Loss Of Primordial Follicles Research Articles

Sleep Deprivation Triggers the Excessive Activation of Ovarian Primordial Follicles via β2 Adrenergic Receptor Signaling.

Sleep deprivation (SD) is observed to adversely affect the reproductive health of women. However, its precise physiological mechanisms remain largely elusive. In this study, using a mouse model of SD, it is demonstrated that SD induces the depletion of ovarian primordial follicles, a phenomenon not attributed to immune-mediated attacks or sympathetic nervous system activation. Rather, the excessive secretion of stress hormones, namely norepinephrine (NE) and epinephrine (E), by overactive adrenal glands, has emerged as a key mediator. The communication pathway mediated by the KIT ligand (KITL)-KIT between granulosa cells and oocytes plays a pivotal role in primordial follicle activation. SD heightened the levels of NE/E that stimulates the activation of the KITL-KIT/PI3K and mTOR signaling cascade in an β2 adrenergic receptor (ADRB2)-dependent manner, thereby promoting primordial follicle activation and consequent primordial follicle loss in vivo. In vitro experiments further corroborate these observations, revealing that ADRB2 upregulates KITL expression in granulosa cells via the activation of the downstream cAMP/PKA pathway. Together, these results reveal the significant involvement of ADRB2 signaling in the depletion of ovarian primordial follicles under sleep-deprived conditions. Additionally, ADRB2 antagonists are proposed for the treatment or prevention of excessive activation of primordial follicles induced by SD.

Primary oocytes with cellular senescence features are involved in ovarian aging in mice

In mammalian females, quiescent primordial follicles serve as the ovarian reserve and sustain normal ovarian function and egg production via folliculogenesis. The loss of primordial follicles causes ovarian aging. Cellular senescence, characterized by cell cycle arrest and production of the senescence-associated secretory phenotype (SASP), is associated with tissue aging. In the present study, we report that some quiescent primary oocytes in primordial follicles become senescent in adult mouse ovaries. The senescent primary oocytes share senescence markers characterized in senescent somatic cells. The senescent primary oocytes were observed in young adult mouse ovaries, remained at approximately 15% of the total primary oocytes during ovarian aging from 6 to 12 months, and accumulated in aged ovaries. Administration of a senolytic drug ABT263 to 3-month-old mice reduced the percentage of senescent primary oocytes and the transcription of the SASP factors in the ovary, in addition, led to increased numbers of primordial and total follicles and a higher rate of oocyte maturation. Our study provides experimental evidence that primary oocytes, a germline cell type that is arrested in meiosis, become senescent in adult mouse ovaries and that senescent cell clearance reduced primordial follicle loss and mitigated ovarian aging phenotypes.

Investigating the potential role of α-SNAP in preventing chemotherapy-induced ovarian dysfunction: Insights from cellular and animal models

BackgroundThe phosphoinositide 3-kinase/Akt/mammalian target of rapamycin complex 1 (PI3K/Akt/mTORC1) pathway plays a crucial role in the activation of primordial follicles. However, excessive activation and the loss of primordial follicles can lead to ovarian dysfunction. The alpha-soluble N-ethylmaleimide sensitive factor attachment protein (α-SNAP) protein has been implicated in PI3K/Akt/mTORCl signaling, suggesting its potential involvement in follicle activation. Thus, this study aimed to explore the role of α-SNAP in the activation of the PI3K/Akt/mTORC1 signaling pathway and its ability to mitigate the effects of cisplatin on ovarian function, using both in vitro and in vivo models. MethodsWe transfected KGN human ovarian granulosa cells (GCs) with small interfering RNA (siRNA) targeting α-SNAP to investigate the effects of α-SNAP inhibition on GC proliferation and apoptosis, as well as on the activity of the PI3K/Akt/mTORC1 pathway. In a mouse model, α-SNAP siRNA was delivered via an adeno-associated virus before treatment with cisplatin to assess its effects on follicle activation and ovarian function. Follicle counts at various growth stages, western blotting, and immunohistochemistry analyses were conducted to detect the expression of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR. Additionally, the serum concentrations of anti-Müllerian hormone (AMH) were measured through an enzyme-linked immunosorbent assay. ResultsIn vitro, α-SNAP depletion prevented GC proliferation by inhibiting the PI3K/Akt/mTORC1 pathway, thereby indicating its role in the regulation of cell growth. In vivo, α-SNAP knockdown attenuated the cisplatin-induced overactivation of primordial follicles by suppressing the PI3K/Akt/mTORC1 signaling pathway and partially restoring AMH levels. In addition, the expression and distribution patterns of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR varied across different follicular growth stages, suggesting a protective effect against chemotherapy-induced ovarian damage. ConclusionsInhibiting α-SNAP may attenuate GC proliferation by suppressing the PI3K/Akt/mTORC1 pathway, thereby mitigating the overactivation and loss of primordial follicles induced by cisplatin. Targeting α-SNAP may emerge as a novel strategy to prevent ovarian damage resulting from chemotherapy. However, these conclusions warrant repeated testing, and the mechanistic underpinnings of α-SNAP must be further elucidated in the future.

Trajectory of primordial follicle depletion is accelerated in obese mice in response to 7,12-dimethylbenz[a]anthracene exposure†.

Both obesity and exposure to environmental genotoxicants, such as 7,12-dimethylbenz[a]anthracene, negatively impair female reproductive health. Hyperphagic lean KK.Cg-a/a (n = 8) and obese KK.Cg-Ay/J (n = 10) mice were exposed to corn oil as vehicle control (CT) or 7,12-dimethylbenz[a]anthracene (1mg/kg/day) for 7d intraperitoneally, followed by a recovery period. Obesity increased liver and spleen weight (P < 0.05), and 7,12-dimethylbenz[a]anthracene exposure decreased uterine weight (P < 0.05) in obese mice. Primordial follicle loss (P < 0.05) caused by 7,12-dimethylbenz[a]anthracene exposure was observed in obese mice only. Primary (lean P < 0.1; obese P < 0.05) and secondary (lean P < 0.05, obese P < 0.1) follicle loss initiated by 7,12-dimethylbenz[a]anthracene exposure continued across recovery. Reduced pre-antral follicle number in lean mice (P < 0.05), regardless of 7,12-dimethylbenz[a]anthracene exposure, was evident with no effect on antral follicles or corpora lutea number. Immunofluorescence staining of DNA damage marker, γH2AX, did not indicate ongoing DNA damage but TRP53 abundance was decreased in follicles (P < 0.05) of 7,12-dimethylbenz[a]anthracene-exposed obese mice. In contrast, increased (P < 0.05) superoxide dismutase was observed in the corpora lutea of 7,12-dimethylbenz[a]anthracene-exposed obese mice and reduced (P < 0.05) TRP53 abundance was noted in preantral and antral follicles of 7,12-dimethylbenz[a]anthracene-exposed obese mice. This study indicates that obesity influences ovotoxicity caused by a genotoxicant, potentially involving accelerated primordial follicle activation and hampering normal follicular dynamics.

Mathematical recapitulation of the end stages of human ovarian aging.

Ovarian aging in women can be described as highly unpredictable within individuals but predictable across large populations. We showed previously that modeling an individual woman's ovarian reserve of primordial follicles using mathematical random walks replicates the natural pattern of growing follicles exiting the reserve. Compiling many simulations yields the observed population distribution of the age at natural menopause (ANM). Here, we have probed how stochastic control of primordial follicle loss might relate to the distribution of the preceding menopausal transition (MT), when women begin to experience menstrual cycle irregularity. We show that identical random walk model conditions produce both the reported MT distribution and the ANM distribution when thresholds are set for growing follicle availability. The MT and ANM are shown to correspond to gaps when primordial follicles fail to grow for 7 and 12 days, respectively. Modeling growing follicle supply is shown to precisely recapitulate epidemiological data and provides quantitative criteria for the MT and ANM in humans.

Sodium thiosulfate does not protect ovarian reserve from cisplatin-induced gonadotoxicity†.

Cisplatin, a platinum-containing alkylating agent, is used in the treatment of various tumors owing to its potent antitumor activity. However, it causes permanent and adverse effects, particularly hearing loss and depletion of ovarian reserve. Until recently, there were no clinically available protective agents to mitigate the adverse side effects of cisplatin-induced cytotoxicity. In 2022, sodium thiosulfate (STS) was approved by the Food and Drug Administration for mitigating hearing loss in children and adolescents undergoing cisplatin treatment. Consequently, our investigation aimed to determine if STS could protect ovarian reserve against cisplatin-induced gonadotoxicity. In an ex vivo culture, the cisplatin-only group exhibited a loss of primordial follicles, while post-STS administration after cisplatin exposure effectively protected primordial follicles. However, when post-STS was administrated either 6 or 4h after cisplatin exposure, it did not confer protection against cisplatin-induced gonadotoxicity in postnatal day 7 or adolescent mouse models. Immunofluorescence assays using γH2AX and cPARP revealed that oocytes within primordial follicles exhibited DNA damage after cisplatin exposure, irrespective of post-STS administration. This underscores the rapid and heightened sensitivity of oocytes to gonadotoxicity. In addition, oocytes demonstrated an increased expression of pCHK2 rather than pERK, suggesting that the pathway leading to oocyte death differs from the pathway observed in the inner ear cell death following cisplatin exposure. These results imply that while the administration of STS after cisplatin is highly beneficial in preventing hearing loss, it does not confer a protective effect on the ovaries in mouse models.

ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice.

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

O-273 Impact of ovarian tissue transplantation on extracellulaire matrix remodelling

Abstract Study question Does ovarian tissue transplantation affect ovarian extracellular matrix (ECM) components? Summary answer Ovarian tissue transplantation affects ECM components in a time- and spatial-dependent manner What is known already Human ovarian ECM is subjected to a specific pattern of remodeling during woman’s life. Such changes have a crucial influence on folliculogenesis. Ischemia-reperfusion injury after ovarian tissue transplantation also leads to changes in the ECM, such as the occurrence of fibrotic areas. However, there is no in-depth characterization of this remodeling yet or any study on how this change could affect the preantral follicle population, notably primordial follicle activation. Study design, size, duration Frozen-thawed human ovarian tissue was collected from reproductive- age women (N = 6). In each case, ovarian tissue was divided into 4 fragments: one for non-grafted controls (D0) and three for grafting to immunodeficient mice for 3, 7 and 21 days (D3, D7 and D21, respectively). After grafting, ovarian ECM components were investigated and compared. Participants/materials, setting, methods To assess the impact of ovarian tissue transplantation on ECM remodeling, collagen, elastin, fibrilin-1, EMILIN1, and glycosamoniglycans (GAGs) were investigated at different time points (D0, D3, D7, and D21). Both global (ECM from the entire ovarian tissue fragment) and perifollicular (ECM immediately surrounding preantral follicles) ECM were examined by histological staining and immunofluorescence. Computer-assisted quantification of histological staining and immunolabeling was carried out. Main results and the role of chance Collagen content from global ECM significantly decreased from D0 to D7 (p &amp;lt; 0.05) and D21 (p &amp;lt; 0.01). However, no difference was found in the perifollicular collagen concentration. Compared to D0, in the global ECM, thin collagen fibers decreased significantly on D3 (p &amp;lt; 0.05). On the other hand, thick collagen fibers tended to increase from D0 to D3. In the perifollicular area of primordial follicles, thin collagen fibers decreased significantly from D0 to D7 (p &amp;lt; 0.01) and D21 (p &amp;lt; 0.05), while thick fibers slightly increased from D0 to D3 and decreased significantly up to D21 (p &amp;lt; 0.01). In the global ECM, GAGs’ content did not change significantly after transplantantion. However, they significantly increased around primordial and primary follicles from D0 to D3 and D7 (p &amp;lt; 0.001) and decreased from D7 to D21 (p &amp;lt; 0.001). While elastin content did not change after transplantation in the whole tissue, it increased significantly surrounding primary follicles on D3 (p &amp;lt; 0.05) and then diminished around primordial and primary follicles from D3 to D7 and D21 (p &amp;lt; 0.001). Fibrilin-1 concentration significantly decreased at D21 (p &amp;lt; 0.05) in the whole tissue and surrounding primordial and primary follicles. EMILIN1 increased significantly at D3 (p &amp;lt; 0.01) and D7 (p &amp;lt; 0.001) and decreased significantly from D7 (p &amp;lt; 0.001) and D21 (p &amp;lt; 0.01) around preantral follicles. Limitations, reasons for caution While there is an evident remodeling of the ECM, including the one immediately around primordial and primary follicles, due to the cryopreservation and/or transplantation procedure, we cannot ascertain if it could influence folliculogenesis. For this, the assessment of follicle activation parameters (e.g., mTOR, FOXO-1, YAP) is ongoing. Wider implications of the findings At the best of our knowledge this is the first study to describe ovarian ECM remodeling after ovarian tissue transplantation. These data may open new insights for a better understanding of the phenomena occurring in the ovarian follicle microenvironment and develop new strategies to prevent primordial follicle loss after transplantation. Trial registration number Not applicable

BNIP3-mediated autophagy via the mTOR/ULK1 pathway induces primordial follicle loss after ovarian tissue transplantation.

To explore the underlying mechanism of primordial follicle loss in the early period following ovarian tissue transplantation (OTT). BNIP3 was selected through bioinformatic protocols, as the hub gene related to autophagy during OTT. BNIP3 and autophagy in mice ovarian grafts and in hypoxia-mimicking KGN cells were detected using immunohistochemistry, transmission electron microscopy (TEM), western blotting, qPCR, and fluorescence staining. The regulatory role played by BNIP3 overexpression and the silencing of KGN cells in autophagy via the mTOR/ULK1 pathway was investigated. Ultrastructure examination showed that autophagic vacuoles increased after mice ovarian auto-transplantation. The BNIP3 and autophagy-related proteins (Beclin-1, LC3B, and SQSTM1/p62) in mice ovarian granulosa cells of primordial follicle from ovarian grafts were altered compared with the control. Administration of an autophagy inhibitor in mice decreased the depletion of primordial follicles. In vitro experiments indicated that BNIP3 and autophagy activity were upregulated in KGN cells treated with cobalt chloride (CoCl2). The overexpression of BNIP3 activated autophagy, whereas the silencing of BNIP3 suppressed it and reversed the autophagy induced by CoCl2 in KGN cells. Western blotting analysis showed the inhibition of mTOR and activation of ULK1 in KGN cells treated with CoCl2 and in the overexpression of BNIP3, and the opposite results following BNIP3 silencing. The activation of mTOR reversed the autophagy induced by BNIP3 overexpression. BNIP3-induced autophagy is crucial in primordial follicle loss during OTT procedure, and BNIP3 is a potential therapeutic target for primordial follicle loss after OTT.

TAp63 determines the fate of oocytes against DNA damage.

Cyclophosphamide and doxorubicin lead to premature ovarian insufficiency as an off-target effect. However, their oocyte death pathway has been debated. Here, we clarified the precise mechanism of ovarian depletion induced by cyclophosphamide and doxorubicin. Dormant oocytes instead of activated oocytes with high PI3K activity were more sensitive to cyclophosphamide. Checkpoint kinase 2 (CHK2) inhibitor rather than GNF2 protected oocytes from cyclophosphamide and doxorubicin, as cyclophosphamide up-regulated p-CHK2 and depleted primordial follicles in Abl1 knockout mice. Contrary to previous reports, TAp63 is pivotal in cyclophosphamide and doxorubicin-induced oocyte death. Oocyte-specific Trp63 knockout mice prevented primordial follicle loss and maintained reproductive function from cyclophosphamide and doxorubicin, indicated by undetectable levels of BAX and cPARP. Here, we demonstrated that TAp63 is fundamental in determining the signaling of oocyte death against DNA damage. This study establishes the role of TAp63 as a target molecule of adjuvant therapies to protect the ovarian reserve from different classes of chemotherapy.

Association of Serum Anti-Mullerian Hormone Level with Age in Subfertile Patients

Introduction: Infertility affects approximately 10%-15% of reproductive-aged couples. Ovarian reserve describes the number of good-quality oocytes remaining within the ovaries. As a women ages, her ovarian reserve declines, principally due to apoptotic loss of primordial follicles. Serum AMH level is being considered a possible testing method for determining ovarian reserve. The aim of the study was to observe any association between serum Anti- Mullerian Hormone (AMH) level with patient age among subfertile patients. Methods: This cross-sectional observational study was conducted at the Department of Obstetrics and Gynaecology, Infertility Unit, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. The study duration was 14 months, from October 2011 to December 2012. The present study was conducted with 86 women of the reproductive age group with subfertility. Result: There was a gradual linear decline of AMH observed with an increment of age. The mean value of FSH and LH gradually increased in the higher age groups of 40-45 years than in the lower age group of 21-30 years. The mean BMI levels were almost similar in all age groups, and no remarkable difference could be discerned. The difference in AMH levels among the different age groups was statistically significant. a statistically significant negative correlation between age and serum AMH was observed, while a significant positive correlation was observed between FSH and age. Conclusion: As the age of a women advances, the AMH level decreases. The serum AMH is negatively correlated with age and serum FSH is positively correlated with age.

Early postnatal alterations in follicular stress response and survival in a mouse model of Classic Galactosemia

Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1–3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Our group uses a GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as “burnout.” These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions.

Hyperoside protects against cyclophosphamide induced ovarian damage and reduced fertility by suppressing HIF-1α/BNIP3-mediated autophagy

Ovarian damage and infertility are the main side effects of chemotherapy for women of childbearing age with cancer. The main objective of this study was to investigate the protective effects and mechanisms of hyperoside against cyclophosphamide (Cy) -induced ovarian damage and reduced fertility. This study consists of two parts: in vivo experiments using Cy intraperitoneal injections to simulate clinical chemotherapy sessions and in vitro experiments using 4-HC, a precursor of an activated form of Cy, to intervene in human granulosa-like cell line (KGN). We found that Cy disrupted the estrous cycle in mice, resulting in decreased serum Anti-Mullerian hormone (AMH) levels, loss of primordial follicles, primary follicle and secondary follicle, increased atretic follicles, and diminished ovarian reserve function. Cy prolonged the time between mating and pregnancy in mice and increased the number of absorbed embryos. Western Blot analysis demonstrate that Cy activated key proteins of HIF-1α/BNIP3-associated autophagy both in vivo and in vitro, while in vivo experiments we also found that 4-HC increased KGN cell apoptosis, damaged mitochondrial membrane potential, and activated autophagic flow. Co-treatment with hyperoside diminished follicular depletion of the primordial follicles, decreased follicular atresia, prevented Cy-induced excessive hypoxia and autophagy activation, increased mitochondrial membrane potential, thereby increasing follicular reserve and rescuing fertility in Cy-treated mice. It suggests that HIF-1α/BNIP3-mediated autophagy is an essential mechanism by which Cy impairs ovarian function and fertility in mice, by blocking this activation, hyperoside shows potential as an ovarian protectant that may be capable of preserving fertility in women undergoing chemotherapy.

Effects of chemotherapy on ovaries of pregnant mice.

It is unknown if future fertility is compromised by the administration of chemotherapy during pregnancy. The aim of this study was to identify if chemotherapy affects the maternal ovaries during pregnancy and whether these effects depend on type of chemotherapy and duration of exposure. Pregnant 8-week-old female BL6 mice were exposed to 6 different single chemotherapeutic agents (carboplatin, cisplatin, paclitaxel, epirubicin, doxorubicin, or cyclophosphamide) or saline at gestational day (GD) 13.5. The mice were sacrificed at GD 15.5 or GD 18.5. Ovaries were assessed by histopathology and immunohistochemistry. Follicle count was determined per follicle stage and per treatment modality. Maternal ovarian damage was demonstrated by the presence of apoptosis and necrosis in preantral follicles. The extent of this damage depends upon type of chemotherapy and duration of exposure (2 or 5days). After short exposure, 81% of ovaries showed histopathologic signs of damage compared to 36% after long exposure, which might suggest a transient effect. Loss of primordial follicles (PMFs) was observed after both short and long exposure, with a reduction of more than 70%. Evidence of DNA damage, as demonstrated by phospho-H2AX expression, was present in 23% (range 0-89%) of PMFs exposed to chemotherapy, but only in the short exposure group. Overall, the least damage was seen after administration of paclitaxel. Despite physiological ovarian function suppression during gestation, chemotherapy-induced damage of the ovaries occurs in pregnant mouse models, potentially affecting future fertility.

Continued exposure to D-galactose in postnatal period may inhibit excessive primordial follicle reduction in rats exposed prenatally to D-galactose.

We aimed to compare the ovarian reserve of rats exposed to oral D-galactose during prenatal and early life with rats exposed to D-galactose only during the prenatal period. Fifteen female pregnant Wistar rats were randomly divided into three groups. The first and second groups were fed a D-galactose enriched diet (35%) from the third day of pregnancy to parturition (PP)and the third day to the end of lactation (PL), respectively. The control group (C group) was fed a standard diet. The study population was the female offspring of three groups (PP', PL', and C'), in which some reproductive factors were examined between 45 and 50 days of age. When compared with the PP' group, the number of primordial follicles was significantly higher in the PL' group at PND 45-50 (40 vs. 30; p= .01); however, the antimullerian hormone level was significantly reduced in the PL' group versus control group (-2.2, 95% confidence interval [CI]: -2.83, -1.53 ng/ml p=.000), and follicle-stimulating hormone level significantly increased in PP' group versus control (4.5 mIU/ml, 95% CI: 1.40-7.62, p=.005). There was no significant difference in leukocyte infiltration or antiovarian antibody among the groups. Continued exposure to D-galactose during the lactation period inhibits the primordial follicle loss in rats in terms of producing fewer atretic follicles.

Epigallocatechin-3-gallate attenuates cyclophosphamide-induced damage in mouse ovarian tissue via suppressing inflammation, apoptosis, and expression of phosphorylated Akt, FOXO3a and rpS6

This study was conducted to evaluate the protective effects of epigallocatechin-3-gallate (EGCG) against ovarian toxicity in cyclophosphamide-treated mice and to verify the possible involvement of phosphorylated Akt, FOXO3a and rpS6 in the EGCG actions. Mice received saline solution (i.p.; control) or a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) or mice were pretreated with N-acetylcysteine (150 mg/kg body weight, i.p.; positive control) or with EGCG (5, 25 or 50 mg/kg body weight, i.p.) once daily for three days followed by injection with single dose of cyclophosphamide (200 mg/kg body weight, i.p.). Thereafter, the mice were euthanized, and the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemistry (cleaved caspase-3 and TNF-α) and fluorescence (mitochondrial activity and GSH concentrations) analyses. Furthermore, we examined the participation of p-Akt, p-FOXO3a and p-rpS6 in the protective effects of EGCG in cyclophosphamide-induced ovarian damage by immunohistochemical staining. The results showed that pretreatment with N-acetylcysteine or EGCG at 25 and 50 mg/kg before cyclophosphamide administration preserved the normal follicular morphology, prevented primordial follicle loss, reduced atresia, inflammation, and mitochondrial damage, and increased GSH concentrations compared to the only cyclophosphamide treatment. Additionally, pretreatment with 25 mg/kg EGCG regulated phosphorylated Akt, FOXO3a and rpS6 after cyclophosphamide treatment. In conclusion, short-time pretreatment with 25 mg/kg EGCG can prevent follicle loss in cyclophosphamide-treated mice by reducing oxidative damage, inflammation, and apoptosis, and regulating of p-Akt, p-FOXO3a and p-rpS6.

Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

Cyclophosphaty -45mide (Cyc) chemotherapy in young female cancer patients is associated with an increased risk of premature ovarian insufficiency (POI). This study was designed to investigate the protective role of melatonin (Mel) as an adjuvant against Cyc-induced POI. Female mice received a single intraperitoneal (i.p.) dose of Cyc (75 mg/kg). Mel protection was achieved in mice after i.p. injection of melatonin (50 mg/kg) every 24 h for four consecutive days prior to chemotherapy initiation and for 14 additional days. Ovarian reserve testing, hormonal assays for follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone (AMH), assessment of the oxidative stress status, and measurement of the relative expression of genes in PTEN/AKT/FOXO3a and mitochondrial apoptosis pathways were performed. The results showed that treatment with 50 mg/kg Mel significantly prevented Cyc-induced over-activation of primordial follicles by maintaining the plasma level of AMH and subsequently preventing litter size reduction in mice treated with Cyc chemotherapy. Importantly, Mel treatment significantly prevented ovarian granulosa cell loss by inhibiting the mitochondrial apoptotic pathway. Identifying the protective actions of Mel against Cyc-induced primordial follicle loss has important implications for fertility maintenance in young cancer patients undergoing chemotherapy.

O-265 Luteinizing hormone is able to protect reproductive health in cancer patients

Abstract Study question Is luteinizing hormone (LH) able to protect ovarian follicles against cyclophosphamide (CPM)-induced damage in women? Summary answer LH significantly reduces primordial follicle (PMF) loss in ovarian cortical strips cultured in vitro with phosphoramide mustard (PM), the active metabolite of CPM. What is known already Cancer therapies are cause of premature ovarian insufficiency (POI) in female patients, since they induce a severe reduction of PMFs. For this reason, several compounds have been analyzed as adjuvant therapies to protect the ovarian reserve without interfering with cancer treatment on tumor cells. We recently demonstrated the protective effect of LH against ovotoxicity induced by PM and cisplatin on the ovary of prepuberal and adult mice. These results suggest the possibility to use LH prior or in concomitance with anticancer drugs in order to preserve oocytes in human patients, therefore preventing the early onset of menopause and/or infertility. Study design, size, duration Ovarian cortical tissues were collected from nine patients (age ± SD: 15.33 ± 4.50) who have cryopreserved their tissue before receiving anticancer treatment. For each patient, ovarian cortical strips were thawed and randomly assigned to the experimental conditions: Control (CTRL), PM and PM+LH. LH was added 1 hour before the treatment with PM. Samples were analyzed after 8, 16, 24 and 48 hrs of treatment. Participants/materials, setting, methods Ovarian cortial strips were placed onto culture plate insert and cultured for a maximum of 48 hours, in αMEM with 10% Serum Substitute Supplement with/out 200mIU/ml LH and/or 10μM PM. The samples were processed for: - Histology, for PMFs and primary follicles (PFs) analysis; - Immunohistochemistry, for the expression of protein involved in DNA damage, apoptosis and follicle activation; - Real-Time PCR, for the expression of apoptotic and inflammation related genes. Main results and the role of chance The follicle density in the untreated group varied from 233.03 to 3420.27 PMFs/mm3 between the nine patients analysed. Relative follicular density (%) was performed to analyse statistical differences between the groups. Relative PMFs density (%) was significantly reduced in PM vs CTRL either after 24 and 48 hrs, while this reduction was significantly counteracted by LH (24 hrs: CTRL=76.19±2.12; PM = 44.95±5.06; PM+LH=73.97±11.64. 48 hrs: CTRL=66.47±3.96; PM = 36.76±3.96; PM+LH=55.74±8.72). To investigate the mechanism underlying the observed effects, the expression of markers involved in DNA damage (gH2AX), apoptosis (Cleaved caspase 3, NOXA, PUMA), follicle activation (p-AKT, FOXO3a), cell cycle arrest (p21, Ki67), and inflammation (IL1β, TNFα) were analysed. The results showed that LH did not prevent DNA damage induced by PM, since gH2AX positive oocytes were seen both in PM and PH+LH group at 16h; at 24hrs however we observed a downregulation of the gH2AX expression in the PH+LH group (PM@ 100% vs PM+LH@35%). LH also counteracts the activation of chemotherapy-induced apoptotic processes, by reducing the levels of pro-apoptotic factors such as NOXA, PUMA and CC3, and follicles activation lowering AKT-FOXO3a signaling axis. Moreover, PM treatment creates a proinflammatory microenvironment, as shown by increased IL1β and TNFα gene expression, partially counteracted by LH pretreatment. Limitations, reasons for caution Experiments are carried out in vitro; the functionality of the cortex in xenografts should be evaluated. Careful evaluation of which patients might use this protector is needed. Wider implications of the findings These findings demonstrate that LH is able to reduce PMFs loss in human ovarian cortex exposed to PM. These results encourage thinking about the use of the hormone as a ferto-protector agent in clinical trials to prevent the premature onset of menopause and/or infertility in women undergoing anticancer treatments. Trial registration number not applicable

P-471 Effect of verteporfin, an inhibitor of Hippo signaling pathway, on chemotherapy-induced follicle activation and survival of neonatal mice ovaries in vitro

Abstract Study question Does verteporfin, an inhibitor of Hippo signaling pathway, influence mechanisms involved in chemotherapy-induced follicle activation and survival in vitro? Summary answer Verteporfin acts directly on cell growth and indirectly on phosphatidylinositol-3-kinase (PI3K) signaling pathway, but does not prevent chemotherapy-induced follicle activation, although moderates 4-hydroperoxycylophosphamide (4HC) gonadotoxicity. What is known already Cryopreservation of ovarian tissue is an experimental technique aimed at prepubertal patients in need of chemotherapy. To optimize this method, in vitro culture of cryopreserved tissue is being investigated. However, this process leads to a massive loss of primordial follicles due to their spontaneous activation. Inhibition of PI3K pathway, the major signaling pathway involved in follicle activation, is moderately efficient to prevent chemotherapy-induced follicle activation. Recently, the effect of Hippo pathway disruption due to sectioning on follicle activation has been described, suggesting that verteporfin can inhibit Hippo pathway-induced follicle activation. Study design, size, duration Ovaries collected from post-natal day 3 and/or 4 (PND3/4) mice were used for this study. Whole and cut ovaries were cultured in vitro for 3 hours to validate the effect of verteporfin on Hippo pathway disruption. Whole ovaries were cultured in vitro for 24 and 48 hours to assess the impact of verteporfin and/or chemotherapy (4HC, 10 µM) exposure on primordial follicle activation and survival. At least 3 ovaries were used for each condition. Participants/materials, setting, methods The inhibitory and toxic effects of verteporfin on whole and cut ovaries were investigated by analysing the expression of genes involved in Hippo and PI3K pathways, as well as in apoptosis (RT-qPCR). The effect of co-treatment with verteporfin and 4HC on follicle activation and survival was assessed by histological analysis (immunohistochemistry and TUNEL staining), gene expression (RT-qPCR) and protein analysis (immunostaining and Western Blot) to investigate signaling pathways, cell proliferation and apoptosis. Main results and the role of chance The inhibitory effect of verteporfin on Hippo pathway disruption induced by sectioning was achieved on ovaries exposed to 3 µM of verteporfin despite slightly increasing apoptosis at 3 hours of culture. Verteporfin was able to slightly prevent the effect of chemotherapy on Hippo pathway, but its impact was limited by the time of culture. Surprisingly, verteporfin also acted indirectly on PI3K pathway by significantly decreasing the level of downstream phosphorylated proteins. However, in presence of chemotherapy, verteporfin was not sufficient to prevent the effect of 4HC on this pathway. Alone, verteporfin also significantly decreased granulosa cells proliferation, depending on the time of culture: although reducing cell growth at 24 hours of culture, this effect was not observed after 48 hours of in vitro culture. The inhibitor was not able to prevent apoptosis induced by 4HC at 24 hours of culture. However, at 48 hours of culture, the decrease in the number of positive TUNEL follicles in the co-treatment condition compared to ovaries treated with chemotherapy alone suggested a potential positive effect of verteporfin to prevent chemotherapy-induced gonadotoxicity. Limitations, reasons for caution The main limitation of this study is the experimental model used. Although mouse is a strong and reliable model, it lacks certain characteristics specific to human. Investigating other timepoints of culture and exposition, as well as different doses of verteporfin, might benefit the study. Wider implications of the findings Our study supports Hippo and PI3K pathways interaction during in vitro follicle activation. Despite verteporfin does not appear to be an inhibitor of interest, testing another Hippo inhibitor or combining Hippo and PI3K pathways inhibitors could be investigated to control follicle activation during in vitro culture of ovarian tissue. Trial registration number Not applicable

P-684 The impact of oral sphingosine-1-phosphate analogue on ovarian aging in women with multiple sclerosis

Abstract Study question Is there a protective effect of oral sphingosine-1-phosphate analogue (S1P) on ovarian reserve? Summary answer The use of oral long acting S1P analogue may reduce spontaneous primordial follicle loss and preserve ovarian reserve in women of reproductive age. What is known already The number of primordial follicles decrease by apoptosis with advancing age in women. Seramid pathway is one of the apoptosis regulatory pathways in human ovaries. An increase in the balance between ceramide and S1P in S1P direction, known as cell membrane sphingomyelins, inhibits follicular apoptosis. Fingolimod (Novartis, Germany), a long-acting oral analogue of S1P, is a drug used in the treatment of multiple sclerosis (MS). We have previously found that use of fingolimod might reduce the spontaneous follicular apoptosis and increase the number of residual primordial follicles in a rat model. Study design, size, duration A prospective cohort study. Twenty-one consecutive women in reproductive age to whom fingolimod therapy were commenced for MS at Hacettepe University, Department of Neurology in between 2018 – 2021were included. Participants/materials, setting, methods Regularly menstruating women under &amp;lt;38 years old was included. Serum Anti-Mullerian Hormone (AMH) levels were measured at the beginning of Fingolimod, 6th and 12th months of the treatment. The decrease in serum AMH levels were calculated and compared with standard AMH decrease in healthy women. Main results and the role of chance The mean±standard deviation (SD) age of the 21 patients was 26.95±6.07. The mean±SD of AMH level at the beginning was 2,62±1,64 ng/ml. The respective figure for the 6th month and 12th month were 3,10±2,02 and 1,70±1,70 ng/ml. Overall, there was an increase of 0.6 ng/ml in mean AMH during this period. AMH level decreased in 2 patients, remained unchanged in 2 patients, and increased in 5 patients. Limitations, reasons for caution High (∼50%) loss to follow-up rate is a main limitation. Wider implications of the findings Oral long-acting MS drug fingolimod may have a protective effect on ovarian reserve in human by reducing spontaneous follicle loss rate, studies with larger sample size and longer follow-up period is warranted to demonstrate this effect. Trial registration number not applicable