Researcher: Bildik, Gamze
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Bildik, Gamze
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Publication Metadata only A comprehensive comparative transcriptional and translational analyses of the impact of ovarian response type, stimulation protocol and mode of trigger on the luteal function(Elsevier Science Inc, 2018) Seyhan, A; Yakın, Kayhan; Ata, Mustafa Barış; Öktem, Özgür; Bildik, Gamze; Urman, Cumhur Bülent; Faculty Member; Faculty Member; Faculty Member; Teaching Faculty; Faculty Member; School of Medicine; School of Medicine; School of Medicine; School of Medicine; School of Medicine; Koç University Hospital; 106822; 182910; 102627; N/A; 12147Objective: We aimed to compare molecular characteristics of the luteal granulosa cells between natural vs. stimulated IVF cycles in good and poor-responders. Design: Translational research study. Materials and Methods: Luteinized granulosa cells were obtained from good (n=154) and poor responder (n=64) IVF patients comparable for age, type and dose of gonadotropin and IVF etiology. Good-responders (4-15 oocytes) underwent natural (n=22), GnRH agonist (long protocol n=44) and antagonist IVF cycles triggered with rec-hCG (n=46) or GnRH agonist leuprolide acetate (n=42). Poor-responders fulfilling the Bologna criteria consisted of 64 patients undergoing GnRH antagonist protocol triggered with hCG (n=36) or hCG+GnRH agonist (n=28). Results: In the good-responders, natural cycle (NC) granulosa cells were significantly more viable (88%) compared to the stimulated IVF cycles (66%, 64% and 37% for agonist and antagonist cycles triggered with hCG and agonist respectively, p<0.05). The mRNA expression of steroidogenic enzymes (SCC, stAR, 3B-HSD, 17B-HSD and aromatase), LH receptor and VEGF and in vitro E2 and P productions were comparable between hCG-triggered agonist and antagonist cycles, but significantly higher than NC in the first days of culture. However, on the following days their hormone productions and viability began to decline very rapidly with the most drastic decrease being observed in the agonist triggered cycles. By contrast, NC granulosa cells maintained their viability and produced E 2 and P in increasing amounts in culture up to six days. The expression of anti-apoptotic genes (AKT-1, BCL2-L2) were significantly lower, and pro-apoptotic genes (BAD, BID, BAX, Cas3) were significantly higher in the stimulated cycles particularly in the agonist triggered ones compared to NC granulosa cells. Pulse exposure to cisplatin induced apoptosis only in a small fraction of the cells from the NCs whereas the same exposure caused massive apoptosis in the cells of the stimulated cycles (27% vs. 78% respectively, p<0.01). In the poor-responders both viability and steroidogenic activity of the cells were more severely reduced compared to the antagonist cycles of the good-responders. There were no significant differences between hCG and hCG+agonist triggered cycles in terms of viability, hormone production, VEGF and LH receptor expressions in the luteal granulosa cells. Conclusions Reduced survival and increased apoptosis of luteal granulosa cells leading to defective steroid production in stimulated cycles in comparison to natural ones may at least in part explain why luteal phase is defective and requires exogenous P supplementation for support in these cycles. Also dual trigger does not appear to improve luteal function in the poor-responders.Publication Metadata only The expressions of ovarian steroidogenic enzymes do not increase proportionally after FSH, creating a shunting that promotes progesterone output in the granulosa cells without luteinization(Oxford University Press (OUP), 2016) Seyhan, A.; Keles, I.; Balaban, B.; N/A; N/A; N/A; N/A; Akın, Nazlı; Bildik, Gamze; Urman, Defne; Urman, Cumhur Bülent; Öktem, Özgür; Master Student; Teaching Faculty; Master Student; N/A; Faculty Member; Faculty Member; Graduate School of Health Sciences; School of Medicine; Graduate School of Health Sciences; N/A; School of Medicine; School of Medicine; N/A; N/A; N/A; N/A; 12147; 102627N/APublication Metadata only Molecular evidence against the preventive actions of GnRH agonists in chemotherapy induced damage in human ovary and granulosa cells(2015) Güzel, Y.; Urman, B; N/A; Bildik, Gamze; Akın, Nazlı; Taşkıran, Çağatay; Selek, Uğur; Öktem, Özgür; Teaching Faculty; Master Student; Faculty Member; Faculty Member; Faculty Member; School of Medicine; Graduate School of Health Sciences; School of Medicine; School of Medicine; School of Medicine; Koç University Hospital; N/A; N/A; 134190; 27211; 102627N/APublication Metadata only Sphingosine-1-phosphate protects human ovarian follicles from apoptosis in vitro(Elsevier, 2018) Güzel, Yılmaz; Bildik, Gamze; Öktem, Özgür; Teaching Faculty; Faculty Member; School of Medicine; School of Medicine; N/A; 102627Objective(s): We aimed to analyze if anti-apoptotic agent sphingosine-1-phosphate offers protection against in vitro follicle atresia during culture of human ovarian cortical samples. Study design: A translational research study of ex-vivo and in-vitro models of human ovarian tissue. Material and methods: Ovarian cortical tissue fragments (1 x 0.5 cm) were obtained from young patients (n = 15 mean age +/- SD: 29.4 +/- 2.5) undergoing laparoscopic excision of benign ovarian cysts. The samples were cultured for 4 days in 24-well format culture plate using conventional culture techniques. S1P was added to culture media at 200 and 400 mu M concentrations. At the end of culture period the samples were processed for both histomorphological assessment and detection of apoptosis with immunohistochemistry and western blot methods using apoptosis marker cleaved caspase-3. In vitro estradiol (E-2) and AMH productions of the samples were measured with ELISA. Follicle counts were expressed as the mean number of follicles per mm(2). Results: The mean numbers of primordial and secondary follicles were 3.2 +/- 0.4 and 0.7 +/- 0.2 respectively, in the fresh fixed uncultured samples. After four days of culture their numbers were significantly decreased to 0.8 +/- 0.2 (p < 0.01) and 0.1 +/- 0.05 (p < 0.05) respectively, in the control samples cultured without S1P compared to fresh fixed samples. SIP treatment decreased follicle atresia and significantly higher number of primordials (2.3 +/- 0.3, p < 0.01) and secondary follicles (0.5 +/- 0.1, p < 0.05) survived in the samples after 4 day culture period compared to those cultured without SIR In line with this there was dose-dependent decrease in the protein expression of cleaved caspase-3 on western blot and in the number of apoptotic follicles stained positive for cleaved caspase-3 on immunohistochemistry in the samples incubated with S113 at 200 and 400 mu M concentrations. Furthermore, those samples incubated with SlP produced significantly higher amounts of E2 (2339 +/- 321 vs. 1156 +/- 125 pg/mL respectively, p < 0.01) compared to control samples. Conclusions: These results suggest that S1P promotes follicle survival in human ovarian cortical samples in vitro. (C) 2018 Elsevier B.V. All rights reserved.Publication Metadata only In-vitro AMH production of ovarian tissue samples in culture correlates with their primordial follicle pool(Elsevier, 2020) N/A; N/A; N/A; N/A; N/A; N/A; Vatansever, Doğan; İncir, Said; Bildik, Gamze; Taşkıran, Çağatay; Öktem, Özgür; Faculty Member; Faculty Member; PHD Student; Faculty Member; Faculty Member; School of Medicine; School of Medicine; Graduate School of Health Sciences; School of Medicine; School of Medicine; N/A; 193687; 175430; N/A; 134190; 102627Objective: We aimed to investigate if there is a correlation between in-vitro AMH production and primordial follicle reserve of the ovarian cortical samples in culture. Methods: Seven patients undergoing laparoscopic excision of ovarian dermoid cysts were included in the study. 0.5 x 0.5 cm of ovarian cortical samples embedded within the cyst wall were removed and cultured for one day. Then, the cultured cortical pieces were fixed, paraffin-embedded and serially sectioned for histormorphometric analysis. AMH and estradiol (E-2) production of the samples after one-day culture period were measured in the spent culture media. Primordial follicle density was expressed as the number of primordial follicles per mm(2). Pearson correlation and linear regression analyses were applied. Results: The mean age of the patients was 29.2 +/- 6.8 (ranging from 18 to 36). There was a negative correlation between age and PF density (r=-0.92, %95CI: -0.99 to -0.76, p <0.001). In-vitro AMH level of the cortical samples was significantly associated with age (R-2 = 0.67, p = 0.023), primordial follicle density (R-2 = 0.71, p = 0.015). There was a borderline significance between in-vitro levels of AMH and E-2 level (R-2 = 0.55, p = 0.058). A similar comparison could not be made for secondary follicles (preantral and small antral follicles) because of their rarity in the histological sections analyzed. Conclusions: This histomorphometric study provides evidence that in-vitro AMH production of the ovarian cortical samples reflects primordial follicle pool of the samples. (C) 2020 Elsevier B.V. All rights reserved.Publication Metadata only FSH stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization(Oxford Univ Press, 2017) Alper, Ebru; Balaban, Basak; N/A; Öktem, Özgür; Akın, Nazlı; Bildik, Gamze; Yakın, Kayhan; Urman, Cumhur Bülent; Faculty Member; Master Student; Teaching Faculty; Faculty Member; Faculty Member; School of Medicine; Graduate School of Health Sciences; Graduate School of Health Sciences; School of Medicine; School of Medicine; 102627; N/A; N/A; 106822; 12147STUDY QUESTION: Can granulosa cells produce progesterone (P) in response to FSH stimulation? SUMMARY ANSWER: FSH actively promotes P synthesis and output from granulosa cells without luteinization by up-regulating the expression and increasing enzymatic activity of 3 beta-hydroxysteriod dehydrogenoase (3 beta-HSD), which converts pregnenolone to P. WHAT IS KNOWN ALREADY: Serum P level may rise prematurely prior to ovulation trigger in stimulated IVF cycles and adversely affect implantation and clinical pregnancy rates by impairing endometrial receptivity. STUDY DESIGN, SIZE, DURATION: A translational research study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian cortical samples (n = 15) and non-luteinizing FSH-responsive human mitotic granulosa cell line (HGrC1) were stimulated with rec-FSH at 12.5, 25 and 50 mIU/ml concentrations for 24 and 48 h. FSH receptor expression was knocked-down and up-regulated in the granulosa cells using short hairpin RNA (shRNA) technology and activin-A administration, respectively. The expressions of the steroidogenic enzymes were analyzed at mRNA level by real-time quantitative RT-PCR, and protein level by western blot and immunoprecipitation assay. The enzymatic activity of 3 beta-HSD was measured using a spectrophotometric method. In vitro estradiol (E2) and P productions of the cells before and after FSH stimulation were measured by electrochemiluminescence immunoassay method. MAIN RESULTS and THE ROLE of CHANCE: Stimulation of the HGrC1 cells with FSH resulted in a dose-dependent increase in the mRNA and protein level of 3 beta-HSD. Overall, when all time points and FSH doses were analyzed collectively, FSH significantly up-regulated the mRNA expression of its own receptor (3.73 +/- 0.06-fold, P < 0.001), steroidogenic acute regulatory protein (stAR, 1.7 +/- 0.03-fold, P < 0.01), side-chain cleavage enzyme (SCC, 1.75 +/- 0.03-fold, P < 0.01), aromatase (4.49 +/- 0.08-fold, P < 0.001), 3 beta-HSD (1.68 +/- 0.02-fold, P < 0.01) and 17 beta-hydroxy steroid dehydrogenase (17 beta-HSD, 2.16 +/- 0.02-fold, P < 0.01) in the granulosa cells. Expression of 17 alpha-hydroxylase (17 alpha-OH, 1.03 +/- 0.01-fold P > 0.05) did not significantly change. Similar changes were observed in the protein expression analysis of these enzymes on western blotting after FSH stimulation. FSH significantly increased 3a-HSD, 17 beta-HSD and aromatase in a dose-dependent manner but did not affect 17 alpha-OH. Protein expression of P was increased along with 3a-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3 beta-HSD was significantly enhanced by FSH administration in the HGrC1 cells in a dose-dependent manner. In line with these findings P output (1.05 +/- 0.3 vs. 0.2 +/- 0.1 ng/ml, respectively, P < 0.001) from the samples stimulated with FSH were significantly increased along with E2 (1918 +/- 203 vs. 932 +/- 102 pg/ml, respectively, P < 0.001) compared to unstimulated controls. FSH-induced increase in 3 beta-HSD expression was amplified and reversed in the HGrC1 cells when FSH receptor expression was up-regulated by activin-A and down-regulated with shRNA, respectively. LIMITATIONS and REASONS FOR CAUTION: As only the effect of FSH was studied we cannot extrapolate our findings to the potential effects of HMG and recombinant LH. WIDER IMPLICATIONS of THE FINDINGS: This data provides a molecular explanation for the largely unexplained phenomenon of P rise during the follicular phase of gonadotropin stimulated IVF cycles. Our findings may progress the research to uncover potential mechanisms for preventing premature P rise that appears to be associated with inferior outcomes in women undergoing IVF.Publication Metadata only Luteal phase ovarian stimulation protocol for gynecological cancer patients with time constraints(Lippincott Williams & Wilkins, 2015) Arvas, M; N/A; N/A; N/A; N/A; N/A; Taşkıran, Çağatay; Mısırlıoğlu, Selim; Bildik, Gamze; Akın, Nazlı; Öktem, Özgür; Faculty Member; Doctor / Faculty Member; Teaching Faculty; Master Student; Faculty Member; School of Medicine; School of Medicine; School of Medicine; Graduate School of Health Sciences; School of Medicine; 134190; N/A; N/A; N/A; 102627N/APublication Metadata only Can we use gemcitabine for the treatment of granulosa cell tumor of the ovary?(Lippincott Williams & Wilkins, 2015) Arvas, M; N/A; N/A; N/A; N/A; N/A; Taşkıran, Çağatay; Mısırlıoğlu, Selim; Bildik, Gamze; Akın, Nazlı; Öktem, Özgür; Faculty Member; Doctor / Faculty Member; Teaching Faculty; Master Student; Faculty Member; School of Medicine; School of Medicine; School of Medicine; Graduate School of Health Sciences; School of Medicine; 134190; N/A; N/A; N/A; 102627N/APublication Metadata only A comperative molecular analysis of DNA damage response and apoptosis of malignant granulosa cells after exposure to gemcitabine and cisplatin(Bmj Publishing Group, 2019) Vatansever, Doğan; Bildik, Gamze; Taşkıran, Çağatay; Öktem, Özgür; Faculty Member; Teaching Faculty; Faculty Member; Faculty Member; School of Medicine; School of Medicine; School of Medicine; School of Medicine; 193687; N/A; 134190; 102627Introduction/Background: We aimed to compare gemcitabine vs. cisplatin in terms of DNA damage response, viability/apoptosis of malignant granulosa cells. Methodology: Malignant granulosa tumour cell lines (COV434 and KGN) were used for the experiments. Cell viability, proliferation, DNA damage response and apoptosis were investigated using immunofluorescence staining and immunoblotting. Cell cycle analysis was carried out using flow cytometry. In vitro oestradiol and AMH productions were analysed by ECLIA method. Gemcitabine and cisplatin were used at four different concentrations corresponding to their therapeutic blood levels. Results: Gemcitabine treatment caused DNA damage, cellular stress, inhibited proliferation and activated cell cycle check-point sensors and induced apoptosis as shown by increased expression of g-histone H2AX, p-JNK, Chk-1/Chk-2, cleaved forms of PARP and caspase-3 in the asynchronous cells in a dose dependent manner. As a Result: the proliferation and in vitro AMH and oestrogen production of the cells were decreased at post-exposure 24h. In the cells synchronized at S phase gemcitabine significantly inhibited DNA synthesis and blocked their proliferation. Similar effects were also observed after cisplatin treatment. Exposure of the cells to gemcitabine at G2/M transition abolished the progression of mitosis, caused mitotic arrest and failure to exit mitosis as evidenced by the inhibition of Cyclin B degradation and absence of de-phosphorylation of cdc-2 at Tyr 15 residue. However, such an effect was not observed in the cells synchronized and treated with cisplatin at G2/M. Conclusion: These results may suggest that anti-metabolite chemotherapy drug gemcitabine might have anti-neoplastic actions on granulosa cell tumour.Publication Metadata only Ivf characteristics and the molecular luteal features of random start ivf cycles are not different from conventional cycles in cancer patients(Oxford Univ Press, 2023) Akin, Nazli; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; Esmaeilian, Yashar; Hela, Francesko; Bildik, Gamze; İltümür, Ece; Yusufoğlu, Sevgi; Yıldız, Ceren Sultan; Keleş, İpek; Vatansever, Doğan; Taşkıran, Çağatay; Yakın, Kayhan; Öktem, Özgür; Researcher; PhD Student; Teaching Faculty; Phd Student; N/A; N/A; Doctor; Faculty Member; Faculty Member; Faculty Member; Faculty Member; N/A; Graduate School of Health Sciences; School of Medicine; Graduate School of Health Sciences; N/A; N/A; N/A; School of Medicine; School of Medicine; School of Medicine; School of Medicine; N/A; N/A; N/A; N/A; N/A; N/A; Koç University Hospital; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; 193687; 134190; 106822; 102627Study question: Are the IVF parameters and the steroidogenic luteal characteristics of random-start IVF cycles different from conventional cycles in cancer patients? Summary answer: No; controlled ovarian stimulation cycles randomly started at late follicular phase (LFP) and luteal phase (LP) are totally comparable to those conventional IVF cycles started at early follicular phase (EFP) in terms of the expression of the enzymes involved in cholesterol utilization and steroid hormone biosynthesis pathways, gonadotropin receptor expression and, estradiol (E2) and progesterone (P4) production in addition to the similarities in ovarian response to gonadotropin stimulation, oocyte yield, fertilization rate and embryo development competency in cancer patients. What is known already: Random start ovarian stimulation protocols are commonly employed for oocyte and embryo freezing for fertility preservation in cancer patients with time constraints who do not have sufficient time to undergo ovarian stimulation initiated conventionally at EFP of the next cycle. No data is available regarding the molecular steroidogenic features of these cycles analyzed together with the clinical IVF characteristics in cancer patients. We aimed to address this question in this study to help understand how similar the random start cycles are to the conventional start ones. Study design, size, duration: A clinical translational research study conducted in 62 cancer patients undergoing IVF for fertility preservation between the years 2017 and 2022. Participants/materials, setting, methods: Sixty-two patients who were diagnosed with different types of cancer and underwent ovarian stimulation for oocyte (n = 41) and embryo (n = 21) cryopreservation using GnRH antagonist protocol and human menopausal gonadotropins before receiving cancer treatment/surgery were enrolled in the study. For patients with breast cancer and endometrial cancer the aromatase inhibitor letrozole was used with gonadotropin stimulation. Ovarian stimulation was initiated conventionally at EFP in 22 patients and served as control while it was started at LFP in 20, and mid-LP in the other 20 patients. The luteinized granulosa cells (GCs) were recovered from follicular aspirates during oocyte retrieval procedure and used for the experiments separately for each individual patient. The expression of the enzymes involved in sex steroid biosynthesis (StAR, 3β-HSD, Aromatase) and cholesterol synthesis (3-hydroxy 3-methylglutaryl Co-A reductase (HMG-Co-A reductase)), utilization (hormone sensitive lipase (HSL)), and storage (Acetyl-Coenzyme A acetyltransferase 1 (ACAT-1)), and gonadotropin receptor expression status were analyzed using immunoblotting and RT-PCR methods. Laser confocal immunofluorescence imaging was applied to analyze and compare the expression patterns of the steroidogenic enzymes and their relation with mitochondria. In vitro E2 and P4 production by the cells were compared among the groups. Main results and the role of chance: Baseline demographic and IVF characteristics of the patients undergoing the conventional start and random start IVF cycles were similar. Duration of gonadotropin stimulation was significantly longer in LFP and LP start cycles in comparison to the conventional ones. Ovarian response to gonadotropin stimulation, mature and total oocyte yield, fertilization and Day 5 blastulation rates of the embryos were comparable between the conventional versus random start cycles. When the luteal GCs of these random start cycles were analyzed we could not find any gross differences between these cycles in terms of the viability index and gross light microscopic morphologic features. More detailed analysis of the molecular luteal characteristics of the cells using RT-PCR, immunoblotting methods revealed that the expression profiles of the gonadotropin receptors, and the enzymes involved in sex steroid biosynthesis and cholesterol synthesis/utilization, and the steroidogenic activity of the luteal GCs of the random start cycles are almost identical to those of the conventional start cycles. Confocal image analysis demonstrated similar patterns in the signal expression profiles of the steroidogenic enzymes and their co-localization within mitochondria. Large scale data: N/A. Limitations, reasons for caution: Caution should be exercised when interpreting our data and counseling cancer patients seeking fertility preservation because it is still unclear if previous exposure to cancer drugs, different ovarian pathologies or infertility etiologies, previous ovarian surgery and/or any other underlying diseases that are concomitantly present with cancer may cause a difference between conventional and random start stimulation protocols in terms of IVF parameters, luteal function and reproductive outcome. Relatively low number of patients in each stimulation protocol and pooling of luteal GCs for each patient rather than individual analysis of each follicle and oocyte are additional limitations of our study. Wider implications of the findings: Our findings provide reassurance that random start protocol offers cancer patients an equally good prospect of fertility preservation as conventional IVF. Study funding/competing interest(s): Funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koç University Research Center for Translational Medicine (KUTTAM), equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest. Trial registration number: N/A.