The Activation of Residual Dormant Follicles by Human Chorionic Gonadotropin (HCG) in Vivo: a Novel Treatment for Premature Ovarian Insufficiency

BackgroundWith the influence of factors such as ovarian surgery, high-dose radiotherapy and chemotherapy, environmental degradation, and bad living habits, the occurrence of premature ovarian insufficiency(POI) is getting younger and younger, and many young women's ovaries have entered the aging stage earlier. While many studies have investigated the patients with POI, which is still a challenge in reproductive medicine as the treatments available now are not ideal. POI patients have varying amounts of residual dormant follicles in the ovaries. Therefore, it is critical to further our understanding of primordial follicle activation in order to treat.This study aimed to investigate the activation of residual follicles in POI patients with injection of HCG, whether they could obtain embryos and become pregnant.Methods Four patients with POI were pretreated with dehydroepiandrosterone, Coenzyme Q10, estrogen and medroxyprogesterone. The prescribed amounts of estrogen and medroxyprogesterone were adjusted to maintain the level of FSH at ˂15 mIU/ml and the level of LH˂10 mIU/ml. When the treatments failed to induce the appearance of follicles after 3 months, the patients received treatment with 10000 IU of HCG. Results The residual dormant follicles in POI patients can be activated using our approach to obtain embryos and conceive by injection of HCG. ConclusionsPOI patients may conceive their own genetic children by activating dormant follicles in vivo. These findings may represent a new simple and feasible solution for the treatment of patients with POI to conceive their own genetic children.

ESR2 regulates indian hedgehog signaling in neonatal rat ovary

The transcriptional regulatory function of estrogen receptor β (ESR2) is essential for the regulation of primordial follicle activation (PFA). Increased PFA due to the loss of ESR2 becomes evident as early as postnatal day 8 (PND8). To identify the ESR2-regulated genes that control PFA, we performed RNA-seq analyses of wildtype, and Esr2 knockout (Esr2KO) neonatal rat ovaries collected on PND4, PND6, and PND8. Among the differentially expressed genes in Esr2KO ovaries, indian hedgehog (Ihh) displayed the highest downregulation among the ovary enriched genes. IHH regulated genes including Hhip as well as the steroidogenic enzymes were also downregulated in Esr2KO rat ovaries. Remarkably, the expression of Ihh in Esr2KO ovaries was not upregulated despite the high levels of Gdf9 and Bmp15, which are known regulators of Ihh expression in granulosa cells. Our findings suggest that indian hedgehog signaling in the neonatal rat ovary is dependent on ESR2.

Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1 and ZFX, was investigated with in-situ immunolocalisation in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signalling pathway’s role in primordial follicle activation.

P-444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4-8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections. Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /

P–444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4–8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections. Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings: We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /

O-218 The effect of mTOR activation on human primordial follicle activation during in-situ culture

Study question What is the effect of mTOR pathway activation on human primordial follicles in-situ activation and subsequent development following tissue cryopreservation? Summary answer Temporary treatment of cryopreserved human ovarian tissue with mTOR activators cause the initiation of primordial follicle development and influence steroidogenesis. What is known already In-vitro activation of primordial follicles to produce mature oocytes provides an alternative technique for fertility preservation. The employment of different stimulators of PI3K pathway has been successfully used to activate resting follicles during culture or prior to grafting in patients with premature ovarian insufficiency. The addition of phosphatidic acid (PA) and propranolol (PP), as mTOR stimulators, in the culture medium has promoted primordial follicle activation morphologically in mouse and human ovaries. Molecular and functional evaluations of primordial follicle activation after treatment with the mentioned stimulators has not been conducted. Study design, size, duration Ovarian tissues which were donated from 6 transsexual patients (23-35 years old), were dissected and cryopreserved with slow-freezing technique. After thawing, they were cut into 1x1x1 mm fragments and incubated with different stimulators in three groups: 1) Control (without stimulators), 2) PA (200µM), and 3) PA+PP (200µM & 50µM respectively). In groups two and three, ovarian fragments were cultured for 24 hours in presence of stimulators and then cultured for additional 6 days without stimulators. Participants/materials, setting, methods The cultured ovarian fragments were directly processed for Hematoxylin and Eosin staining and Western blot analysis. The proportion of morphologically normal and degenerated primordial and growing follicles and 17β-estradiol (E2) level in the culture medium were compared after 1 and 7 days of culture to assess follicular development and function. Western blot analysis for phosphorylated and non-phosphorylated status of FOXO3a and RSP6 proteins expression were compared after 24 hours of incubation. Main results and the role of chance The proportion of primordial and growing follicles were not significantly different in the experimental groups after 24 hours of incubation with either of the stimulators. Western blot analyses indicated a significant reduction of FOXO3a in the PA+PP group compared to the control group. The phosphorylation level of RPS6 protein did not significantly change in either of the groups. The proportion of transitional follicles were significantly higher in the PA group compared to other groups after 7 days of culture. The E2 secretion level was significantly higher at the last day of culture compared to day 1 for all groups. At the end of the culture period, E2 levels was significantly higher in both PA and PA+PP groups compared to the control group. Limitations, reasons for caution Due to ovarian fragmentation before culture, the HIPPO pathway downstream molecules should have also been evaluated by western blot, which was not contributed in this study. Wider implications of the findings The results demonstrate the beneficial effect of mTOR signaling to accelerate early primordial follicle recruitment in cryopreserved-thawed human ovarian fragments. Trial registration number not applicable

Current Understandings of Core Pathways for the Activation of Mammalian Primordial Follicles

The mammalian ovary has two main functions—producing mature oocytes for fertilization and secreting hormones for maintaining the ovarian endocrine functions. Both functions are vital for female reproduction. Primordial follicles are composed of flattened pre-granulosa cells and a primary oocyte, and activation of primordial follicles is the first step in follicular development and is the key factor in determining the reproductive capacity of females. The recent identification of the phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway as the key controller for follicular activation has made the study of primordial follicle activation a hot research topic in the field of reproduction. This review systematically summarizes the roles of the PI3K/PTEN signaling pathway in primordial follicle activation and discusses how the pathway interacts with various other molecular networks to control follicular activation. Studies on the activation of primordial follicles have led to the development of methods for the in vitro activation of primordial follicles as a treatment for infertility in women with premature ovarian insufficiency or poor ovarian response, and these are also discussed along with some practical applications of our current knowledge of follicular activation.