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Study 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. [ABSTRACT FROM AUTHOR]

Study Question: Are there any differences in the molecular characteristics of the luteal granulosa cells (GC) obtained from stimulated versus non-stimulated (natural) IVF cycles that may help explain the defective luteal phase in the former?Summary Answer: Luteal GC of stimulated IVF cycles, particularly those of agonist-triggered antagonist cycles, are less viable ex vivo, express LH receptor and anti-apoptotic genes at lower levels, undergo apoptosis earlier and fail to maintain their estradiol (E2) and progesterone (P4) production in comparison to natural cycle GC.What Is Known Already: Luteal function is defective in stimulated IVF cycles, which necessitates P4 and/or hCG administration (known as luteal phase support) in order to improve clinical pregnancy rates and prevent miscarriage. The luteal phase becomes shorter and menstruation begins earlier than a natural cycle if a pregnancy cannot be achieved, indicative of early demise of corpus luteum (premature luteolysis). Supra-physiological levels of steroids produced by multiple corpora luteae in the stimulated IVF cycles are believed to inhibit LH release directly via negative feedback actions on the hypothalamic-pituitary-ovarian axis resulting in low circulating levels of LH and a defective luteal phase. We hypothesized that some defects in the viability and steroidogenic activity of the luteal GC of the stimulated IVF cycles might contribute to this defective luteal phase in comparison to natural cycle GC. This issue has not been studied in human before.Study Design, Size, Duration: A comparative translational research study of ex vivo and in vitro models of luteal GC recovered from IVF patients undergoing natural versus stimulated IVF cycles was carried out. Luteinized GC were obtained from 154 IVF patients undergoing either natural (n = 22) or stimulated IVF cycles with recombinant FSH and GnRH agonist (long) (n = 44), or antagonist protocol triggered conventionally either with recombinant hCG (n = 46) or with a GnRH agonist (n = 42). GC were maintained in vitro for up to 6 days.Participants/materials, Setting, Methods: Cellular viability (YO-PRO-1 staining), the expression of the steroidogenic enzymes, pro-apoptotic genes [Bcl-2-associated death promoter (BAD), Bcl-2-associated X protein (BAX) and Caspase-3 (CASP3)], anti-apoptotic genes [RAC-alpha serine/threonine-protein kinase (AKT-1) and Bcl-2-like protein 2 (BCL2-L2)], LH receptor, vascular endothelial growth factor (VEGF) (using real-time quantitative PCR at mRNA level and western blot immunoprecipitation assay at protein level) and in vitro E2 and P4 production (electrochemiluminescence immunoassay) were compared in GC among the groups.Main Results and the Role Of Chance: Natural cycle GC were significantly more viable ex vivo (88%) compared to their counterparts of the stimulated IVF cycles (66, 64 and 37% for agonist and antagonist cycles triggered with hCG and GnRH agonist respectively, P < 0.01). They were also more capable of maintaining their vitality in culture compared to their counterparts from the stimulated IVF cycles: at the end of the 6-day culture period, 74% of the cells were still viable whereas only 48, 43 and 22% of the cells from the agonist and antagonist cycles triggered with hCG and agonist respectively, were viable (P < 0.01). The mRNA expression of anti-apoptotic genes (AKT-1 and BCL2-L2) was significantly lower, while that of pro-apoptotic genes (BAD, BAX and CASP3) was significantly higher in the stimulated cycles, particularly in the agonist-triggered antagonist cycles, compared to natural cycle GC (P < 0.01 for long protocol and antagonist hCG trigger, P < 0.001 for agonist trigger). The expression of steroidogenic enzymes (stAR, SCC, 3β-HSD and aromatase) and VEGF was significantly higher in the agonist and hCG-triggered antagonist cycles compared to natural cycle GC. Therefore, in vitro E2 and P4 production in cells from the stimulated IVF cycles was significantly higher than their counterparts obtained from the natural cycles in the first 2 days of culture. However, after Day 2, their viability and hormone production began to decline very rapidly with the most drastic decrease being observed in the agonist-triggered cycles. By contrast, natural cycle GC maintained their viability and produced E2 and P4 in increasing amounts in culture up to 6 days. In vitro P production and the mRNA and protein expression of LH receptor, VEGF and 3β-HSD were most defective in the agonist-triggered antagonist cycles compared to natural and agonist and hCG-triggered antagonist cycles. In vitro hCG treatment of a subset of the cells from the agonist-triggered cycles improved their viability, increased E2 and P4 production in vitro and up-regulated the mRNA expression of anti-apoptotic gene BCL-L2 together with steroidogenic enzymes stAR, SCC, 3B-HSD, LH receptor and VEGF.Large Scale Data: Not applicable.Limitations, Reasons For Caution: The limitations include analysis of luteinized GC only might not reflect the in vivo mechanisms involved in survival and function of the whole corpus luteum; GC recovered during oocyte retrieval belong to a very early stage of the luteal phase and might not be representative; effects of ovulation triggered with hCG may not equate to the endogenous LH trigger; the clinical characteristics of the patients may vary among the different groups and it was not possible to correlate stimulation-related molecular alterations in luteal GC with the clinical outcome, as no oocytes have been utilized yet. Therefore, our findings do not conclusively rule out the possibility that some other mechanisms in vivo may also account for defective luteal function observed in stimulated IVF cycles.Wider Implications Of the Findings: Ovarian stimulation is associated with significant alterations in the viability and steroidogenic activity of luteal GC depending on the stimulation protocol and mode of ovulation trigger. Reduced survival and down-regulated expression of 3B-HSD, LH receptor and VEGF leading to compromised steroid production in stimulated cycles, and particularly in the agonist-triggered cycles, may at least in part help explain why the luteal phase is defective and requires exogenous support in these cycles.Study Funding/competing Interest(s): This study was 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. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of mammalian target of rapamycin (mTOR) in human granulosa cell ovarian tumors and the therapeutic effect of rapamycin in COV434 mitotic granulosa cell lines., Material and Methods: A retrospective evaluation of the medical records and pathologic sections of patients with granulosa cell ovarian carcinoma was performed. mTOR and p-mTOR expression was immunohistochemically investigated. A COV434 cell culture were treated with 0.5, 1, 2, and 5 μM rapamycin. Real-time growth curve analysis via xCELLigence system and apoptotic cell analysis via YO-PRO™-1 Iodide were performed to assess the therapeutic effect of rapamycin on cancer cells., Results: A total of twenty patients were evaluated. mTOR staining was detected in 18 (90%) patients. Mild, moderate, intense, and very intense staining was observed in three (15%), eight (40%), six (30%), and one (5%) sample, respectively. The mean mTOR staining ratio was 59±41%. P-mTOR staining was observed in two (10%) patients. One (5%) patient had 5% staining, and one (5%) patient had 100% staining for p-mTOR. Both of the latter patients had very intense staining. Rapamycin caused a dose-dependent growth arrest and induced apoptosis in COV434 mitotic granulosa cells. The real-time growth curves of the cells treated with these drugs were distinguished by a marked reduced slope after exposure for several hours, indicating a rapid onset of apoptosis. Live/dead cell analysis with YO-PRO-1 staining showed that rapamycin induced apoptosis in 24% of the cells when used at 1 μM concentration, whereas the rate increased to 61% and 72% when the cells were treated with 2 μM and 5 μM rapamycin, respectively., Conclusion: mTOR expression is observed in various degrees in 90%, and p-mTOR expression is observed in only 10% of patients with granulosa cell ovarian carcinoma. Rapamycin caused a dose-dependent growth arrest and apoptosis in COV434 mitotic granulosa cells.