Exposure of antral follicles to medroxyprogesterone acetate during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed cycles.

STUDY QUESTION: Does medroxyprogesterone acetate (MPA) exposure in progestin-primed ovarian stimulation (PPOS) cycles cause molecular perturbations in the steroidogenic function and gonadotropin responsiveness of the granulosa cells? SUMMARY ANSWER: PPOS cycles are identical to traditional GnRH antagonist cycles not only for clinical IVF characteristics but also for gonadotropin receptor expression, response to gonadotropins, and steroidogenic function at the molecular level. WHAT IS KNOWN ALREADY: PPOS is increasingly used as an alternative to GnRH antagonists due to the inhibitory effect of progesterone on LH release by reducing GnRH pulsatility at the hypothalamic level. Although a growing body of evidence from clinical studies did not indicate significant differences between PPOS and antagonist protocols for IVF cycle characteristics and obstetrical outcomes, it is still unknown whether exposure of the antral follicle cohort to progesterone or its synthetic derivatives during ovarian stimulation causes any subtle molecular aberrations in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed comparative molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from normal responding IVF patients undergoing PPOS and antagonist cycles. STUDY DESIGN, SIZE, DURATION: A clinical translational research study was conducted with IVF patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 55 normal responding IVF patients who underwent ovarian stimulation with either PPOS using MPA (5 mg twice daily) or GnRH antagonist cetrorelix acetate. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation triggering, respectively. Luteinized mural GCs obtained during the oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging, and hormone assays were used. MAIN RESULTS AND THE ROLE OF CHANCE: Demographic and IVF cycle characteristics of the patients undergoing ovarian stimulation with PPOS and GnRH antagonist were similar, including ovarian response, mature oocyte yield, and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, SCC, 3ß-HSD, 17ß-HSD, aromatase) and the uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase, and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to those of the antagonist cycles. The expression of the receptors for gonadotropins, estrogen, and progesterone hormones was also similar. Basal and hCG-induced increases in 3ß-HSD expression and progesterone production and basal and FSH-induced increases in aromatase expression and E2 output of the GCs from PPOS patients did not exhibit any meaningful differences when compared with GCs from antagonist cycles. Furthermore, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other important genes regulating cumulus expansion, ovulation, and luteal function [Relaxin, ADAMTS-1, and epidermal growth factor (EGF)-like growth factor amphiregulin] in the GCs of the PPOS and antagonist cycles were similar. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Caution should be exercised when interpreting our data which was derived from normally responding patients whose ovulation was triggered with hCG. It is unclear whether the molecular parameters assessed vary according to infertility etiologies, magnitude of ovarian response, mode of trigger, and any other underlying ovarian pathologies or systemic diseases. MPA was the progestin used for PPOS and whether these findings can be generalized to other progestins is unknown. WIDER IMPLICATIONS OF THE FINDINGS: This study provides reassuring molecular evidence that exposure of antral follicle cohorts to MPA during the follicular growth phase does not have any detrimental effects on steroidogenic, ovulatory, and luteal functions when compared with GnRH antagonist 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), and 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.

Patients with gynecological malignancies are similar to other IVF patients without cancer for clinical and molecular reproductive parameters and DNA damage response pattern.

This study intended to investigate if gynecological cancers compromise ovarian function and reduce the success of assisted reproduction techniques (ART). No clinical and molecular data together is available on this issue for gynecological or other organ cancers. Steroidogenic pathways and DNA damage response characteristics of the granulosa cells retrieved from the 39 gynecological cancer patients were analyzed together with their clinical ART characteristics in comparison to 31 control ART patients. Patients with gynecological malignancies were similar to the control IVF patients for the number of mature oocytes retrieved, fertilization rates and embryo development competency. Molecular analyses of the granulosa cells retrieved from these cancer patients did not detect any perturbations in gonadotropin receptor expression and response, sex steroid production, cholesterol utilization/storage and, DNA damage response pattern in comparison to control IVF patients without cancer. This study provides the first reassuring clinical and molecular combined data set that the presence of gynecological malignancy does not appear to have any detrimental effect on clinical IVF cycle characteristics and ovarian functioning at molecular level.

Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis.

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. Previous studies documented the role of autophagy in sex steroid hormone biosynthesis in different animal models and human testis. Here we demonstrate in this study that sex steroid hormones estrogen and progesterone are produced through the same autophagy-mediated mechanism in the human ovary in addition to the human testis. In brief, pharmacological inhibition and genetic interruption of autophagy through silencing of autophagy genes (Beclin1 and ATG5) via siRNA and shRNA technologies significantly reduced basal and gonadotropin-stimulated estradiol (E2), progesterone (P4) and testosterone (T) production in the ex vivo explant tissue culture of ovary and testis and primary and immortalized granulosa cells. Consistent with the findings of the previous works, we observed that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LD)s with lysosome to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis. Gonadotropin hormones are likely to augment the production of sex steroid hormones by upregulating the expression of autophagy genes, accelerating autophagic flux and promoting the association of LDs with autophagosome and lysosome. Moreover, we detected some aberrations at different steps of lipophagy-mediated P4 production in the luteinized GCs of women with defective ovarian luteal function. The progression of autophagy and the fusion of the LDs with lysosome are markedly defective, along with reduced P4 production in these patients. Our data, together with the findings of the previous works, may have significant clinical implications by opening a new avenue in understanding and treatment of a wide range of diseases, from reproductive disorders to sex steroid-producing neoplasms, sex steroid-dependent malignancies (breast, endometrium, prostate) and benign disorders (endometriosis).

IVF characteristics and the molecular luteal features of random start IVF cycles are not different from conventional cycles in cancer patients.

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, 3ß-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.

Cholesterol uptake or trafficking, steroid biosynthesis, and gonadotropin responsiveness are defective in young poor responders.

OBJECTIVE: 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. DESIGN: A translational research study. SETTING: University Hospital Translational Research Center. PATIENT(S): 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. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 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. RESULT(S): 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, 3ß-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. CONCLUSION(S): 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.

Terminal differentiation of human granulosa cells as luteinization is reversed by activin-A through silencing of Jnk pathway.

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.