In vitro evaluation of exocytosis-associated SNARE molecules in human granulosa cells in polycystic ovary syndrome.

PURPOSE: Patients with polycystic ovarian morphology (PCOM) make up 20% cases for assisted reproductive technology (ART). Folliculogenesis is impaired in PCOS. Signaling molecules are involved in follicle development. Dysregulations of intrafollicular environment and signaling molecules are observed in PCOS. Granulosa cells (GCs) and oocytes secrete molecules into follicular fluid by exocytosis of SNAREs. The aim of this study is to evaluate vesicle transport and vesicle fusion proteins (SNAREs) in GCs from PCOS patients who have undergone IVF treatment. METHODS: Follicular fluids were collected from patients who undergo IVF/ICSI with the diagnosis of male factor (n = 10) and PCOS (n = 10) patients. GCs were separated and cultured. Each group of GCs was stimulated with FSH-hCG. The cells were examined under electron microscope. Immunofluorescent labeling was performed on cells for Stx6, SNAP25, StxBP1, FSHr, and KITL. Integrated density was analyzed from images of Stx6, SNAP25, StxBP1, FSHr, and KITL. RESULTS: Intercellular communication occurs by signal molecules; Stx6, SNAP25, and StxBP1 fusion proteins involved in exocytosis were decreased in the GCs of PCOS. There was no increase in in vitro stimulation with FSH-hCG either. In the electron microscope, it was observed that exocytosis of the vesicles was disrupted. CONCLUSIONS: Exocytosis and vesicular dynamics are among the basic physiological functions of human steroidogenic granulosa cells. Follicle development is necessary for production of competent oocytes and ovulation. Understanding the pathophysiology of PCOS at follicular level is important for disease management. According to our findings, deficits in vesicular dynamics of human granulosa cells in may be central to the treatment strategy for PCOS patients.

Evaluation of Dysfunctional HDL by Myeloperoxidase/Paraoxonase Ratio in Unexplained Infertility Patients Undergoing IVF/ICSI.

The relationship between oxidative stress and unexplained infertility (UEI) has not been studied in detail. This is the first study to evaluate dysfunctional high-density lipoprotein (HDL) by the myeloperoxidase (MPO) and paraoxonase (PON) ratio to investigate the role of oxidative stress in UEI. MATERIALS AND METHODS: Patients with UEI (study group, n = 40) and male factor infertility (control group, n = 36) were included in this prospective study. Demographics and laboratory assessments were analyzed. RESULTS: Total dosages of gonadotropin were higher in UEI when compared to the control group (p = 0.033). Number of Grade 1 embryos and the quality of blastocysts were lower in UEI than in the control group (p = 0.024, p = 0.020, respectively), whereas serum MPO/PON ratio was higher in UEI (p = 0.042). Stepwise linear regression analysis revealed that serum MPO/PON ratio levels could significantly predict the duration of infertility (p = 0.012). CONCLUSIONS: Serum MPO/PON ratio increased in patients with UEI, whereas the number of Grade 1 embryos and the quality of blastocysts decreased. Similar clinical pregnacy rates were found in both groups but the ET on day five is associated with higher clinical pregnancy rate in the male factor infertility.

A Lipotoxic Medium Decreases the Number of Lipid Droplets in ß Cells: One Possible Explanation of the ß-Cell Failure in Patients With Hyperlipidemia Receiving Tacrolimus.

OBJECTIVES: Dyslipidemia is a risk factor for post- transplant diabetes mellitus, especially in patients who are taking tacrolimus. Although lipotoxicity of dyslipidemia leads to ß-cell failure, the handling of lipids by ß cells is a mystery in molecular endocrinology. Likewise, lipid droplet homeostasis is appreciated as a key component of lipid metabolism in cells like hepatocytes, but its role in ß cells remains to be elucidated. MATERIALS AND METHODS: To evaluate the morphologic changes in ß cells with special focus on lipid droplets, we evaluated electron micrographs under metabolic stress conditions of glucotoxicity, lipotoxicity, and glucolipotoxicity in isolated rat insulinoma INS-1E ß cells. Cells were treated with palmitic acid (0.5 mM), glucose (33 mM), or both for 16 hours, after which morphologic changes were observed with an electron microscope. RESULTS: Many lipid droplets were observed in the cytoplasm of healthy ß cells in the control group (no treatment). Lipid droplets were also visible in the cytosol, and the cytoplasm was rich in organelles and insulin vesicles under high glucose stimulation. However, after treatment with palmitic acid, almost no lipid droplets were observed. Endocrine vesicles were also depleted, with severe morphologic disruption of other organelles. Under glucolipotoxic conditions, ß cells showed a decreased number of lipid droplets and insulin vesicles compared with controls. CONCLUSIONS: Lipid droplet dynamics seemed important in the homeostasis of ß-cell metabolism. In this preliminary study, healthy ß cells appeared rich in lipid droplets under normal conditions. However, lipotoxicity depleted and glucolipotoxicity decreased the number of lipid droplets in ß cells. Because dyslipidemia causing lipotoxicity is one of the most frequent metabolic problems in transplant patients and increases risk of posttransplant diabetes mellitus, understanding the mystery of lipid droplets in ß cells and the pathophysiology of diabetes in transplant patients is important, especially for those taking tacrolimus.

A new isolation and culture method for granulosa cells.

Granulosa cells are the cell population who have an increasing importance in the female genital system and reproduction. Thus, nowadays in vitro studies to address these cells are also gaining importance and attracts researcher's attention. The aim of our study is to develop a more feasible, low-cost granulosa cell isolation and culture method compared to methods defined so far. Granulosa cells were isolated from follicular fluids obtained from both healthy women donors (n = 19) and polycystic ovary syndrome (n = 15) applied to in vitro fertilization treatment process. Granulosa cells were isolated by using Lymphosep® separation fluid that was not used for this purpose before. The isolated cells were cultured in suitable culture dishes with a mixture of BIO-AMFTM-1 and DMEM/F12 in the first seeding and only complete DMEM/F12 in the following feeds. Complete medium contains only 5% fetal calf serum, 4% L-glutamine and 1% penicillin-streptomycin-amphotericin. The new methods we have developed in granulosa cell isolation and in vitro culture have been successful. Reduction in supplement types and amount; improved the proliferation rate of the granulosa cells in culture. Our new methods of isolation and cell culture for granulosa cells from healthy women, have been also successful in samples of polycystic ovarian patients. With these developed methods, granulosa cells, which belong to humans and have an important role in the ovary, could be isolated and subsequently be maintained to reproduce (proliferate) more easily and cheaper.