Neonatal Ovaries Research Articles

The effect of bisphenol A on the Notch (Notch2 and Jagged2) signaling pathway in the follicular development of the neonatal rat ovary

ABSTRACT The formation of primordial follicles determines the pool size of follicles in the ovary, and is crucial for female reproductivity. Oocyte nest breakdown, and the formation of primordial follicles, largely depend upon the communication between oocytes and the surrounding pregranulosa cells. The neurogenic locus notch homolog protein (Notch) signaling pathway is the key player for this cell-to-cell communication, and is responsible for primordial folliculogenesis. However, different endocrine disruptors, including bisphenol A (BPA; a plasticizer and a constituent of reusable plastic containers) may affect the Notch signaling pathway, and might induce ovary dysfunction via Notch signaling. Consequently, we investigated the possible influence of BPA treatment on the proportional distribution of the follicular stages, follicle numbers, levels of apoptosis, and on Notch2 and Jagged2 expressions in the ovary. BPA was administered at doses of either 50 µg/kg/day or 50 mg/kg/day, at different time intervals, during neonatal and fetal periods in vivo. After collecting the ovaries from the various experimental groups, follicles were counted, and frequency of apoptosis was determined by TUNEL assay. In addition, Notch2 and Jagged2 expressions were assessed by immunohistochemical staining and qPCR. In summary, BPA treatment affected the follicle numbers and apoptosis level, and Notch2 and Jagged2 expressions varied with follicular stage. It was also observed that these parameters were dose and time dependent with respect to BPA exposure.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and β1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

Perinatal bisphenol S exposure exacerbates the oxidative burden and apoptosis in neonatal ovaries by suppressing the mTOR/autophagy axis

Bisphenol S (BPS) is an emerging environmental endocrine disruptor capable of crossing the placental barrier, resulting in widespread exposure to pregnant women due to its extensive usage. However, the impact of perinatal maternal exposure to BPS on reproductive health in offspring and the underlying molecular mechanism remain underexplored. In this study, gestational ICR mice were provided with drinking water containing 3.33 mg/L BPS to mimic possible human exposure in some countries. Results demonstrated that BPS accelerated the breakdown of germ-cell cysts and the assembly of primordial follicles in neonates, leading to oocyte over-loss. Furthermore, the expression levels of folliculogenesis-related genes (Kit, Nobox, Gdf9, Sohlh2, Kitl, Bmp15, Lhx8, Figla, and Tgfb1) decreased, thus compromising oocyte quality and disrupting early folliculogenesis dynamics. BPS also disrupted other aspects of offspring reproduction, including advancing puberty onset, disrupting the estrus cycle, and impairing fertility. Further investigation found that BPS exposure inhibited the activities and expression levels of antioxidant-related enzymes in neonatal ovaries, leading to the substantial accumulation of MDA and ROS. The increased oxidative burden exacerbated the intracellular apoptotic signaling, manifested by increased expression levels of pro-apoptotic markers (Bax, Caspase 3, and Caspase 9) and decreased expression levels of anti-apoptotic marker (Bcl2). Concurrently, BPS inhibited autophagy by increasing p-mTOR/mTOR and decreasing p-ULK1/ULK1, subsequently down-regulating autophagy flux-related biomarkers (LC3b/LC3a and Beclin-1) and impeding the degradation of autophagy substrate p62. However, the imbalanced crosstalk between autophagy, apoptosis and oxidative stress homeostasis was restored after rapamycin treatment. Collectively, the findings demonstrated that BPS exposure induced reproductive disorders in offspring by perturbing the mTOR/autophagy axis, and such autophagic dysfunction exacerbated redox imbalance and promoted excessive apoptosis. These results provide novel mechanistic insights into the role of autophagy in mitigating BPS-induced intergenerational reproductive dysfunction.

The Impacts of Periconceptional Alcohol on Neonatal Ovaries and Subsequent Adult Fertility in the Rat.

Maternal exposures during pregnancy can impact the establishment of the ovarian reserve in offspring, the lifetime supply of germ cells that determine a woman's reproductive lifespan. However, despite alcohol consumption being common in women of reproductive age, the impact of prenatal alcohol on ovarian development is rarely investigated. This study used an established rat model of periconceptional ethanol exposure (PCEtOH; 12.5% v/v ethanol) for 4 days prior to 4 days post-conception. Ovaries were collected from neonates (day 3 and day 10), and genes with protein products involved in regulating the ovarian reserve analyzed by qPCR. Adult offspring had estrous cycles monitored and breeding performance assessed. PCEtOH resulted in subtle changes in expression of genes regulating apoptosis at postnatal day (PN) 3, whilst those involved in regulating growth and recruitment of primordial follicles were dysregulated at PN10 in neonatal ovaries. Despite these gene expression changes, there were no significant impacts on breeding performance in adulthood, nor on F2-generation growth or survival. This contributes additional evidence to suggest that a moderate level of alcohol consumption exclusively around conception, when a woman is often unaware of her pregnancy, does not substantially impact the fertility of her female offspring.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Cyanidin-3-O-glucoside Mitigates the Ovarian Defect Induced by Zearalenone via p53-GADD45a Signaling during Primordial Follicle Assembly.

Zearalenone (ZEN) is well known as a kind of endocrine disruptor whose exposure is capable of causing reproductive toxicity in animals. Cyanidin-3-O-glucoside (C3G) is a derivative of cyanidin and owns multiple biofunctions, and prior efforts have suggested that C3G has therapeutic actions for reproductive diseases. In this article, a ZEN exposure model during primordial follicle assembly was constructed using the in vitro culture platform of neonatal mouse ovaries. We investigated the protective effect of C3G on ZEN-induced ovarian toxicity during primordial follicle assembly in mice, as well as its potential mechanism. Interestingly, we observed that C3G could effectively protect the ovary from ZEN damage, mainly by restoring primordial follicle assembly, which upregulated the expression of LHX8 and SOHLH1 proteins and relieved ZEN-induced DNA damage. Next, to explore the mechanism by which C3G rescued ZEN-induced injury, we performed RNA sequencing (RNA-seq). The bioinformatic analysis illustrated that the rescue pathway of C3G was associated with p53-Gadd45a signaling and cell cycle. Then, western blotting and flow cytometry results revealed that C3G restored the expression levels of cyclin-dependent kinase 6 (CDK6) and cyclin D2 (CCND2) and regulated the ovarian cell cycle to normal. In conclusion, our findings manifested that C3G could alleviate ZEN-induced primordial follicle assembly impairment by restoring the cell cycle involved in p53-GADD45a signaling.

The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation.

MicroRNA-338-3p helps regulate ovarian function by affecting granulosa cell function and early follicular development

BackgroundFollicular development in mammalian ovaries is a complex and dynamic process, and the interactions and regulatory-feedback loop between the follicular microenvironment, granulosa cells (GCs), and oocytes can affect follicular development and normal ovary functions. Abnormalities in any part of the process may cause abnormal follicular development, resulting in infertility. Hence, exploring the pathogenesis of abnormal follicular development is extremely important for diagnosing and treating infertile women.MethodsRNA sequencing was performed with ovarian cortical tissues established in vitro. In situ-hybridization assays were performed to study microRNA-338-3p (miR-338-3p) expressed in GCs and oocytes. In vitro culture models were established with GCs and neonatal mouse ovaries to study the biological effects of miR-338-3p. We also performed in vivo experiments by injecting adeno-associated virus vectors that drive miR-338-3p overexpression into the mouse ovarian bursae.ResultsSequencing analysis showed that miR-338-3p was expressed at significantly higher levels in ovarian cortical tissues derived from patients with ovarian insufficiency than in cortical tissues derived from patients with normal ovarian function; miR-338-3p was also significantly highly expressed in the GCs of patients with diminished ovarian reserve (P < 0.05). In situ-hybridization assays revealed that miR-338-3p was expressed in the cytoplasm of GCs and oocytes. Using in vitro culture models of granulosa cells, we found that miR-338-3p overexpression significantly suppressed the proliferation and oestradiol-production capacity of GCs (P < 0.05). In vitro culture models of neonatal mouse ovaries indicated that miR-338-3p overexpression suppressed the early follicular development in mouse ovaries. Further analysis revealed that miR-338-3p might be involved in transforming growth factor β-dependent regulation of granulosa cell proliferation and, thus, early follicular development. Injecting miR-338-3p-overexpression vectors into the mouse ovarian bursae showed that miR-338-3p down-regulated the oocyte mitochondrial membrane potential in mice and disrupted mouse oestrous cycles.ConclusionmiR-338-3p can affect early follicular development and normal ovary functions by interfering with the proliferation and oestradiol production of GCs. We systematically elucidated the regulatory effect of miR-338-3p on follicular development and the underlying mechanism, which can inspire new studies on the diagnosis and treatment of diseases associated with follicular development abnormalities.

Maternal exposure to cetylpyridinium chloride impairs oogenesis by causing mitochondria disorder in neonates

Cetylpyridinium Chloride (CPC) is a common disinfectant with potential mitochondrial toxicity. However, the effects of CPC on female reproduction remains unclear. In the present study, pregnant mice were exposed to environmentally relevant doses of CPC for 3 days, the effects were evaluated in the female offspring. Maternal exposure to CPC caused loss of oocytes in neonatal ovaries. TEM analysis of neonatal ovaries showed CPC caused aberrant mitochondrial morphology including vacuolated and disorganized structure, reduced functional cristae. In addition, CPC decreased mitochondrial membrane potential in neonatal oocytes. Seahorse analysis showed that CPC hampered mitochondrial reserve, manifested as reduced spare respiratory capacity. Furthermore, CPC damaged mitochondrial function and impaired developmental competence of MII oocytes, suggesting a persisting impact into adulthood. In summary, this is the first known demonstration that maternal exposure to CPC caused mitochondrial disorders in neonatal ovaries and had long-term effects on fertility of the female offspring.

Determination of the ovine ovarian reserve during the prenatal and neonatal periods

Objective: To determine the ovine ovarian histomorphology and follicular staging at various age periods in Awassi breed. Methods: Ovaries were collected from prenatal fetuses [gestational age (95±5) days], neonatal (day 0), and prepubertal ewe lambs (two and four months of age); each age group included six animals. Ovaries (n=12, each group) were dissected and processed for hematoxylin and eosin staining. Stained sections (n=24, each group) were imaged and utilized for histomorphology assessment, follicle measurement, and classification. Results: Prenatal ovaries were mainly enriched with primordial follicles accompanied by a lower proportion of primary follicles. In addition to primordial and primary follicles, neonatal ovaries demonstrated a proportion of centrally located multilayered and antral follicles. In comparison with neonatal ovaries, the proportion of multilayered and antral follicles was significantly higher in the ovaries of two-month-old lambs; conversely, the proportion of peripherally situated primordial follicles dramatically declined compared to that of earlier age of lamb. Although there was no statistical variation in the sizes of primordial follicles across groups, the mean diameter of the primary follicle in the prenatal ovaries was substantially smaller than in postnatal ovaries. Compared to the neonatal ovaries, the size of the multilayered and antral follicles in the prepubertal ovaries was substantially larger. Conclusions: The earliest follicular developmental stages were established prenatally whereas the advanced growth stages started in the neonatal period and greatly increased in the prepubertal period.

In vitro exposure to benzo[a]pyrene damages the developing mouse ovary.

Females are born with a finite number of oocytes, collectively termed the ovarian reserve, established within the developing fetal ovary. Consequently, maternal exposure to reproductive toxicants can have harmful effects on the future fertility of her unborn female fetus. The chemical benzo[a]pyrene (B[a]P) is a prominent component of cigarette smoke. Despite it being a known ovotoxicant, around 8% of women in Europe smoke during pregnancy. The purpose of this research was to examine the effect of B[a]P on the developing ovary, using the mouse as a model and with experiments carried out in vitro. B[a]P-exposure to the fetal ovary prior to follicle formation reduced the number of germ cells and subsequently, the number of healthy primordial follicles, by up to 76%; however, while proliferation of germ cells was not affected, the germ cells contained higher levels of DNA double-strand breaks. Exposure to B[a]P also affected the proportion of oocytes progressing through prophase I of meiosis. B[a]P exposure to neonatal mouse ovaries, after follicle formation, resulted in an 85% reduction in the number of healthy follicles, with a corresponding increase in apoptotic cell death and reduction in somatic cell proliferation. Although there was a trend towards a higher level of oxidative stress in B[a]P-exposed ovaries, this was not statistically significant; likewise, the antioxidant melatonin failed to protect against the B[a]P-induced ovarian damage. Together, the results here demonstrate that B[a]P-exposure damages the developing ovary, both before and shortly after follicle formation, an effect that could lead to a subsequent decrease in fertility.

CAMP controls the balance between dormancy and activation of primordial follicles in mouse ovaries.

In mammalian ovaries, the balance between dormancy and activation of primordial follicles determines the female fecundity and endocrine homeostasis. Recently, several functional molecules and pathways have been reported to be involved in the activation of primordial follicles. However, the homeostasis regulatory mechanisms of primordial follicle activation are still scant. Our previous study has proved that a relatively higher concentration of cyclic AMP (cAMP) is required for primordial follicle formation. Here, we identified that cAMP also plays a vital role in the balance between dormancy and activation of primordial follicles. Our results showed that the concentration of cAMP remained stable in neonatal mouse ovaries, which is due to ADCY3, the synthetase of cAMP, and PDE3A, the hydrolytic enzyme of cAMP, were synchronously increased during the activation of primordial follicles in mouse ovaries. Once the concentration of cAMP in neonatal ovaries was either elevated or reduced in vitro, the activation of primordial follicles was either accelerated or decelerated accordingly. In addition, a higher concentration of cAMP in the ovaries of puberty mice improved primordial follicle activation in vivo. Finally, cAMP promoted primordial follicle activation via canonical mTORC1-PI3K signaling cascades and PKA signaling. In conclusion, our findings reveal that the concentration of cAMP acts as a key regulator in balancing the dormancy and activation of primordial follicles in the mouse ovary.

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility

BackgroundHigh-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown.MethodsWe investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments.ResultsIn HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age.ConclusionsThe depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

In vivo promotion of primordial follicle activation by stem cell factor treatment in mice with premature ovarian insufficiency and advanced age.

Dormant primordial follicles (PFs) are the most abundant reproductive resource in mammalian ovaries. With advances in the mechanism of study of the regulation of PF activation, PFs have been used to improve fertility in clinical practice. As a central controlling element of follicle activation signaling, the pre-granulosa cell-secreted stem cell factor (SCF; also known as KIT ligand, KITL), which initiates the growth of dormant oocytes, is an ideal natural activator that stimulates follicle activation. However, no systematic study has been conducted to identify the activating effect of SCF in vivo and in vitro. In this study, by combining an in vitro whole ovary culture system and several mouse models, we provide a series of experimental evidence that SCF is an efficient activator for improving PF activation in mouse ovaries. Our in vitro study showed that SCF increased phosphatidylinositol 3-kinase (PI3K) signaling and PF activation ratio in neonatal ovaries. In vivo ovarian non-invasive topical administrations of SCF to the ovaries efficiently improved follicle activation and development, oocyte retrieval ratio and fertility in inducible premature ovarian insufficiency mouse models and aged mice. Our study suggests that SCF is an efficient growth factor that can be applied to improve PF activation.

Perfluorooctanoic acid (PFOA) promotes follicular growth and alters expression of genes that regulate the cell cycle and the Hippo pathway in cultured neonatal mouse ovaries

Perfluorooctanoic acid (PFOA) is a synthetic chemical resistant to biodegradation and is environmentally persistent. PFOA is found in many consumer products and is a major source of water contamination. While PFOA has been identified as a contaminant of concern for reproductive health, little is known about the effects of PFOA on ovarian follicular development and growth. Recent evidence indicates that the Hippo pathway is an important regulator of ovarian physiology. Here, we investigated the effects of PFOA on ovarian folliculogenesis during the neonatal period of development and potential impacts on the Hippo signaling pathway. Post-natal day 4 (PND4) neonatal ovaries from CD-1 mice were cultured with control medium (DMSO <0.01% final concentration) or PFOA (50 μM or 100 μM). After 96 h, ovaries were collected for histological analysis of folliculogenesis, gene and protein expression, and immunostaining. Results revealed that PFOA (50 μM) increased the number of secondary follicles, which was accompanied by increases in mRNA transcripts and protein of marker of proliferation marker Ki67 with no impacts on apoptosis markers Bax, Bcl2, or cleaved caspase-3. PFOA treatment (50 μM and 100 μM) stimulated an upregulation of transcripts for cell cycle regulators Ccna2, Ccnb2, Ccne1, Ccnd1, Ccnd2, and Ccnd3. PFOA also increased abundance of transcripts of Hippo pathway components Mst1/2, Lats1, Mob1b, Yap1, and Taz, as well as downstream Hippo pathway targets Areg, Amotl2, and Cyr61, although it decreased transcripts for anti-apoptotic Birc5. Inhibition of the Hippo pathway effector YAP1 with Verteporfin resulted in the attenuation of PFOA-induced follicular growth and proliferation. Together, these findings suggest that occupationally relevant levels of PFOA (50 μM) can stimulate follicular activation in neonatal ovaries potentially through activation of the Hippo pathway.

Dysfunctional Ovarian Stem Cells Due to Neonatal Endocrine Disruption Result in PCOS and Ovarian Insufficiency in Adult Mice.

Polycystic ovarian syndrome (PCOS) is a common global cause of anovulatory infertility but underlying etiology leading to PCOS still remains elusive. Fetal and perinatal endocrine disruption reportedly affects germ cell nests (GCN) breakdown, meiosis, and primordial follicle (PF) assembly with unassembled oocytes in neonatal ovaries. We recently reported that very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) express ERα, ERβ and FSHR, undergo distinct cyclic changes and neo-oogenesis encompassing GCN formation, meiosis, and primordial follicle (PF) assembly on regular basis in adult mice ovaries and these GCN are arrested in pre-meiotic or early meiotic stage in aged ovaries. Present study was undertaken to evaluate whether neonatal exposure to endocrine disruption (estradiol E2 or diethylstilbestrol DES) affects ovarian stem cells and their differentiation (neo-oogenesis) and PF assembly in adult 100 days old ovaries. Neonatal exposure to E2 resulted in typical features of PCOS including hyperandrogenism, infertility, increased stromal compartment, absent corpus lutea, and cystic follicles whereas DES treated ovaries showed rapid recruitment of follicles in young ovaries and multi-ovular/cystic follicles. Ovary surface epithelial cells smears showed large numbers of growth-arrested GCN in zygotene/pachytene with increased expression of Mlh-1 and Scp-1 suggesting defects at synapsis and recombination stages during prophase-1 of meiosis. Being immortal and expression of ERα and ERβ makes VSELs directly vulnerable to carry developmental endocrine insults to adult life. Dysfunction of VSELs/OSCs possibly results in oocyte defects observedin our study in PCOS/POI besides the widely reported defects in granulosa cells.

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-603 Presence of an asymmetrical response to ovarian stimulation in patients with low ovarian reserve

Abstract Study question Does ovarian reserve decline with a symmetrical manner between right and left ovaries in poor responders (POR) with diminished ovarian reserve (DOR)? Summary answer Asymmetrical ovarian response to ovarian stimulation with the left-side dominance was found in POR with DOR. What is known already Ovarian follicles are produced during fetal stage and not regenerated after birth. Thus, the number of ovarian follicles declines with age, resulting in infertile POR with DOR. In the morphometric study of human neonatal ovaries, no significant difference was found in the number of follicles between the right and left ovaries in the same individual. A previous study demonstrated that there is a difference in the number of follicles between right and left ovaries in patients with normal ovarian reserve with the right-side dominance, suggesting the asymmetrical activation and growth of follicles. Study design, size, duration A retrospective analysis was conducted in patients with POR with DOR based on the Bologna Criteria. Inclusion criteria was patients who received more than five times of ovarian stimulations followed by oocyte retrievals. Data were obtained from a total of 265 participants who received IVF-ET treatments from April 2015 to March 2021 after receiving written informed consents under an approval from the institutional ethical committee. Patients with the history of previous ovarian surgery were excluded. Participants/materials, setting, methods The enrolled patients were received ovarian simulation under short or GnRH antagonist protocols for oocyte retrieval. We collected the data of retrieved oocyte number as well as the outcome of IVF from medical chart. We defined the right-left asymmetry of ovarian reserve (%) based on the number of retrieved oocytes from dominant side ovary per total number of retrieved oocytes. Statistical significance was determined using Dunnett or chi-square tests, with P &amp;lt; 0.05 being statistically significant. Main results and the role of chance The average age of participants was 37.2±5.99 years of age exhibiting low serum AMH levels (average 0.09±0.20 ng/ml). We analyzed 2,181 cycles of ovarian stimulation (average 8.3±3.9 cycles/patient). The number of retrieved oocytes were 3, 882 in total cycles (average 12.8±7.1/patient). Among participants, 22 cases (8.4%) showed left and right equal in the number of retrieved oocytes, whereas &amp;gt;70% asymmetry was observed in 107 cases (40.7%) and &amp;gt;80% asymmetry was detected in 60 cases (22.8%). In 18 cases (6.9%), oocytes were collected from one side ovary only showing 100% asymmetry. In the cases with &amp;gt;70 and 100% asymmetry, the left-side dominance was 1.3-fold and 5.0-fold higher than right-side dominance, respectively. In cases with 100% asymmetry, there was no difference in the number of cryopreserved high-quality embryos between left and right sides of ovary. Limitations, reasons for caution Although we enrolled POR with DOR patients who received ovarian stimulations more than five times, the duration of ovarian stimulation was different among patients. It affects the numbers of ovarian stimulation cycles and retrieved oocytes in each patient. Wider implications of the findings Considering the finding of right-side dominance in the number of follicles with normal ovarian reserve, the activation and development of follicles might be accelerated in the right side due to asymmetric blood supply to the ovaries, and thus follicles are likely remained in the left-side ovary with low ovarian reserve. Trial registration number not applicable

Single-cell transcriptomics of staged oocytes and somatic cells reveal novel regulators of follicle activation.

In briefProper development of ovarian follicles, comprised of an oocyte and surrounding somatic cells, is essential to support female fertility and endocrine health. Here, we describe a method to isolate single oocytes and somatic cells from the earliest stage follicles, called primordial follicles, and we characterize signals that drive their activation.Primordial follicles are the first class of follicles formed in the mammalian ovary and are comprised of an oocyte surrounded by a layer of squamous pre-granulosa cells. This developmental class remains in a non-growing state until individual follicles activate to initiate folliculogenesis. What regulates the timing of follicle activation and the upstream signals that govern these processes are major unanswered questions in ovarian biology. This is partly due to the paucity of data on staged follicle cells since isolating and manipulating individual oocytes and somatic cells from early follicle stages are challenging. To date, most studies on isolated primordial follicles have been conducted on cells collected from animal-age- or oocyte size-specific samples, which encompass multiple follicular stages. Here, we report a method for collecting primordial follicles and their associated oocytes and somatic cells from neonatal murine ovaries using liberase, DNase I, and Accutase. This methodology allows for the identification and collection of follicles immediately post-activation enabling unprecedented interrogation of the primordial-to-primary follicle transition. Molecular profiling by single-cell RNA sequencing revealed that processes including organelle disassembly and cadherin binding were enriched in oocytes and somatic cells as they transitioned from primordial to the primary follicle stage. Furthermore, targets including WNT4, TGFB1, FOXO3, and a network of transcription factors were identified in the transitioning oocytes and somatic cells as potential upstream regulators that collectively may drive follicle activation. Taken together, we have developed a more precise characterization and selection method for studying staged-follicle cells, revealing several novel regulators of early folliculogenesis.

Fetal exposure of Aristolochic Acid I undermines ovarian reserve by disturbing primordial folliculogenesis

The primordial follicle pool established in early life determines the ovarian reserve in the female reproductive lifespan. Premature exhaustion of primordial follicles contributes to primary ovarian insufficiency (POI), that is dependent by the initial size of the primordial follicle pool and by the rate of its activation and depletion. AAI, a powerful nephrotoxin with carcinogenic potential, is present in the Aristolochiaceae species, which can release AAI into soil as a persistent pollutant. In order to assess the potential risk of Aristolochic Acid I (AAI) exposure on mammalian oogenesis, we uncovered its adverse effect on primordial folliculogenesis in the neonatal mouse ovary and its effect on female fertility in adulthood. Pregnant mice were orally administrated with doses of AAI without hepatic or renal toxicity during late-gestation. Ovaries from offspring of administered female displayed gross aberrations during primordial folliculogenesis. Also, unenclosed oocytes in germ-cell cysts showed increased DNA damage. Furthermore, several key factors, including NANOS3, SOX9, KLF4, that govern early gonad’s differentiation were abnormally expressed in the exposed ovary, while the follicle formation was partially restored by knockdown of Nanos3 or sox9. In adulthood, these aberrations evolved into a significant reduction in offspring number and impaired ovarian reserve. Together, our results show that AAI influences primordial folliculogenesis and, importantly, affected female fertility. This study shows that administration of drugs herbs or consumption of vegetables that contain AAs during pregnancy may adversely influence the fertility of offspring.