JNK Inhibition Modulates the Cytoskeleton, Hypoxia, and Neurogenesis on the Protein Level in Glioblastoma Cells and Astrocytes: An Immunofluorescence Study.

AIM: To evaluate the effects of c-Jun N-terminal kinase (JNK) inhibition and signal blocking on hypoxia (hypoxia-inducible factor 1-alpha (HIF-1α)), differentiation and neurogenesis (bone morphogenetic protein (BMP4)), and the cytoskeleton (F-actin) in glioblastoma multiforme cells (GBMCs). MATERIAL AND METHODS: We evaluated the differences between GBMCs and astrocytes in terms of the abovementioned parameters and assessed them with the aim of studying human GBMCs (U-87 MG) and astrocytes (SVG p12). The cells were exposed to different doses of the JNK inhibitor, SP600125, for 24, 48, and 72 hours. HIF-1α, BMP4, and F-actin expressions were evaluated using immunofluorescence image analysis. RESULTS: The half-maximal inhibitory concentration value for SP600125 was determined to be 10 µM at 24 hours of exposure. After SP600125 administration, elevated levels of HIF-1α and BMP4 were detected in GBMCs and astrocytes. F-actin level only increased in GBMCs after SP600125 administration. CONCLUSION: JNKs are important for cell proliferation, differentiation, survival, and death; thus, research on JNKs has become important for the treatment of many human diseases, especially brain tumors, Parkinson's disease, and Alzheimer's disease. The results of this study involving immunofluorescence techniques should be investigated and supported by studies that involve comprehensive molecular techniques.

GnRH agonist administration as luteal support on the transfer day of single blastocyst in dual-triggered cycles.

OBJECTIVES: Luteal phase support with gonadotropin-releasing hormone agonist (GnRH-a) has been considered in terms of its potential beneficial effects on in vitro fertilisation (IVF) cycles. In our study, we assessed the effectiveness of single-dose GnRH-a administration in dual-triggered cycles on pregnancy outcomes. MATERIAL AND METHODS: Eighty women who underwent intra cytoplasmic sperm injection (ICSI) cycle and had fresh blastocyst transfer were divided into two groups in terms of luteal phase support. The study group (Group A) consisted of patients (n = 40) who received a single-dose GnRH-a injection (0.1 mg of triptorelin acetate) subcutaneously 6 days after oocyte retrieval in addition to 600 mg daily of micronised progesterone, and the control group (Group B) comprised of patients (n = 40) taking 600 mg micronised progesterone daily from the first day after oocyte retrieval. GnRH-a and human chorionic gonadotropin (hCG; dual trigger) were administered to all patients. Comparison of the clinical pregnancy and live birth rates was our main goal. RESULTS: There was no significant difference between the two groups in terms of ß-hCG positivity rates, clinical pregnancy rates and live birth rates (p value for beta-hCG = 0.25, clinical pregnancy = 0.80, live birth = 0.45). CONCLUSIONS: Our study demonstrated that in dual triggered cycles administration of a single dose of GnRH-a on the transfer day of a single blastocyst in addition to routine luteal phase support with progesterone does not statistically increase implantation, clinical pregnancy or live birth rates.

Does Follicle-Stimulating Hormone Receptor Polymorphism Status Affect In vitro Fertilization-Intracytoplasmic Sperm Injection Results and Live Birth Rate? A Retrospective Study.

Background: Follicle-stimulating hormone (FSH) plays a key role in fertility and shows its effect through the FSH receptor (FSHR), which is localized in cells. Aims: The aim of this study was to examine pregnancy outcomes and responses to controlled ovarian stimulation according to FSHR polymorphism types. Study Setting and Design: The study was retrospective, and included patients who applied to the University of Health Sciences Tepecik Training and Research Hospital in vitro fertilization (IVF) Unit during 2018 and 2019. Materials and Methods: Patients who underwent IVF-intracytoplasmic sperm injection and at the same time studied FSHR gene polymorphism in the genetic unit of our hospital were included in the study. Statistical Analysis: The Kruskal-Wallis test was used for multiple comparisons of continuous variables. The Chi-square test was used for categorical variables between groups. Results: A total of 143 patients who met our criteria were included in the study. 14% (n = 20) of the patients are also homozygous natural (Asn/Asn) type; 44.7% (n = 64) of the heterozygous mutant (Asn/Ser) type; 41.3% (n = 59) of them were homozygous mutant (Ser/Ser) type. There was no statistically significant difference between the groups in terms of pregnancy rate per started cycle, ongoing pregnancy per started cycle, ongoing pregnancy per embryo transfer and live birth per embryo transfer. A significant difference was observed between peak E2 and peak progesterone levels between Asn/Ser and Ser/Ser groups, and the levels of these hormones were lower in the Ser/Ser group (P = 0.018 and P = 0.016, respectively). Ovarian responses were classified as poor (≤3 oocytes), normal (4-20 oocytes) and hyperresponse (≥20 oocytes) according to the oocyte count. Accordingly, the number of patients with poor response was higher in the Ser/Ser group (P = 0.011). Conclusions: Ser/Ser polymorphism is characterised by a poor ovarian response. Despite this, polymorphisms in the FSHR gene do not seem to affect the results of pregnancy per started cycle, ongoing pregnancy per started cycle, ongoing pregnancy per embryo transfer and live birth per embryo transfer.

Letrozole as an adjunct treatment in antagonist cycles that previously failed in poor responders.

OBJECTIVES: To investigate whether adding letrozole in the early follicular phase of a gonadotropin-releasing hormone (GnRH) antagonist (GA) stimulation cycle improves in vitro fertilization (IVF) outcomes in poor responder patients. MATERIAL AND METHODS: To be included in this study, patients had to have had at least one previous GA cycle and a subsequent GA cycle with added early follicular phase letrozole (LzGA). A total of 41 poor responder patients were identified based on the Bologna criteria. RESULTS: The LzGA group had a lower dosage of follicular stimulating hormone (FSH) (p = 0.001), the duration of stimulation days (p = 0.015) and the duration of GnRH antagonist stimulation days (p = 0.033) when compared with controls. Comprehensive analysis of the cycle characteristics showed that the number of oocytes retrieved, the number of MII oocytes retrieved, the number of fertilized oocytes, and the fertilization rate were significantly higher in the LzGA cycle (p = 0.041, p = 0.019, p = 0.008, p = 0.01, respectively). The rate of cycle cancellation was lower in the LzGA group (24.4%) than in the GA group (48.8%), (p < 0.001). Although LzGA administration demonstrated a trend toward improved implantation and clinical pregnancy rates, this was an insignificant trend (p = 1.000, p = 0.177, respectively). CONCLUSIONS: Adjunctive letrozole administration seems to restore an IVF cycle by improving the cycle characteristics and reducing the total gonadotrophin dosage.

Relationship between testicular histopathology and the success of testicular sperm extraction in patients with non-mosaic Klinefelter syndrome.

OBJECTIVE: To investigate the effect of testicular histopathology on the success of microscopic testicular sperm extraction (mTESE) and the factors that could predict the success of mTESE in patients with non-mosaic Klinefelter syndrome (KS). MATERIAL AND METHODS: Forty-one KS patients diagnosed with non-obstructive azoospermia (NOA) who had undergone mTESE at our clinic were included in the study. The patients were divided into 5 groups according to the histopathology results: hyalinisation of tubules (HT), sertoli cell only (SCO), early maturation arrest (EMA), late maturation arrest (LMA), and hypospermatogenesis (HS). The groups were compared with regard to age, duration of infertility, hormone profile, testicular volume, and sperm retriveal rate. The clinical features of the patients with mTESE from whom sperm could or could not be obtained were also compared with the aim of investigating the predictive value of testicular histopathology and the other variables for prediction of the success of mTESE. RESULTS: Sperm could be obtained through mTESE in 13 out of 41 patients (31.7%). A statistically significant difference was determined between the groups with regard to the rate of sperm collection. No significant difference was determined between the histopathology groups with regard to the other variables. A statistically significant difference was determined between the groups from whic sperm could be collected or not with regard to age, Johnsen criteria, SCO, EMA and LMA variables. Multi-variate analysis revealed that age and Johnsen score were the independent variables predictive for success of mTESE. CONCLUSION: The present study has revealed that impairment in testicular histopathology negatively affects the success of mTESE and that it is a predictive factor for the success of mTESE in patients with KS. Increased patient age was also determined to negatively affect the success of mTESE and the operation was demonstrated to be more successful before 34 years of age.

Evaluation of chromosomal abnormalities and Y-chromosome microdeletions in 1696 Turkish cases with primary male infertility: A single-center study.

OBJECTIVE: The aim of this study was to determine the frequencies of chromosomal abnormalities and Y-chromosome microdeletions in Turkish cases with primary male infertility in a single center. MATERIAL AND METHODS: Chromosomal abnormalities and Y-chromosome microdeletions were investigated in 1696 cases with primary male infertility between 2012 and 2017. Karyotype analyzes and Y-chromosome microdeletions analyzes [azoospermia factor (AZF) regions] were performed in all cases by using standard cytogenetic methods and the multiplex polymerase chain reaction method, respectively. RESULTS: Chromosomal abnormalities were found in 142 cases (8.4%; 142/1696). Y-chromosome microdeletions were detected in 46 cases (2.7%; 46/1696). Y-chromosome microdeletions in the AZFc region were found in 20 of 46 cases (43%). CONCLUSION: This study is one of the few were a large number of cases was studied in Turkey. It indicates that cytogenetic and Y-chromosome microdeletion studies should be conducted in cases with primary male infertility prior to selecting assisted reproductive techniques.

The higher heparin-binding epidermal growth factor (HB-EGF) in missed abortion.

OBJECTIVE: Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in the female reproductive tract. HB-EGF has a function in the menstruation cycle, implantation, decidualization, placenta development, and also inhibition of apoptosis. This study aims to investigate a possible role of HB-EGF in missed abortion. MATERIALS AND METHODS: Decidual and placental tissue samples were obtained from women with unwanted pregnancy as the control group and from women with missed abortions as the patient group. Immunohistochemistry was utilized to compare HB-EGF expression of fibroblast and decidual cells in uterine decidual stroma and fibroblasts and mesenchymal cells in placental villous stroma; the TUNEL technique was used to detect apoptotic cells within the decidual and placental tissues of the two groups. RESULTS: It was demonstrated that HB-EGF expression in both uterine decidual stroma and placenta stroma was increased in the missed abortion group (142.70 ± 12.80; 116.10 ± 14.16, respectively), compared with the normal pregnancy group (101.60 ± 14.18; 81.60 ± 10.74, respectively). It was also shown that there was no difference in TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labelling) positive cells between the uterine decidual stroma (11.4 ± 3%; 13.6 ± 3%, respectively), placental villous stroma (13.7 ± 3%; 15.9 ± 3%, respectively), and cytotrophoblast-syncytiotrophoblast cells (7.3 ± 2; 9.8 ± 3, respectively) of the two groups. CONCLUSION: This data supports the hypothesis that increased HB-EGF expression in a missed abortion may prevent the discharge of the dead fetus.

The role of selectins in the first trimester pregnancy loss.

OBJECTIVE: There are no well-defined findings about reasons for first trimester abortion in some pregnancy cases. Selectins are cell adhesion proteins which are important for blastocyst implantation in the decidua. The goal of the study was to investigate the role of selectins in first trimester pregnancy loss by immunohistochemistry. STUDY DESIGN: Decidual and placental tissue samples have been obtained from the women with unwanted pregnancy as the control group (n = 40) and missed abortion (n = 40) as the study group. Immunohistochemistry technique has been used to compare P, L and E-selectin expression of the fibroblast and the decidual cells in uterine decidual stroma; and fibroblasts and mesenchymal cells in placental villous stroma. Immunostaining for P, L, E-Selectin has been evaluated semiquantitatively by HSCORE analysis. RESULTS: Decidual cells, for E and L-selectin showed stronger staining in the study group than controls, and the difference was statistically significant (p = 0.007, p = 0.007). P-selectin showed stronger staining in the control group, but the difference was not as significant as the E and L-selectins (p = 0.04). In the placenta, cytotrophoblasts and syncytiotrophoblasts showed stronger staining for P, E, L-selectins for the control group (p < 0.007, p = 0.001 and p < 0.001, respectively). CONCLUSION: Strong expression of each of the three investigated selectins in healthy pregnancy villi shows their contribution to implantation and strong placentation. There is a need for better understanding of the functions of adhesive molecules in these events to reveal unknown causes for pregnancy loss.

Effect of raloxifene and atorvastatin in atherosclerotic process in ovariectomized rats.

AIM: The goal of this study was to investigate the combined effects of raloxifene and atorvastatin in aged ovariectomized rats during endothelial dysfunction and atherosclerotic process. MATERIAL AND METHODS: This study was conducted on 28 Wistar albino female rats randomly divided into four groups. All groups were ovariectomized and one group was kept as the control group (OVX). For four weeks, the remaining three groups were treated with the statin atorvastatin (OVX+AV), the selective estrogen receptor modulator raloxifene (OVX+RL), and both atorvastatin and raloxifene (OVX+RL+AV), respectively. At the end of the treatment period, all rats were sacrificed and thoracic aortas excised, and endothelial cells were immunohistochemically stained for markers in the atherosclerotic process, such as inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). RESULTS: Compared to the ovariectomized group, the iNOS level was significantly increased in the OVX+RL group (P=0.002), but contrarily decreased in the groups OVX+AV (P=0.002) and OVX+RL+AV (P=0.002). eNOS levels in the groups OVX+AV (P=0.002) and OVX+RL+AV (P=0.002) were significantly lower than that in the OVX group. When compared to the OVX group, significant reductions in ET-1 and TNF-α levels were found in all treatment groups. A significant decrement in MCP-1 level was found in the OVX+AV group (P=0.002). CONCLUSION: In aged ovariectomized rats, the administration of both raloxifene and atorvastatin significantly decreased the levels of ET-1 and TNF-α on endothelial cells. Combined treatment with these drugs shortly after menopause might play a potential preventive role in the early stages of atherosclerosis development.

The use of ultrasound during intrauterine insemination in unexplained infertility may improve pregnancy outcomes.

OBJECTIVE: To investigate the role of ultrasound guidance in intrauterine insemination (IUI). MATERIALS AND METHODS: A retrospective study was conducted. The data was collected from the records of 197 couples with unexplained infertility who underwent IUI with a total of 267 IUI cycles in the in vitro fertilization center of our hospital between January 2009 and December 2010. RESULTS: Of the 267 IUI cycles, 145 were carried out as US-guided, while 122 cycles IUI were performed with a blind procedure. In the US-guided IUI and blinded IUI groups, the pregnancy rates were 23.4 and 13.9%, respectively. The difference between the groups was statistically significant (p = 0.049), thereby indicating that US guidance improves pregnancy rates. In the US-guided IUI group, 9.7% of the cases were difficult, while in the blinded IUI group, 26.2% were difficult and the difference between the groups was also statistically significant (p < 0.001). CONCLUSION: US guidance in IUI improves pregnancy rates and reduces the frequency of difficult IUI.

Defining the macroscopic and microscopic findings of experimental focal brain ischemia in rats from a forensic scientist's point of view.

Approximately 10% of all deaths in the world occur as a result of stroke. Determination of the time schedule of the pathologic events in a stroke patient is invaluable for a forensic specialist. The aim of this study was to define the schedule of the macroscopic and microscopic changes that occurred in a rat model of permanent focal ischemia for providing useful clues for the evaluation of stroke patients. Male Wistar rats weighing 250 to 350 g were used in this study. Permanent focal brain ischemia was applied by the suture occlusion method. The animals were divided into 7 experimental groups (n = 6) with time schedules including 1.5, 3, 6, 12, 24, 72 hours, and the sham. Brains were harvested at the end of the determined time schedule. Lesions in the frontoparietal cortex were evaluated macroscopically first and later hematoxylin eosin stained sections from the infarct core were investigated microscopically. Macroscopically, enlargement of the ipsilateral hemisphere was mild at 6 hour, apparent at 12 and 24 hours, and mild again at 72 hours. Microscopically, ischemic changes were apparent even at 1.5 hour. Red neurons and infiltration of the parenchyma with neutrophil leukocytes were observed at 12 hours. Pannecrosis and massive leukocyte infiltration were observed at 72 hours. Macroscopic and microscopic findings obtained from a rat model may provide clues for determination of the time-dependent changes due to brain ischemia in human subjects. Finally, the benefits of determination of time course of pathologic changes in the brain for forensic scientists were discussed.

Antenatal administration of granulocyte-macrophage colony-stimulating factor increases fetal lung maturation and endothelial nitric oxide synthase expression in the fetal rat lung.

OBJECTIVES: We investigated how maternal administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) induced fetal lung maturation compared with dexamethasone and whether maternal administration of GM-CSF and dexamethasone influenced the fetal lung eNOS expression. STUDY DESIGN: Thirty pregnant rats were divided into three groups of 10 rats each to receive GM-CSF, dexamethasone or saline solution at 16 days of gestation. Lung maturation using bronchial area and immunohistochemical lung airway epithelium and the vascular endothelial eNOS expression, using H Scores, were evaluated at 18 and 20 days of gestation. The statistical analysis was done with the Kruskal-Wallis test for comparisons of more than two groups and the Mann-Whitney U-test as a post hoc test using SPSS for windows release 10.0. Values of p>0, 0.05 were considered significant. RESULTS: On the 20th day of gestation both GM-CSF and dexamethasone injections caused a significant increase in fetal lung bronchial area, as compared with the controls (24.9%, 36.8%, 13.4%, respectively, p=0.001). eNOS immunoreactivity was observed in the endothelium of large pulmonary vessels and large and small airway epithelium on the 18th and 20th day of gestation. Maternal GM-CSF and dexamethasone increased lung eNOS expression in the airway epithelium when compared to controls. CONCLUSION: Maternal administration of GM-CSF induced fetal lung maturation and this effect may be mediated, at least partly, by an increase in the eNOS expression.

Pretreatment with pro- and synbiotics reduces peritonitis-induced acute lung injury in rats.

BACKGROUND: To study whether enteral pretreatment with a synbiotic composition of lactic acid bacteria and bioactive fibers can reduce peritonitis-induced lung neutrophil infiltration and tissue injury in rats. MATERIALS AND METHODS: Rats were divided into five groups, and subjected to induction of peritonitis-induced lung injury using a cecal ligation and puncture model (CLP). All animals were pretreated for 3 weeks prior the CLP by daily gavage with either (1) a synbiotic composition (10(10) CFU of Pediococcus pentosaceus 5-33:3, 10(10) CFU of Leuconostoc mesenteroides 77:1, 10(10) CFU of L. paracasei subspecies paracasei, 10(10) CFU of L. plantarum 2362 plus fermentable fibers), (2) fermentable fibers alone, (3) nonfermentable fibers, (4) a probiotic composition (10(10) CFU of P. pentosaceus 5-33:3, 10(10) CFU of L. mesenteroides 77:1, 10(10) CFU of L. paracasei subsp. paracasei, 10(10) CFU of L. plantarum 2,362), or (5) a heat-killed probiotic composition. All animals were killed 24 hours after CLP and lung tissue samples were studied for degree of neutrophil infiltration and levels of tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta. In addition the lung wet-to-dry tissue weight ratio, the myeloperoxidase activity, and malondialdehyde content were also assessed. RESULTS: No mortality was encountered in any of the groups. Histologic signs of lung injury (number of neutrophils and TNF-alpha, IL-1beta staining) were observed in all groups except the synbiotic and probiotic treated groups. Myeloperoxidase activity and malondialdehyde content were significantly lower in the two lactobacillus- pretreated groups, with no difference between them. Heavy infiltration of lung tissue with neutrophils was observed only in fiber-treated (302.20 +/- 7.92) and placebo-treated (266.90 +/- 8.92) animals. This was totally abolished in the synbiotic-treated group (34.40 +/- 2.49). Lung edema (wet-to-dry lung weight ratio) was significantly reduced in the synbiotic-treated group (4.92 +/- 0.13 vs. 5.07 +/- 0.08 and 5.39 +/- 0.10, respectively). CONCLUSION: Three weeks of preoperative enteral administration of a synbiotic composition reduced peritonitis-induced acute lung injury in rats in a CLP model.