane (or acetone) at 40°C for 4 h to introduce methacryloyl groups via the reaction of sulfonate groups and epoxy groups. Mechanical strength and column efficiency of the resulting monoliths were evaluated through the separation of a series of alkylbenzenes in acetonitrile-water (50:50, v/v) eluent over the flow rate range of 50-750 μL/min (corresponding to 1.7-25.5 mm/s). The poly(LMA-co-EDMA) monolith provided acceptable column efficiency of 2000 theoretical plates/10 cm (HETP value of 50 μm) for amylbenzene (separation factor k=40) and low flow resistance of 0.5 MPa/10 cm at a normal flow rate of 50 μL/min. The methacryloylated PEEK tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 15 MPa, allowing rapid separation at a 15-20 fold higher flow rate than normal.

A proteomic analysis of human follicular fluid: comparison between fertilized oocytes and non-fertilized oocytes in the same patient

PurposeHuman follicular fluid constitutes the microenvironment of follicles and includes various biological active proteins that can affect follicle growth and oocyte fertilization. Conducting proteomic evaluations of human follicular fluid may be helpful for identifying potential biomarkers possibly possessing a predictive value for oocyte quality and the success of in vitro fertilization.MethodWe performed proteomic profiling of human follicular fluids containing oocytes that were fertilized and resulted in pregnancy and follicular fluids containing oocytes that were not fertilized in the same patients undergoing intracytoplasmic sperm injection using the LTQ Orbitrap coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analyses.ResultsWe identified a total of 503 proteins in human follicular fluids containing fertilized and non-fertilized oocytes obtained from 12 patients. We also found that 53 proteins exhibited significantly different spectral counts between the two groups, including heparan sulfate proteoglycan perlecan, which showed significant upregulation in the follicular fluids containing fertilized oocytes in comparison with that observed in the follicular fluids containing non-fertilized oocytes.ConclusionOur results suggest a possibility that proteins identified by LC/MS/MS in follicular fluid might not only be involved in folliculogenesis, but also function as biomarkers possessing predictive potential for oocyte maturation and the success of IVF when their expression levels are significantly different between fertilized and non-fertilized oocytes, although no distinctive biomarkers were identified in the current study.

Establishment of a Human Nonluteinized Granulosa Cell Line that Transitions from the Gonadotropin-Independent to the Gonadotropin-Dependent Status

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis

OBJECTIVE To understand the role of ectopic endometriotic stromal cells in ovarian endometriosis (OEM) and the associated risks for infertility and carcinogenesis. DESIGN Analyses of secreted proteins and gene expression using immortalized eutopic/ectopic endometrial(-otic) stromal cells from OEM. SETTING University. PATIENT(S) Women with and without OEM. INTERVENTION(S) Samples of endometrial(-otic) tissue from women with or without OEM. MAIN OUTCOME MEASURE(S) Immunohistochemical analysis of oxidative stress in OEM, gene expression profiles, and the identification of secreted proteins by mass spectrometry in immortalized endometrial(-otic) stromal cells. RESULT(S) 4-Hydroxy-2-nonenal-modified proteins and carboxymethyllysine were abundant in the stroma, rather than epithelia, of OEM patients, indicating the presence of oxidative stress. Immortalized ectopic endometriotic stromal cells exhibited high IRP1/IRP2/HIF-1β expression and contained lower amounts of iron and copper than their eutopic counterparts. Expression profiles, in combination with protein identification, revealed that complement component 3 (C3) and pentraxin-3 (PTX3) are the major proteins secreted from immortalized ectopic endometriotic stromal cells. Complement-3/PTX3 promoted the secretion of various cytokines by THP1 macrophage cells and thus supported M1 differentiation. CONCLUSION(S) Immortalized ectopic endometriotic stromal cells in OEM predominantly secrete C3 and PTX3 and exhibit a differential regulation of iron metabolism.

Three-dimensional CT Angiography Is Useful for Diagnosis of Postabortion Uterine Hemorrhage: 3 Case Reports and Review of the Literature

Uterine hemorrhage is a major complication associated with abortion. There are various causes of postabortion uterine hemorrhage. The objective of this article is to estimate the efficacy of three-dimensional computed tomography (3D-CT) angiography in the diagnosis of this condition. We present 3 case reports of women with massive genital bleeding after abortion. 3D-CT angiography clearly demonstrated the 3-D features of the feeding artery, the draining vein, and the surrounding normal structures. The diagnosis in patient 1 was a uterine arteriovenous malformation, in patient 2 was a placental polyp mimicking a uterine arteriovenous malformation, and in patient 3 was a placental polyp. Patients were all successfully treated with uterine artery embolization or transcervical resection of the placental polyp. We conclude that 3D-CT angiography is useful for making a differential diagnosis and for preoperative planning in patients with postabortion uterine hemorrhage.

Assessment of the predictive value of follicular fluid insulin, leptin and adiponectin in assisted reproductive cycles.

Purpose. To assess the correlation of intrafollicular insulin, leptin and adiponectin levels with assisted reproductive technologies (ART) outcome. Methods. This was a retrospective study of 46 patients undergoing in vitro fertilisation/intracytoplasmic sperm injection. Follicular fluid (FF) samples collected at oocyte retrieval were assayed for insulin, leptin and adiponectin levels using enzyme-linked immunosorbent assay, and correlations with ART outcome were analysed. Results. There was no significant correlation between intrafollicular insulin, leptin and adiponectin levels. There was a significant difference in the concentration of insulin (P = 0.007), but not leptin or adiponectin, between pregnant (n = 20) and non-pregnant (n = 26) cycles. Only two pregnancies was observed in the 12 cycles in which the concentration of insulin was greater than 7 mU/l in FF, while 18 pregnancies was observed in the 34 cycles in which the concentration of insulin was less than 7 mU/l (P = 0.043). The significantly high concentration of insulin in FF was observed in non-pregnant cycles of patients with polycystic ovary syndrome (PCOS). Conclusions. Our results suggest the possible involvement of intrafollicular insulin in folliculogenesis. Insulin resistance-related substances may affect the reproductive process in patients with PCOS.

Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues

PurposeInflammatory mediators, including chemokines, may play crucial roles in the development of endometriosis. Therefore, we investigated the expression and localization of CXCL16 and its receptor, CXCR6, in ovarian endometriotic tissues. We also examined whether CXCL16 induces IL-8 production in endometriotic stromal cells.MethodsWe performed immunohistochemical and Western blotting analyses of in vivo and in vitro samples. IL-8 production was assayed using an ELISA.ResultsBoth CXCL16 and CXCR6 were expressed by endometriotic epithelial cells and stromal cells, but not normal ovarian stroma. A Western blotting analysis using primary cultured endometriotic stromal cells showed a constant expression of CXCL16 and CXCR6 in the proliferative phase, secretory phase and during gonadotropin-releasing hormone agonist therapy. CXCL16 induced IL-8 production in several endometriotic stromal cells in vitro.ConclusionsCXCL16 and CXCR6 might be involved in the pathophysiology of endometriosis through regulation of the inflammatory response.

Successful fertility management of a patient with factor V deficiency: planned transfusion of fresh frozen plasma under infertility treatment

OBJECTIVE To report the case of a patient with factor V deficiency who achieved pregnancy with the planned transfusion of fresh frozen plasma (FFP) while monitoring follicle development and ovulation induction using gonadotropin. DESIGN Case report. SETTING University hospital. PATIENT(S) A 28-year-old nulliparous female. INTERVENTION(S) Medical management including infertility treatment. MAIN OUTCOME MEASURE(S) Clinical follow-up. RESULT(S) A patient with factor V deficiency experienced repeated ovulation-related hemoperitoneum following the withdrawal of oral contraceptive pills. The monitoring of follicle development and ovulation induction using gonadotropin followed by FFP transfusion was useful to avoid hemoperitoneum. Pregnancy was achieved within a relatively short period using intrauterine insemination. CONCLUSION(S) Planned prophylactic FFP administration and intervention with infertility treatment might be useful to minimize the risk of ovulation-related hemoperitoneum in patients with factor V deficiency.