Effect of single versus grouped culture of human cumulus-oocyte complexes in PCOS women treated with biphasic in vitro maturation: A sibling oocyte pilot study.

Purpose: This study investigated the differences in the maturation rate of single versus grouped cumulus-oocyte complexes (COCs) culture methods for capacitation in vitro maturation (CAPA-IVM) in women with polycystic ovary syndrome (PCOS). Methods: This study was performed at My Duc Phu Nhuan Hospital, Vietnam from October 1, 2020 to October 24, 2021. Women aged 18-37 years with a diagnosis of PCOS were recruited. COCs from each woman were randomly divided into two groups: single or grouped culture during CAPA-IVM culture. The primary outcome was the maturation rate. Results: A total of 322 COCs from 15 eligible women included were randomly assigned to the two study groups. The maturation rate was comparable between the single and grouped culture groups (61.3% vs. 64.8%; p = 0.56). There were no significant differences in the number of 2-pronuclei fertilized oocytes, number of day-3 embryos, and number of good-quality embryos in the two culture method groups. In the single culture group, COCs morphology was associated with the day-3 embryo formation rate but not the maturation rate. Conclusions: Comparable oocyte maturation and embryology outcomes between single and grouped COCs culture utilizing sibling COCs derived from women with PCOS suggest the feasibility of both methods for CAPA-IVM culture.

Epigenetic variation in neonatal tissues in infants conceived using capacitation-in vitro maturation vs. in vitro fertilization.

OBJECTIVE: To investigate alterations of the global DNA methylation profile in placenta, cord blood, and neonatal buccal smears in infants conceived using in vitro maturation (IVM) with a prematuration step (capacitation-IVM [CAPA-IVM]) vs. in vitro fertilization (IVF). DESIGN: Analysis of data from the offspring of participants in a randomized controlled trial. SETTING: Private clinic. PATIENTS: Forty-six women with polycystic ovary syndrome and/or high antral follicle count and their offspring (58 newborns). INTERVENTION(S): Women with polycystic ovary syndrome and/or a high antral follicle count participating in the clinical trial were randomized to undergo CAPA-IVM or conventional IVF. MAIN OUTCOME MEASURE(S): At delivery, biological samples including cord blood, placental tissue, and a neonatal buccal smear were collected. Genome-wide DNA methylation was determined using the Illumina Infinium MethylationEPIC BeadChip. Variability in methylation was also considered, and mean variances for the two treatment categories were compared. RESULTS: In neonatal buccal smears, there were no significant differences between the CAPA-IVM and conventional IVF groups on the basis of the CpG probe after linear regression analysis using a significant cut-off of false-discovery rate <0.05 and |Δß|≥0.05. In cord blood, only one CpG site showed a significant gain of methylation in the CAPA-IVM group. In the placenta, CAPA-IVM was significantly associated with changes in methylation at five CpG sites. Significantly more variable DNA methylation was found in five probes in the placenta, 54 in cord blood, and two in buccal smears after IVM of oocytes. In cord blood samples, 20 CpG sites had more variable methylation in the conventional IVF vs. IVM group. Isolated CpG sites showing differences in methylation in cord blood were not associated with changes in gene expression of the overlapping genes. CONCLUSION(S): Capacitation-IVM appeared to be associated with only a small amount of epigenetic variation in cord blood, placental tissue, and neonate buccal smears. CLINICAL TRIAL REGISTRATION NUMBER: NCT03405701 (www. CLINICALTRIALS: gov).

Mouse oocytes restore antral stage partial mechanical denudation in vitro.

In brief: Partially denuded mouse cumulus-oocyte complexes restore likely functional transzonal projections in culture, under meiotic inhibition, with no detectable impact on oocyte competence. This proof-of-concept study constitutes positive premises for improving the developmental competence of human capacitation (CAPA)-in vitro maturation (IVM) oocytes with inadequate somatic cell connections. Abstract: In vitro oocyte culture might be the sole option for fertility preservation in some patients. This relies on constant oocyte-somatic bidirectional communication, and its precocious disruption alters oocyte competence. In non-human chorionic gonadotropin-triggered human in vitro maturation (IVM), retrieval of cumulus-oocyte complexes (COCs) by needle aspiration from the targeted small follicles (2-8 mm) leads to the collection of some partially denuded (PD) COCs with poor developmental competence. Hypothetically, re-establishing connectivity in these COCs could rescue oocyte quality. To test this, we used a well-characterized mouse preantral follicle culture system. On day 8, at antral stage, in part of the follicles, the oocytes were mechanically denuded while in other follicles in vitro grown oocytes were replaced with age matched fully stripped in vivo grown ones. The denuded oocytes were cultured on top of the somatic compartment until day 12, when oocyte-somatic reconnection was assessed. Furthermore, to better mimic the current biphasic IVM setup, fully surrounded (FS) COCs were collected from 19- to 21- day-old unprimed mice. Following partial mechanical denudation, COCs were cultured under meiotic inhibition for 2-4 days, to test oocyte-cumulus cell (CC) reconnection. Meiotic and developmental competence endpoints were compared between reconnected and FS-cultured COCs. We concluded that (i) in vivo- and in vitro- grown antral oocytes reconnect with in vitro-grown somatic companions; (ii) PD-COCs restore the FS morphology in culture, under meiotic inhibition; and (iii) oocyte quality from reconnected and intact cultured COCs is comparable. These observations encourage translational work to rescue partially denuded oocytes in human IVM.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

PURPOSE: Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. METHODS: Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. RESULTS: Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. CONCLUSIONS: Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

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.

Characterization of carbohydrate metabolism in in vivo- and in vitro-grown and matured mouse antral follicles†.

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence.

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.

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

PURPOSE: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFß superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. METHODS: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. RESULTS: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. CONCLUSION: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

Positive effects of amphiregulin on human oocyte maturation and its molecular drivers in patients with polycystic ovary syndrome.

STUDY QUESTION: Does use of medium containing amphiregulin improve meiotic maturation efficiency in oocytes of women with polycystic ovary syndrome (PCOS) undergoing in vitro maturation (IVM) preceded by a capacitation culture step capacitation IVM (CAPA-IVM)? SUMMARY ANSWER: Use of medium containing amphiregulin significantly increased the maturation rate from oocytes retrieved from follicles with diameters <6 or ≥6 mm pre-cultured in capacitation medium. WHAT IS KNOWN ALREADY: Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence. Amphiregulin added to the meiotic trigger has been shown to improve outcomes of IVM in a range of mammalian species. STUDY DESIGN, SIZE, DURATION: This prospective, randomized cohort study included 30 patients and was conducted at an academic infertility centre in Vietnam from April to December 2019. Patients with PCOS were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the first stage, sibling oocytes from each patient (671 in total) were allocated in equal numbers to maturation in medium with (CAPA-AREG) or without (CAPA-Control) amphiregulin 100 ng/ml. After a maturation check and fertilization using intracytoplasmic sperm injection (ICSI), all good quality Day 3 embryos were vitrified. Cumulus cells (CCs) from both groups were collected at the moment of ICSI denudation and underwent a molecular analysis to quantify key transcripts of oocyte maturation and to relate these to early embryo development. On return for frozen embryo transfer (second stage), patients were randomized to have either CAPA-AREG or CAPA-Control embryo(s) implanted. Where no embryo(s) from the randomized group were available, embryo(s) from the other group were transferred. The primary endpoint of the study was meiotic maturation efficiency (proportion of metaphase II [MII] oocytes; maturation rate). MAIN RESULTS AND THE ROLE OF CHANCE: In the per-patient analysis, the number of MII oocytes was significantly higher in the CAPA-AREG group versus the CAPA-Control group (median [interquartile range] 7.0 [5.3, 8.0] versus 6.0 [4.0, 7.0]; P = 0.01). When each oocyte was evaluated, the maturation rate was also significantly higher in the CAPA-AREG group versus the CAPA-Control group (67.6% versus 55.2%; relative risk [RR] 1.22 [95% confidence interval (CI) 1.08-1.38]; P = 0.001). No other IVM or embryology outcomes differed significantly between the two groups. Rates of clinical pregnancy (66.7% versus 42.9%; RR 1.56 [95% CI 0.77-3.14]), ongoing pregnancy (53.3% versus 28.6%; RR 1.87 [95% CI 0.72-4.85]) and live birth (46.7% versus 28.6%; RR 1.63 [95% CI 0.61-4.39]) were numerically higher in the patients who had CAPA-AREG versus CAPA-Control embryos implanted, but each fertility and obstetric outcome did not differ significantly between the groups. In the CAPA-AREG group, there were significant shifts in CC expression of genes involved in steroidogenesis (STAR, 3BHSD), the ovulatory cascade (DUSP16, EGFR, HAS2, PTGR2, PTGS2, RPS6KA2), redox and glucose metabolism (CAT, GPX1, SOD2, SLC2A1, LDHA) and transcription (NRF2). The expression of three genes (TRPM7, VCAN and JUN) in CCs showed a significant correlation with embryo quality. LIMITATIONS, REASONS FOR CAUTION: This study included only Vietnamese women with PCOS, limiting the generalizability. Although 100 ng/ml amphiregulin addition to the maturation culture step significantly improved the MII rate, the sample size in this study was small, meaning that these findings should be considered as exploratory. Therefore, a larger patient cohort is needed to confirm whether the positive effects of amphiregulin translate into improved fertility outcomes in patients undergoing IVM. WIDER IMPLICATIONS OF THE FINDINGS: Data from this study confirm the beneficial effects of amphiregulin during IVM with respect to the trigger of oocyte maturation. The gene expression findings in cumulus indicate that multiple pathways might contribute to these beneficial effects and confirm the key role of the epidermal growth factor system in the stepwise acquisition of human oocyte competence. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED; grant number FWO.106-YS.2017.02) and by the Fund for Research Flanders (FWO; grant number G.OD97.18N). L.N.V. has received speaker and conference fees from Merck, grants, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring. T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme and Ferring. J.S. reports speaker fees from Ferring Pharmaceuticals and Biomérieux Diagnostics and grants from FWO Flanders, is co-inventor on granted patents on CAPA-IVM methodologies in USA (US10392601B2), Europe (EP3234112B1) and Japan (JP 6806683 registered 08-12-2020) and is a co-shareholder of Lavima Fertility Inc., a spin-off company of the Vrije Universiteit Brussel (VUB, Brussels, Belgium). NA, TDP, AHL, MNHN, SR, FS, EA and UDTH report no financial relationships with any organizations that might have an interest in the submitted work in the previous three years, and no other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: NCT03915054.

Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence†.

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A two-dimensional attachment mouse secondary follicle culture system was used to assess the oocyte's capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral (PA) and early antral (EA) follicles was performed. Established endpoints were the oocyte's potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from PA and EA cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections with no significant differences in meiotic and developmental competence compared with those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as nonhuman chorionic gonadotropin triggered in vitro maturation.

Glucose metabolism characterization during mouse in vitro maturation identifies alterations in cumulus cells†.

In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side-effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared with conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM-a novel two-step IVM method-has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells (CCs) are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared with their in vivo counterparts. However, their CCs exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM CCs are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

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.

hCG Improves Luteal Function and Promotes Progesterone Output through the Activation of JNK Pathway in the Luteal Granulosa Cells of the Stimulated IVF Cycles†.

Human chorionic gonadotropin (hCG) is a luteotropic hormone that promotes the survival and steroidogenic activity of corpus luteum (CL) by acting through luteinizing hormone receptors (LHRs) expressed on luteinized theca and granulosa cells (GCs). Therefore, it is used to support luteal phase in in vitro fertilization (IVF) cycles to improve clinical pregnancy rates and prevent miscarriage. However, the molecular mechanism underlying this action of hCG is not well characterized. To address this question, we designed an in vitro translational research study on the luteal GCs obtained from 58 IVF patients. hCG treatment at different concentrations and time points activated c-Jun N-terminal kinase (JNK) pathway and significantly increased its endogenous kinase activity along with upregulated expression of steroidogenic enzymes (steroidogenic acute regulatory protein (stAR), 3ß-Hydroxysteroid dehydrogenase (3ß-HSD)) in a dose-dependent manner in the luteal GCs. As a result, in vitro P production of the cells was significantly enhanced after hCG. When JNK pathway was inhibited pharmacologically or knocked-down with small interfering RNA luteal function was compromised, P4 production was declined along with the expression of stAR and 3ß-HSD in the cells. Further, hCG treatment after JNK inhibition failed to correct the luteal defect and promote P4 output. Similar to hCG, luteinizing hormone (LH) treatment improved luteal function as well and this action of LH was associated with JNK activation in the luteal GCs. These findings could be important from the perspective of CL biology and luteal phase in human because we for the first time identify a critical role for JNK signaling pathway downstream LHR activation by hCG/LH in luteal GCs. SUMMARY SENTENCE: JNK signaling pathway plays a central role in the upregulated expression of the steroidogenic enzymes StAR and 3b-HSD and augmented progesterone production by hCG/LH in human luteal granulosa cells.

Luteal granulosa cells from natural cycles are more capable of maintaining their viability, steroidogenic activity and LH receptor expression than those of stimulated IVF cycles.

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, 3ß-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 3ß-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.

Endogenous c-Jun N-terminal kinase (JNK) activity marks the boundary between normal and malignant granulosa cells.

Granulosa cell tumor of the ovary (GCT) is a very rare tumor, accounting for only 2% of all ovarian tumors. It originates from sex cords in the ovary and can be divided into adult (95%) and juvenile (5%) types based on histologic findings. To date, no clear etiologic process has been identified other than a missense point mutation in the FOXL2 gene. Our previous works showed that c-Jun N-terminal kinase (JNK) pathway plays critical role in cell cycle progression and mitosis of normal and immortalized granulosa cells and follicle growth in rodent ovaries. These findings led us to investigate the role of JNK pathway in the granulosa cell tumor of the ovary. We used two different GCT cell lines (COV434 and KGN) and fresh GCT samples of adult and juvenile types obtained from the patients during surgery. We have discovered that endogenous kinase activity of JNK is markedly enhanced in the GCT samples and cell lines, whereas it was almost undetectable in mitotic non-malignant human granulosa cells. The inhibition of JNK pathway in GCT cell lines with two different pharmacologic inhibitors (SP600125 and AS601245) or siRNA resulted in a dose-dependent reduction in in vitro cell growth, increased apoptosis and diminished estradiol and AMH productions. JNK inhibition was also associated with a decrease in the number of cells positive for mitosis marker phospho-histone H3Ser 10 in the asynchronous cells; and diminished EdU uptake during S phase and cell cycle arrest at G2/M-phase transition in the synchronized cells. Ex vivo treatment of patient-derived GCT samples with JNK inhibitors for 24 h significantly decreased their in vitro growth and estradiol and AMH productions. Furthermore, in human GCT xenograft model, in vivo tumor growth was significantly reduced and plasma AMH levels were significantly decreased in SCID mice after administration of JNK inhibitors and siRNA. These findings suggest that targeting JNK pathway may provide therapeutic benefit in the treatment of granulosa cell tumors for which currently no curative therapy exists beyond surgery.

FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization.

STUDY 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 3ß-hydroxysteriod dehydrogenoase (3ß-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 3ß-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 electro-chemiluminescence 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 3ß-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), 3ß-HSD (1.68 ± 0.02-fold, P < 0.01) and 17ß-hydroxy steroid dehydrogenase (17ß-HSD, 2.16 ± 0.02-fold, P < 0.01) in the granulosa cells. Expression of 17α-hydroxylase (17α-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 3ß-HSD, 17ß-HSD and aromatase in a dose-dependent manner but did not affect 17α-OH. Protein expression of P was increased along with 3ß-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3ß-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 3ß-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. STUDY FUNDING/COMPETING INTEREST(S): Funded by the School of Medicine and the Graduate School of Health Sciences of Koc University. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: None.

Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

STUDY QUESTION: Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER: The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY: Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION: This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE: The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION: Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS: GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells.

STUDY QUESTION: Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER: Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY: It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION: This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS: For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION: This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.