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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.

To investigate whether poor ovarian response in young patients undergoing in vitro fertilization simply involves lesser follicle growth due to diminished ovarian reserve or whether there are intrinsic perturbations in the ovary.A translational research study.University Hospital Translational Research Center.A total of 40 patients undergoing in vitro fertilization (20 normal and 20 poor responders) with ovarian stimulation using a gonadotropin-releasing hormone antagonist and recombinant follicle-stimulating hormone were included in the study.None.Luteal granulosa cells obtained during oocyte retrieval procedures were used for the experiments. Cell culture, quantitative real-time polymerase chain reaction, immunoblotting, confocal time-lapse live-cell imaging, and hormone assays were used.We tracked the steroidogenic pathway starting from the very initial step of cholesterol uptake to the final step of estradiol and progesterone production in luteal granulosa cells and identified some previously unknown intrinsic defects in the poor responders. Most notably, the expression of low-density lipoprotein receptors was significantly down-regulated and the uptake of cholesterol and its cytoplasmic accumulation and transportation to mitochondria were substantially delayed and reduced in the poor responders. Further, the expression of the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, and aromatase as well as gonadotropin receptors was defective, and the response of the cells to exogenous follicle-stimulating hormone and human chorionic gonadotropin was blunted, leading to compromised basal and gonadotropin-stimulated estradiol and progesterone production in the poor responders.This study demonstrates that poor ovarian response in young individuals should not simply be regarded as lesser follicle growth due to diminished ovarian reserve because the underlying pathogenetic mechanisms appear to be much more complex.

Molecular mechanisms underlying luteinization (terminal differentiation of granulosa and theca cells after ovulation) and luteolysis (demise of corpus luteum) are poorly understood in human ovary. Here we report that activin-A, after binding to its cognate receptors induces a functional luteolytic state and reverses luteinization phenotype by downregulating the expression of the steroidogenic enzymes, LH receptor and VEGF and reducing estradiol (E2) progesterone (P4) production and upregulating FSH receptor and cyclin D1 expression in human primary luteinized granulosa cells. Further, this action of activin-A involves downregulation of JNK signaling pathway and is opposite to that of human chorionic gonadotropin (hCG), which acts as a luteotropic hormone and improves luteal function through the activation of JNK pathway in the same cell type. Reversal of luteinization phenotype in luteal granulosa cells by activin-A potentially makes this hormone an attractive candidate for use under certain clinical situations, where induction of luteolysis and rapid reduction of endogenous sex steroid levels are beneficial such as ovarian hyperstimulation syndrome (OHSS), in which the ovaries hyper-respond to gonadotropin stimulation by producing too many growing follicles along with development of ascites, pleural effusion, and hemo-concentrations as a result of increased vascular permeability and leakage of intravascular volume into third spaces. Our work unveils a previously undefined role for activin-A and JNK signaling pathway in human corpus luteum biology, that might have a direct clinical impact in assisted reproductive technologies., School of Medicine; the Graduate School of Health Sciences of Koç University; Koç University Research Center for Translational Medicine (KUTTAM)

Study question Do all ovarian endometriomas have steroidogenic function and express estrogen and progesterone receptors? Summary answer No, they are heterogenous for the steroidogenic function and the expression of the estrogen and progesterone receptors. What is known already Excessive ectopic estrogen production and up-regulation of estrogen receptor- β, which drives inflammation together with aberrant progesterone signaling leading to impaired decidualization and establishment of ectopic endometrial implants together with down-regulated progesterone receptor (PR) expression are the cardinal molecular features of the disease. However, several fundamental questions still remain to be answered as to whether all ovarian endometriomas carry these molecular aberrations and are steroidogenically active; and if so, the amount of sex steroids they produce correlate with the level of expression of steroidogenic enzymes. We aimed to address these questions in the current study. Study design, size, duration A molecular research study on the surgical specimens collected between April 2020 and December 2020. Seven histopathologically confirmed benign endometriotic cyst capsules obtained from the patients undergoing laparoscopic excision of unilateral ovarian endometriomas without deep infiltrating endometriosis during early follicular phase were used in the study. Participants/materials, setting, methods The mean age±SD (range) of the patients were 32.8±4.9 (30–39). The mean endometrioma size was 5±1.2cm (5–7.5 cm). The samples were cut into equal size pieces of 0.5x0.5cm size and cultured for one day to measure their E2and P4production; and analyzed for the expression of steroidogenic enzymes with quantitative immunoblotting and for the expression of FSH-R, ER and PR with real-time qRT-PCR methods. Luteinized granulosa cells and ovarian cortex were set as references. Main results and the role of chance StAR expression was consistently observed in all samples. However, we noticed significant discrepancies among the samples regarding their steroidogenic function and the expression of aromatase and 3 β-HSD enzymes. E2 production exhibited significant variation (from 5 to 1177pg/mL) from sample to sample despite comparable levels of aromatase expression. ER- βup-regulation as a cardinal molecular feature of endometriosis, was observed in all but one samples (1.46 to 5.48 folds, p Limitations, reasons for caution These results need to be confirmed in studies with larger sample size and different types of endometriotic lesions. Wider implications of the findings: The regulation of steroidogenic activity of endometriomas cannot simply be explained by the expression level of the steroidogenic enzymes, underscoring the importance of other mechanisms that post-translationally regulate their enzymatic activity and metabolism of estrogen and progesterone. PR is not always down-regulated and FSH-R is commonly up-regulated in ovarian endometriomas. Trial registration number Not applicable

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 Koc 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.

We aimed to provide a comparative characterization of DNA damage response elements, survival/apoptosis and cell cycle progression of the malignant granulosa cells exposed to gemcitabine and cisplatin. Malignant granulosa tumor cell lines COV434 and KGN were used for the experiments. Cell viability, proliferation, DNA damage response and apoptosis were investigated. Cell cycle progression was assessed. In vitro estradiol (E2) and AMH productions of the cells were measured. Exposure of asynchronous malignant granulosa cells to gemcitabine caused growth arrest, induced DNA damage and activated cellular stress pathways, cell cycle checkpoint sensors and triggered apoptosis as evidenced by increased expression of phospho-p38, γ-histone H2AX, phospho-Chk-1/phospho-Chk-2, and cleaved forms of PARP and caspase-3 in a dose dependent manner. In vitro E2 and AMH productions of the cells were decreased along with reduction in viable cell mass. Cisplatin treatment produced a similar response but it was associated with JNK activation rather than p38. When the cells were synchronized and treated with gemcitabine at G2/M transition, the degradation of cyclin B1 and dephosphorylation of cdc-2 at Tyr 15 residue did not occur, resulting in cycle arrest. Similar effects on cell cycle progression was also observed in cisplatin. However, it was associated with JNK activation and higher expression of γ-histone H2AX and cleaved forms of caspase-3 and PARP, indicative of more extensive DNA damage and apoptosis in the cells. This descriptive study provides evidence that gemcitabine exerts cytotoxic effects and causes perturbations in cell cycle progression of malignant granulosa cells.

SummaryAccording to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.

SummaryFor decades, scientists have considered that female mammals are born with a lifetime reserve of oocytes in the ovary, irrevocably fated to decline after birth. However, controversy in the matter of the possible presence of oocytes and granulosa cells that originate from stem cells in the adult mammalian ovaries has been expanded. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of neo-oogenesis, who claim that germline stem cells (GSCs) exist in the ovarian surface epithelium (OSE) or the bone marrow (BM). Differentiation of ovarian stem cells (OSCs) into oocytes, fibroblast-like cells, granulosa phenotype, neural and mesenchymal type cells and generation of germ cells from OSCs under the contribution of an OSC niche that consists of immune system-related cells and hormonal signalling has been claimed. Although these arguments have met with intense suspicion, their confirmation would necessitate the revision of the current classic knowledge of female reproductive biology.

The possible presence of oocyte and granulosa cells originated from stem cells in the adult mammalian ovaries was claimed by some studies which will lead to major changes in reproductive biology and infertility treatments. Purpose of this research is to investigate the possible existence and the location of the potential stem cells in mouse ovaries. In this study, the ovaries from 2-week (pre-puberty) and 8-week (adult) old BALB-C mice were used. For the investigation of the presence of possible stem cells, the expression profiles of three well known stem cell markers, Oct-4, Nanog and Sox2 were determined in the ovaries of two different age groups by real time quantitative RT-PCR (qRT-PCR). Protein expression levels and their localization in the ovary cells were immunohistochemically evaluated on fresh-frozen ovary tissue sections by using monoclonal antibodies specific to Sox2, Nanog and Oct-4. The gene expression levels of Oct-4 and Nanog were found to be significantly differentiated between 2-week old and 8-week old mice whereas no significant difference was observed in the expression level of Sox2 between two age groups. Immunohistochemistry results showed the presence of both Sox2 and Oct-4 protein in the cytoplasm of ovarian epithelial cells, granulosa cells, oocytes and theca cells. Nanog protein was observed only in the nucleus of the oocytes and furthermore the expression of Nanog was higher in eight weeks old samples compared to two weeks old ones according to qRT-PCR results. These results suggest for the first time that Nanog protein is expressed both in adult and pre-puberty mouse ovaries and locate at the nucleus of the oocytes and to the best of our knowledge this is the first study that shows the differential expression of Oct-4, Nanog and Sox2 in pre-puberty and adult mouse ovaries by qRT-PCR. Collectively, our results may suggest that both pre-puberty and adult mice ovaries accommodate cells carrying stem cell features.