Atretic Follicles Research Articles

Expression of visfatin in the ovarian follicles of prepubertal and mature gilts and in vitro effect of gonadotropins, insulin, steroids, and prostaglandins on visfatin levels

Recent studies have demonstrated that visfatin participates in the regulation of female reproduction. Due to the lack of data concerning the level of visfatin in the ovarian follicles of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localisation of visfatin and the follicular fluid concentration in the ovarian follicles of prepubertal and mature gilts. We also aimed to examine the in vitro effects of gonadotropins (LH, FSH), insulin, progesterone (P4), oestradiol (E2), prostaglandin E2 (PGE2) and F2α (PGF2α) on visfatin levels. In the present study, we have demonstrated that visfatin expression is dependent on the maturity of the animals and the stage of ovarian follicle development. Visfatin signal was detected in individual follicular compartments from primordial to antral follicles and even in atretic follicles. We have shown that the expression of visfatin in granulosa cells was higher than in theca cells. The level of visfatin is upregulated by LH, FSH, E2, and P4 and downregulated by insulin, while prostaglandins have modulatory effects, dependent on the dose and type of ovarian follicular cells. To summarise, our research has shown that visfatin is widely expressed in the ovarian follicles of prepubertal and mature pigs, and its expression is regulated by gonadotropins, insulin, steroids, and prostaglandins, suggesting that visfatin appears to be an important intra‐ovarian factor that could regulate porcine ovarian follicular function.

MicroRNA-129-1-3p attenuates autophagy-dependent cell death by targeting MCU in granulosa cells of laying hens under H2O2-induced oxidative stress

The present study aimed to investigate the mechanism of microRNA-129-1-3p (miR-129-1-3p) in regulating hydrogen peroxide (H2O2)-induced autophagic death of chicken granulosa cell by targeting mitochondrial calcium uniporter (MCU). The results indicated that the exposure of hens' ovaries to H2O2 resulted in a significant elevation in reactive oxygen species (ROS) levels, as well as the apoptosis of granulosa cells and follicular atresia. This was accompanied by an upregulation of glucose-regulated protein 75 (GRP75), voltage-dependent anion-selective channel 1 (VDAC1), MCU, mitochondria fission factor (MFF), microtubule-associated protein 1 light chain 3 (LC3) I, and LC3II expression, and a downregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitofusin-2 (MFN2) expression. In hens' granulosa cells, a luciferase reporter assay confirmed that miR-129-1-3p directly regulates MCU. The induction of oxidative stress through H2O2 resulted in the activation of the permeability transition pore, an overload of calcium, depolarization of the mitochondrial membrane potential, dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAMs), and ultimately, autophagic cell death. The overexpression of miR-129-1-3p effectively mitigated these H2O2-induced changes. Furthermore, miR-129-1-3p overexpression in granulosa cells prevented the alterations induced by H2O2 in the expression of key proteins that play crucial roles in maintaining the integrity of MAMs and regulating autophagy, such as GRP75, VDAC1, MFN2, PTEN-induced kinase 1 (Pink1), and parkin RBR E3 ubiquitin-protein ligase (Parkin). Together, these in vitro- and in vivo-based experiments suggest that miR-129-1-3p protects granulosa cells from oxidative stress-induced autophagic cell death by downregulating the MCU-mediated mitochondrial autophagy. miR-129-1-3p/MCU calcium signaling pathway may act as a new target to alleviate follicular atresia caused by oxidative stress in laying hens.

Effect of long-term hypoxia on the reproductive systems of female and male yellow catfish (Pelteobagrus fulvidraco).

This study investigated the effects of different levels of hypoxia on the reproductive system of yellow catfish. Yellow catfish (Pelteobagrus fulvidraco) were exposed to three dissolved oxygen concentration levels: normoxia (6.5 ± 0.2 mg/L), moderate hypoxia (MH, 3.8 ± 0.3 mg/L) and severe hypoxia (SH, 1.9 ± 0.2 mg/L) for 30 days. The gonadosomatic index of males, not females, significantly decreased in the SH group. In the SH group, for the females, the ratio of vitellogenic follicles significantly decreased, whereas the number of atretic follicles significantly increased. In male fish, a significantly reduced number of spermatozoa was observed in both the MH and SH groups. Elevated apoptosis levels in the testes and ovaries were observed only in the SH group. Serum 17β-estradiol and vitellogenin levels in females and testosterone levels in males significantly decreased in the SH group. The concentration of 11-ketotestosterone in males significantly decreased in both the MH and SH groups. In female fish, dysregulated expression of the hypothalamic-pituitary-gonadal (HPG) axis, steroidogenesis genes, and hepatic genes related to vitellogenesis were observed only in the SH group. However, in male fish, moderate hypoxia altered the expression of HPG genes, including gnrh1, lhcgr, and amh. Moreover, the MH group significantly altered the expression of steroidogenesis genes like star, 17β-hsd, and cyp17a1. The results of this study suggest that severe hypoxia can cause reproductive defects in female and male yellow catfish. Moreover, the reproductive system of male yellow catfish is more sensitive to moderate hypoxia than that of female catfish. Our findings contribute to our understanding of the response of the teleost reproductive system to long-term hypoxia.

Aristolochic acid I exposure triggers ovarian dysfunction by activating NLRP3 inflammasome and affecting mitochondrial homeostasis.

Aristolochic acids are widely distributed in the plants of Aristolochiaceae family and Asarum species. Aristolochic acid I (AAI) is the most frequent compound of aristolochic acids, which can accumulate in the soil, and then contaminates crops and water and enters the human body. Research has shown that AAI affects the reproductive system. However, the mechanism of AAI's effects on the ovaries at the tissue level still needs to be clarified. In this research, we found AAI exposure reduced the body and ovarian growth in mice, decreased the ovarian coefficient, prevented follicular development, and increased atretic follicles. Further experiments showed that AAI upregulated nuclear factor-κB and tumor necrosis factor-α expression, activated the NOD-like receptor protein 3 inflammasome, and led to ovarian inflammation and fibrosis. AAI also affected mitochondrial complex function and the balance between mitochondrial fusion and division. Metabolomic results also showed ovarian inflammation and mitochondrial dysfunction due to AAI exposure. These disruptions reduced the oocyte developmental potential by forming abnormal microtubule organizing centers and expressing abnormal BubR1 to destroy spindle assembly. In summary, AAI exposure triggers ovarian inflammation and fibrosis, affecting the oocyte developmental potential.

Comparison the therapeutic effects of bone marrow CD144+ endothelial cells and CD146+ mesenchymal stem cells in POF rats.

Premature ovarian insufficiency (POI) is a challenging issue in terms of reproduction biology. In this study, therapeutic properties of bone marrow CD146+ mesenchymal stem cells (MSCs) and CD144+ endothelial cells (ECs) were separately investigated in rats with POI. POI rats were classified into control POI, POI + CD146+ MSCs, and POI + CD144+ ECs groups. Enriched CD146+ MSCs and CD144+ ECs were directly injected into ovarian tissue (15 × 104 cells/10 μL) in relevant groups. After 4 weeks, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were measured in blood samples. Ovarian tissues were collected and subjected to Hematoxylin-Eosin and Masson's trichrome staining. The expression of angp-2, vegfr-2, smad-2, -4, -6, and tgf-β1 was studied using qRT-PCR analysis. Histopathological examination indicated an increased pattern of atretic follicles in the POI group related to the control rats (P<0.0001). Data indicated that injection of POI + CD146+ MSCs and CD144+ ECs in POI rats reduced atretic follicles and increased the number of normal follicles (P<0.01). Along with these changes, the content of blue-colored collagen fibers was diminished after cell transplantation. Besides, cell transplantation in POI rats had the potential to reduce increased FSH, and LH levels (P<0.05). In contrast, E2 content was increased in POI + CD146+ MSCs and POI + CD144+ ECs groups compared to control POI rats, indicating restoration of follicular function. CD144+ (smad-2, and -4) and CD146+ (smad-6) cells altered the activity of genes belonging TGF-β signaling pathway. Unlike POI + CD146+ MSCs, aberrant angiogenesis properties were significantly down-regulated in POI + CD144+ ECs related to the control POI group (P<0.05). The transplantation of bone marrow CD146+ and CD144+ cells can lead to the restoration of ovarian tissue function in POI rats via modulating different mechanisms associated with angiogenesis and fibrosis.

Vertical transmission of porcine circovirus-like virus P1 in BALB/c mice

BackgroundPorcine circovirus-like virus P1 is the animal virus with the smallest genome discovered so far, and it has become widely distributed in the Chinese mainland in recent years.ResultsIn this study, a BALB/c mouse model was used to reveal P1 infection in female reproductive systems and the vertical transmission of the virus. The female reproductive system, including the ovary and uterus, was harvested on day 14 postinfection and examined for pathological lesions. One-day-old mice without colostrum born from infected or uninfected mothers were collected, and P1 virus distribution in the different organs was investigated. During the trials, all the mice showed no clinical symptoms or gross lesions. However, stillbirth did occur in groups infected with the P1 virus. P1 nucleic acid was detected in the heart, liver, spleen, lung, kidney, and brain tissues of 1-day-old mice born from infected mice. Microscopic lesions in P1-infected female mice were characterized by necrosis of the ovarian follicular granulosa cells and abscission, follicular atresia, necrosis of the endometrial epithelial and uterine glandular epithelial cells, and hyperplasia of the squamous endometrial epithelium. The spermatocytes in the seminiferous tubules of the infected male mice were disorderly arranged, and the germ and Sertoli cells were shed, necrotic, and decreased in number. Immunohistochemical results identified P1-positive particles in the nucleus and cytoplasm of cells from the ovary and uterus of female mice.ConclusionsThis study shows that the P1 virus could cause pathological damage to the reproductive system of female mice and could be transmitted vertically.

Ovarian Histology and Histopathology of Olive Barb, Puntius sarana Exposed to Endocrine Disrupting Chemical (17-Α Methyl Testosterone) in Laboratory Condition

17-α methyl testosterone is a major endocrine disrupting chemical (EDCs), reported harmful effect on endocrine system of aquatic animals. To study the impact of 17-αMT on olive barb (Puntius sarana) gonad, sixty fishes were collected for in vitro experiment. Hormonal treatments were carried out 90 days with three distinct hormonal dosages (T1:40 mg/kg; T2:60 mg/kg and T3:90 mg/kg) mixed with pelleted feed and no hormone in control tank (C). Each 30 days intervals, histological analysis was performed to observe alterations in the gonad due to the effect of 17α-MT. Four gamete developmental stages of female P. sarana ovary were identified through microscopic examination named immature, previtellogenic, vitellogenic and mature stage. Mild to moderate histological abnormalities has been observed in ovary for different treatments. T1 showed apparently normal histology except some alterations like empty follicle (ef), lipid droplet (ld), vitelline envelop breakdown (veb), but pronounced alterations were detected at higher concentration T2 and T3, increasing with longer exposures for instance degenerated nucleus (dn), atretic follicle (af), thickened vitelline envelop (tve), residual oocyte (roc). This preliminary study revealed more profound responses to EDCs exposure in the olive barbs which could serve as a model bio-indicator in ecotoxicological study.

Rubus chingii Hu relieved the polycystic ovary syndrome with enhanced insulin sensitivity through inhibiting TXNIP/NLRP3 inflammasome signaling

Background Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders in gynecology with severe metabolic abnormalities. Therefore, identifying effective treatments and drugs for PCOS is important. We aimed to investigate effect of the traditional Chinese medicine (TCM) Rubus chingii Hu (R. chingii) on ovarian function and insulin resistance (IR) of PCOS rat models, and to explore the underlying mechanisms Methods A PCOS rat model was established by subcutaneous injection of dehydroepiandrosterone (DHEA) solution for 20 days. PCOS rats were randomly divided into a control group (CON), model group (MOD), metformin group (MET), TCM R. chingii group (RCG), and RCG + Ad-TXNIP groups. After 28 days of treatment, the samples were collected for subsequent experiments. Results R. chingii treatment alleviated hormone imbalance and IR while improving ovarian pathology in the PCOS model. R. chingi inhibited the activation of the thioredoxin-interacting protein (TXNIP)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in the ovarian tissue of PCOS rats. Furthermore, TXNIP overexpression hindered the protective effect of R. chingii intervention in PCOS rats, as evidenced by the increase of homeostasis model assessment of insulin resistance (HOMA-IR), luteinizing hormone (LH), testosterone (T), C-reactive protein (CRP) levels, and atretic follicles. Conclusion R. chingii intervention improved ovarian polycystic development by suppressing the TXNIP/NLRP3 inflammasome, which may be an effective treatment for PCOS.

FGF12 regulates cell cycle gene expression and promotes follicular granulosa cell proliferation through ERK phosphorylation in geese

The granulosa cells play an important role in the fate of follicular development or atresia in poultry. Fibroblast growth factor 12 (FGF12) is downregulated in atretic follicles and may be involved in regulating granulosa cell survival in previous studies, but its molecular mechanism remains unclear. In this study, FGF12 overexpression and knockdown models of goose granulosa cells were constructed to investigate its function. The downstream expression of the cell cycle pathway was analyzed by qPCR. Granulosa cell proliferative activity and apoptosis were detected by CCK8 and TUNEL. Protein phosphorylation levels of ERK and AKT were measured using Western blotting to analyze the key pathway of FGF12 regulation of granulosa cell proliferation. ERK protein phosphorylation inhibitor was added for further verification. After overexpression of FGF12, cell proliferation activity was increased, the expressions of cell cycle pathway genes CCND1, CCNA2, MAD2, and CHK1 were upregulated, the apoptosis of granulosa cell was decreased, and Caspase 3 gene and protein expression were downregulated. After the knockdown of FGF12, cell proliferation activity decreased, the expression of downstream genes in the cell cycle pathway was downregulated, the apoptosis of granulosa cells was increased, and the Bcl-2 gene and protein were downregulated. Overexpression of FGF12 promoted the synthesis of P4 and upregulates the expression of the STAR gene. Overexpression of FGF12 promoted ERK protein phosphorylation but did not affect AKT phosphorylation. The addition of ERK phosphorylation inhibitors resulted in the elimination of the increase in cell proliferative activity caused by FGF12 overexpression. In conclusion, FGF12 could promote proliferation and inhibit apoptosis of goose granulosa cells by increasing ERK phosphorylation.

Berberine protects cyclophosphamide and busulfan-induced premature ovarian insufficiency in mouse model

Premature ovarian insufficiency (POI) is a clinical syndrome that declines ovarian function in women. Berberine (BBR) is a compound with anti-inflammatory, antioxidant, and anti-apoptotic activities. However, the role of BBR on POI is still unknown. In this study, we investigated the role of BBR on ovarian function decline by establishing a POI mouse model using cyclophosphamide (CTX) and busulfan (BU). Our results showed that POI was attenuated by BBR, which was evidenced by enhanced body weight and ovarian weight, improved morphology of ovary, increased the number of healthy follicles, decreased the production of atretic follicles and restored serum hormone levels, including estradiol, anti-Müllerian hormone and follicle-stimulating hormone. In addition, we showed that germ cell function markers, mouse vasa homologue (MVH) and octamer-binding transcription factor 4 (OCT4) were enhanced by BBR, at both protein and mRNA levels. Furthermore, our results revealed that BBR inhibited inflammation and oxidative stress by reducing nuclear factor kappa B (NF-κB) and enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. Taken together, we demonstrate that BBR can effectively improve ovarian function in POI mice, which is mainly mediated by reducing oxidative stress and inflammatory response. Our study also provides new strategy for POI treatment.

Ovarian Histology of Removed Ovaries After Gender-Affirming Testosterone Therapy.

Introduction. Assigned female at birth transgender people go through a gender-affirming hormone therapy using testosterone. We aimed to define the histological changes in the removed ovaries of these patients and investigate the correlation of these changes to factors like chronological age and duration of hormone therapy. Methods. The ovaries of 84 patients who had at least 6 months of testosterone therapy before surgery were examined. Tunica albuginea thickness, cortical thickness, and number of different stages of follicles were recorded. Results. The mean age was 27.2 ± 4.9 years. Testosterone duration 25.8 ± 13.1 months. The mean tunica albuginea thickness was 356.4 ± 152.6 µm. The mean cortical thickness was 799.6 ± 245.6 µm. The number of primordial (C1) follicles was 18.03 ± 13.6 and antral (C3) follicles was 3.1 ± 1.9 per cm². When grouped as using therapy under or over 2 years the groups did not have differences in histological findings. Hormone duration did not correlate with histological findings except for a positive correlation with atretic follicle number. However, age was negatively correlated with number of follicles at all stages except atretic follicles and positively correlated with cortical thickness (P < .05). Conclusion. Testosterone therapy induces multifollicularity, stromal hyperplasia, and luteinization in some patients. Hormone duration did not correlate with ovarian histology whereas chronological age did suggesting an effect of age on ovarian reserve rather than duration of hormone therapy.

Treatment of porcine ovarian follicles with tert-butyl hydroperoxide as an ovarian senescence model in vitro.

Ovarian aging is the main reason of female reproductive problems. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing the reproductive performance. Follicles were divided into five groups for in vitro culture based on the duration of stimulation with tert-butyl hydroperoxide (t-BHP)-control group and groups 1 h, 2 h, 6 h, and 12 h. The results revealed that the ratio of progesterone (P4) to estradiol (E2) was increased after 24 and 36 h of follicle culture, shifting follicles toward atresia (P < 0.05). Stimulated by 200 μM t-BHP, follicles showed progressive aging phenotype. Senescence-associated β-galactosidase staining (SA-β-Gal) showed a significant increase in the number of positive cells (P < 0.05). Reactive oxygen species were also significantly upregulated (P < 0.05). t-BHP treatment for 6 h induced significant increases in Caspase 3, P53, and Foxo1 mRNA and protein levels (P < 0.05) and significant decreases in SOD mRNA and protein levels (P < 0.05). Transcriptome sequencing analysis of the follicles showed that the aged and treatment groups were clustered together in hierarchical clustering. Correlation analysis indicated significant changes at the transcriptome level in the treatment groups versus the control group. The common differentially expressed genes in the treatment groups were enriched in three growth-factor signaling pathways associated with cell proliferation and apoptosis (P53, mTOR, and MAPK). In conclusion, induction of follicular senescence by treatment with 200 μM t-BHP for 6 h is an effective in vitro model to simulate ovarian senescence in sows.

Investigation of the Mechanisms and Experimental Verification of Yulin Formula in the Treatment of Diminished Ovarian Reserve via Network Pharmacology.

The aim of this study is to examine, using network pharmacology analysis and experimental validation, the pharmacological processes by which Yulin Formula (YLF) reduces cyclophosphamide-induced diminished ovarian reserve (DOR). First, information about the active components, associated targets, and related genes of YLF and DOR was gathered from open-access databases. The primary targets and pathways of YLF to reduce DOR were predicted using studies of functional enrichment from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Protein-Protein Interaction (PPI) networks. Second, we built a cyclophosphamide-induced diminished ovarian reserve (DOR) rat model to verify the primary target proteins implicated in the predicted signaling pathway to explore the mechanism of improve ovarian function of YLF. 98 targets met the targets of the 82 active ingredients in YLF and DOR after searching the intersection of the active ingredient targets and DOR targets. Fourteen targets, including AKT and Caspase-3 among others, were hub targets, according to the PPI network study. The PI3K/AKT pathway was revealed to be enriched by numerous targets by the GO and KEGG enrichment studies, and it was used as a target for in vivo validation. Animal studies showed that YLF administration not only reduced the number of atretic follicles, the proportion of TUNEL-positive ovarian cells, the rate of apoptosis of granulosa cells (GCs) and the proportion of abnormal mitochondria in DOR rats, but also reversed the high expression of Caspase-3, Caspase-9, BAX, cytochrome C, PI3K and P-AKT, improving the ovarian reserve in cyclophosphamide (CTX)-induced DOR rats. Our research results predicted the active ingredients and potential targets of YLF-interfering DOR by an integrated network pharmacology approach, and experimentally validated some key target proteins participated in the predicted signaling pathway. A more comprehensive understanding of the pharmacological mechanism of YLF for DOR treatment was obtained.

Amniotic fluid-derived exosomes attenuated fibrotic changes in POI rats through modulation of the TGF-β/Smads signaling pathway

In the current study, we investigated the regenerative effects of amniotic fluid exosomes (AF-Exos) in a rat model for premature ovarian insufficiency (POI). POI is a condition characterized by a decrease in ovarian function that can lead to infertility. We induced POI by administering cyclophosphamide (CTX) for 15 consecutive days, and then transplanted AF-Exos directly into both ovarian tissues. Four weeks later, we measured the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2), and performed histopathological evaluations using H & E and Masson’s trichrome staining. We also monitored the expression of genes related to the TGF-β signaling pathway using real-time PCR and examined the fertility rate of POI rats after AF-Exos therapy. Histological analysis showed an increase in atretic follicles and a decrease in healthy follicle count after POI induction. Four weeks post-AF-Exos intervention, the healthy follicle count increased (p < 0.01) while the atretic follicle count decreased (p < 0.001). In parallel, the deposition of collagen fibers also decreased following AF-Exos transplantation. The concentrations of FSH and LH hormones in sera remained unchanged after injection of AF-Exos, while E2 levels increased (p < 0.05). The expression of Smad-4 (p < 0.01) and Smad-6 (p < 0.05) was upregulated in POI rats that received AF-Exos, while Smad-2, TGF-β1, TNF-α, and IL-10 remained statistically unchanged. Our records showed a notable increase in litter number after AF-Exos compared to the non-treated POI rats. These results suggest that AF-Exos transplantation has the potential to restore ovarian function through the TGF-β/Smads signaling pathway in POI rats.

P-426 In vitro and in vivo toxicity of carboplatin and paclitaxel regimen on BRCA-mutated ovarian tissue fragments

Abstract Study question Does carboplatin and paclitaxel regimen have a toxic cumulative impact on the ovarian reserve of young breast cancer patients with germline Breast Cancer (BRCA) mutation? Summary answer Based on follicle classification and survival through apoptosis and DNA repair mechanism recruitment analyses, gonadotoxic but no cumulative effect was observed in BRCA-mutated ovarian tissue. What is known already Ovarian function recovery and fertility issues remain major concerns for young patients diagnosed with cancer. Breast cancer is the first cancer diagnosed among women under 40 years and around 10% of them are BRCA germline mutation carriers. Beside their predisposition to breast and ovarian cancers, BRCA carriers might be more sensitive to chemotherapy-induced gonadotoxicity due to the role of BRCA in DNA repair mechanism. Current therapies for BRCA-mutated breast cancer patients often include carboplatin and paclitaxel, drugs that are both considered as moderately gonadotoxic. However, the effect of combined therapy in BRCA-mutated ovarian tissue is unknown. Study design, size, duration Ovarian tissue cryopreserved for fertility preservation before chemotherapy and donated for research by breast cancer patients with or without BRCA1/2 mutation (&amp;lt; 35 years at diagnosis) were used in this study. Two models were investigated: 3 days in vitro culture with carboplatin (10 µg/mL) and/or paclitaxel (1 µM) and ovarian tissue xenograft into mice followed by 3 weeks injections of carboplatin (50mg/kg/week) and paclitaxel (10mg/kg every 3 days). Control conditions were performed for each model. Participants/materials, setting, methods Ovarian tissue from 3 BRCA-mutated and 3 non-mutated patients were thawed and exposed in vitro and in vivo to chemotherapy. Follicle density (number of follicles/mm²) as well as ratios of quiescent/activated and healthy/atretic follicles were evaluated following haematoxylin-eosin staining. Apoptosis and DNA repair mechanisms were analysed using TUNEL/GDF9 co-staining and phosphorylated H2A histone family member X (γH2AX)/ZP-3 co-staining, respectively. Genes expression level will be evaluated on isolated follicles to assess the activation process. Main results and the role of chance First, the impact of in vitro culture and in vivo xenotransplantation was evaluated by follicle counting and classification. A decrease of follicular density (5 to 95% irrespective of patient, model, nor condition) and an increase of activated and atretic follicles was observed after both in vitro and in vivo experiments in all conditions. However, a slight increase of quiescent follicle pool was observed in the in vitro control condition, with no difference between mutated and non-mutated fragments. Then, immunofluorescence staining was performed to evaluate follicle damage. For quantification, 30 to 150 follicles per patient were counted within each condition and classified as positive if at least one cell was stained. Apoptosis was assessed with TUNEL/GDF9 co-staining. In both models, the ratio of positive follicles was higher in fragments treated with chemotherapy (40-60%) compared to control conditions (15-20%). Recruitment of DNA repair complex was analysed using γH2AX/ZP3 co-staining. In both models, an increase in positive follicles ratio was observed in treated fragments (15-40%) compared to control conditions (5-10%). Interestingly, the positive follicles ratio was lower in BRCA-mutated (15-18%) than in non-mutated fragments (25-40%) after 3-days in vitro culture, irrespective of the treatment used. Limitations, reasons for caution Reasons of caution include the high inter-variations between patients as well as intra-variations in one patient regarding follicular density within ovarian tissue. Moreover, BRCA-mutated patients included (27.67 ± 2.08) are younger than control ones (33.67 ± 1.53). Ongoing experiments with additional patients should support the results already obtained. Wider implications of the findings Carboplatin and paclitaxel combined exposure in vitro or in vivo does not seem to have a cumulative deleterious impact on ovarian reserve. Ovarian tissue with BRCA-mutation did not appear to be more sensitive to chemotherapy exposure. Further investigations focusing on DNA repair mechanisms will be performed in both models. Trial registration number Not applicable

P-637 pseudo-targeted metabolomics of follicular fluid reveals ovarian GPD1L-mediated glycerophospholipid metabolism dysfunction in patients with biochemical premature ovarian insufficiency

Abstract Study question Does the metabolic profile of follicular fluid from patients with biochemical premature ovarian insufficiency (bPOI) differ from that of healthy women? Summary answer The metabolic signature of follicular fluid reveals a significant decrease of phosphatidic acid (PA), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) in bPOI patients. What is known already Metabolites in follicular fluid have attracted extensive attention as they are important components of the microenvironment during follicular development and oocyte maturation. Studies on follicular fluid metabolomics may enhance the understanding of the pathogenesis of ovarian aging, particularly premature ovarian insufficiency. Although a variety of metabolites have been suggested to be related to cell senescence, their specific roles in regulating follicular atresia remain unclear. Study design, size, duration An observational study of follicular fluid samples collected from 120 women undergoing in vitro fertilization and embryo transfer was conducted. All enrolled women were classified into two groups: one with 60 bPOI patients and the other with 60 healthy controls. Each group was further subdivided into two subgroups (young and aged group, 30 women for each section). Participants/materials, setting, methods The follicular fluids of mature follicles (diameter over 17 millimeters) were isolated and collected for pseudo-targeted metabolic analysis. The intensities of captured metabolites were utilized for further bioinformatic analysis. The correlation between the significant differential metabolites screened and their clinical outcomes were analyzed. In vitro experiments were used to demonstrate the cellular function of mainly regulated signaling pathway. Main results and the role of chance A total of 608 metabolites were identified in 120 follicular fluid samples. There were significant differences in 143 metabolites between the bPOI and healthy controls. As revealed by metabolic pathway analysis, glycerophospholipid metabolism was the most enriched pathway in bPOI follicular fluids. The intensities of several glycerophospholipid-associated metabolites PA, PC, lysoPC and lysoPE were positively correlated with the ovarian reserve indicator anti-Müllerian hormone (AMH), female age, as well as the expression level of glycerol-3-phosphatedehydrogenase 1-like (GPD1L), a key enzyme of this pathway, in granulosa cells. Furthermore, knockdown of GPD1L induced mitochondria dysfunction and granulosa cell apoptosis via GPD1L/PKCε/ATF2/NEAT1 pathway, as well as led to follicular atresia and impaired the quality of oocytes. Limitations, reasons for caution The validity of differential intensified metabolites we detected as biomarkers for clinical diagnosis needs to be confirmed in a large-scale study. And the multiple effects of glycerophospholipid metabolic pathway posed on follicular development need to be study in-depth. Wider implications of the findings Our results suggested that a highly coordinated follicular metabolism exhibits a strong relationship with the quality of follicles. An understanding of the mechanisms underlying ovarian aging will provide a theoretical basis, as well as new ideas for POI diagnosis, treatment and intervention. Trial registration number Not applicable.

P-466 The effect of SARS-CoV-2 infection or vaccination on controlled ovarian stimulation and in vitro fertilization: A multicenter retrospective cohort study

Abstract Study question Are the ovarian response to controlled ovarian stimulation (COS) and embryo development after in vitro fertilization (IVF) affected by SARS-CoV-2 infection and immunization? Summary answer Both SARS-CoV-2 infection and SARS-CoV-2 mRNA or inactivated vaccinations do not affect ovarian responsiveness to COS or embryo development following IVF. What is known already SARS-CoV-2 infects the target cell by binding to angiotensin-converting enzyme 2 (ACE2) via its surface spike protein. ACE2 receptors are present in the vagina, uterus, fallopian tubes, and ovaries of the female reproductive system. They regulate ovarian steroidogenesis, follicular development, oocyte maturation, and follicle atresia. The virus downregulates ACE2 levels, resulting in decreased conversion of Angiotensin II (ANGII) to Ang (1-7) and increased accumulation of (ANGII). This may impair reproductive fertility. However, initial studies conclude that both SARS-CoV-2 infection and vaccination would not affect reproductive outcomes, but it is necessary to develop new studies that confirm their applicability. Study design, size, duration A retrospective multicentric cohort study of 428 patients who underwent COS for oocyte donation between January 1st, 2018 and July 31th, 2022 were screened for eligibility and followed up to September 18th, 2022. Oocyte donation cycles were utilized because this model is an excellent choice for controlling known female characteristics Participants/materials, setting, methods Eligibility criteria were donors between 18 to 32 years. Patients were categorized into the vaccinated group if they had received SARS-CoV-2 vaccines (BNT162b2 mRNA Pfizer-Biontech, mRNA-1273 Moderna or inactivated SARS-CoV-2 vaccine Sinovac). The control group was chosen from medical records before March 2020 ensure they were neither infected or vaccinated. Demographic, cycle characteristics, and laboratory outcomes were compared. Normally, distributed data were compared across groups by univariate ANOVA. All P-values were considered significant at &amp;lt; 0.05. Main results and the role of chance In total, 428 patients were included in the study, with 12 recovering from SARS–CoV-2 infection (Group 1), 92 reporting vaccination (Group 2), and 324 patients who underwent COS before March 2020 assigned to the non-exposed group (Group 3). The mean time from recovery to oocyte aspiration was 122 days (range 39-315 days) and 213 days from the first vaccine dose to oocyte aspiration (range 17–249 days). The study's participants had an average age of 24.8 years. Age was found to be normally distributed and did not differ significantly between the three groups (P-value 0,953). Similarly, ovarian reserve markers, as measured by AFC and AMH, did not differ between groups (P-values of 0,787 and 0,423 respectively). No significant differences were observed regarding stimulation duration and total gonadotropin dose. The number of retrieved oocytes, MII oocytes, fertilized oocytes, and good-quality day 5 embryos were also similar between the three groups. Comparing oocyte retrieval rate, mature oocyte rate, normal fertilization rate, and blastocyst formation rate consistently revealed no statistically significant differences. Limitations, reasons for caution The main limitation is its retrospective design. Other limitations include the small sample size of recovered patients, the absence of sperm analyses, and data of live birth rates and neonatal outcomes. Wider implications of the findings SARS-CoV-2 infection and vaccination seem unrelated to detrimental effects on ovarian response or embryo development. These findings contribute to the growing body evidence that supports vaccination in women trying to conceive. Further prospective studies with larger cohort sizes and longer follow-ups are warranted to validate our conclusion. Trial registration number NCT05562479

P-430 Characterization of secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue

Abstract Study question Can we obtain secondary follicles from cultured cryopreserved-thawed human cortical tissue? Summary answer We obtained a comparable percentage of secondary follicles after culture of cryopreserved-thawed human cortical tissue to that reported after culture of fresh human cortical tissue. What is known already The complete in vitro maturation of oocytes starting from primary oocytes (present in unilaminar follicles) until mature MII oocytes has been achieved previously by two different multi-step culture protocols. These protocols were applied to culture fresh ovarian cortical tissue from adult cisgender donors. However, the efficiency of these two protocols for growing unilaminar follicles to secondary follicles starting from cryopreserved cortical tissue from cisgender patients has not been investigated. As the ovarian tissue available for fertility preservation is cryopreserved, it is important to understand the potential of cryopreserved unilaminar follicles to mature in vitro. Study design, size, duration Cryopreserved ovarian cortical tissue from 4 cisgender adult donors was used for in vitro culture. According to the existing culture protocols, cortical ovarian tissue was cultured either for 8 days using the first step medium (Telfer’s medium) reported by M.McLaughlin 2018 (doi: 10.1093/molehr/gay002) or for 7 and 21 days using the first step medium (Xu’s medium) reported by Xu 2021 (doi: 10.1093/humrep/deab003). Participants/materials, setting, methods Ovarian cortical tissue obtained from adult cisgender donors undergoing oophorectomy for fertility preservation purposes was cryopreserved before chemotherapy. The cryopreserved ovarian cortical tissue was thawed and cut into small pieces. Several pieces were fixed immediately (day 0), the others were either cultured in Telfer’s medium or Xu’s medium. After culture, follicle survival, growth and morphology were assessed by histology and immunofluorescence. Main results and the role of chance We performed quantification of the different follicular stages (primordial, primary, secondary and atretic) after culture and observed that the percentage of secondary follicles increased independently of the culture media used. However, the ovarian cortical tissue cultured using Telfer’s protocol resulted in a higher percentage of secondary follicles and lower percentage of atretic follicles compared to that using Xu’s protocol. After culture, the ovarian cortical tissue was further immunostained for TUNEL, PCNA, COLLIV, STAR, AMH and KRT19. We observed that secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium showed more proliferative (PCNA+) FOXL2+ granulosa cells and less apoptotic (TUNEL+) stromal cells. By contrast, ovarian cortical tissue cultured in Xu’s medium showed less proliferative (PCNA+) FOXL2+ granulosa cells and more apoptotic (TUNEL+) stromal cells. Moreover, the expression of AMH was high in granulosa cells of secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium and low in Xu’s medium. Finally, in both Telfer’s and Xu’s medium cultures the expression of KRT19 was low in granulosa cells of secondary follicles present in ovarian cortical tissue. Limitations, reasons for caution The number of donors was limited and the study lacked fresh ovarian cortical tissue as control. Only the first step in Telfers’ and Xu’s protocol was performed, hence further culture is required to determine whether complete maturation of oocytes in vitro is possible starting from cryopreserved human cortical tissue. Wider implications of the findings Our study showed evidence of follicular growth in cryopreserved-thawed human ovarian cortical tissue after a period of in vitro culture. This is an important first step to achieve in vitro maturation of oocytes from cryopreserved-thawed human ovarian cortical tissue that could be used for clinical applications. Trial registration number NOT APPLICABLE

P-408 The ovarian tissues outside of the radiation field could be damaged by radiation-induced abscopal effects in mice: an experimental study.

Abstract Study question Could irradiation-induced abscopal effects (RIAEs) induce structural and functional damage in the distal ovary in female mice, and what is its mechanism? Summary answer The RIAEs can induce an inflammatory reaction and inhibit the growth and development of ovarian follicles in mice leading to a decrease in ovarian reserve. What is known already The irradiated tumor could induce cell and tissue injury in the organs distant from the radiation site, referred to as the radiation-induced abscopal effects (RIAEs). Many pieces of research have shown out-of-field tumor regression effects of radiation therapy. However, RIAEs is a double-edged sword and may cause serious side effects on normal tissues. Premature ovarian insufficiency (POI) is one of the main side effects of radiotherapy, but the relationship between RIAE and POI has not been clarified. Study design, size, duration C57BL/6J mice were used to establish a RIAEs model by irradiating the thorax and the abdomens were shielded. Sixteen C57BL/6J female mice were randomly allocated to sham and experimental groups (n = 8/group) according to the presence or absence of irradiation with an 8 Gy X-ray on the local area of the chest every day for three days. After irradiation for twenty-one days, The effect and possible mechanism of RIAEs on non-irradiated ovarian were discussed. Participants/materials, setting, methods After irradiation, the estrous cyclicity, serum steroid hormones, and pro-inflammatory factors were compared between groups. Furthermore, RNA-seq was used to detect the expression of transcriptional levels in ovarian tissues in both groups. The differentially expressed genes (DEGs) were screened and analyzed by gene ontology-biological process (GO_BP) between irradiated and sham groups. The expression and localization of spermatogenesis-and oogenesis-specific basic helix-loop-helix-containing protein 1(SOHLH1) and neutrophil elastase (NE) in ovarian tissues were detected by immunohistochemistry(IHC). Main results and the role of chance Compared with mice in the sham group, the irradiation group had disordered estrous cycles, reduced primordial follicles (P&amp;lt;0.001)] and growing follicles (P&amp;lt;0.001), significantly increased atretic follicles (P&amp;lt;0.001). Levels of serum estradiol [(70.28 ± 5.27) pmol/L] and AMH [(104.00±6.98) ng/L] in the irradiation group were significantly lower than those in the sham group [estradiol (97.58±7.25) pmol/L, P=0.016; AMH [(129.70±8.39) ng/mL, P=0.046], but FSH levels in the irradiation group were not significantly different from those in the sham group (P=0.996). Compared with the sham group, Serum levels of TNF-α [(488.30±36.20) ng/L vs. (31.61±12.89) ng/L, P&amp;lt;0.001] and IL-1β [(62.37±2.50) ng/L vs. (52.75±2.06) ng/L, P=0.018)] in the irradiation group were significantly increased. Serum levels of IL-6 in the irradiation group were also increased compared with the sham group, but the difference was not statistically significant (P=0.301). The results of GO_BP analysis showed that down-regulated DGEs were mainly involved in the process of follicular development, and up-regulated DGEs were involved in the inflammation process. The IHC results showed that the positive expression area of SOHLH1 in the irradiation group was significantly lower than in the sham group (P=0.005). In comparison, the positive expression area of NE was significantly higher than that of the Sham group(P=0.024). Limitations, reasons for caution This study was performed using a short, 3-day irradiation treatment condition. Therefore, the results need to be cautiously interpreted concerning radiation-associated chronic toxicity. Moreover, further studies in mouse tumor models are required to determine the clinical relevance of the role of RIAE. Wider implications of the findings Although the precise molecular mechanisms of these distal effects on the genital system are still unclear, the current discovery might pave the way for establishing novel fertility preservation protocols. The research offers hope to women with POI related to cancer therapy through the modulation of abscopal effects associated with radiotherapy. Trial registration number not applicable

P-422 Ovarian tissue vitrification as a low-technology and cost-effective protocol for female fertility preservation: a bovine study

Abstract Study question Which are the potential effects of vitrification on ovarian tissue, isolated follicles and short-term cultured follicles? Summary answer Vitrification-warming protocol using ethylene glycol as a cryoprotectant causes no severe damage to the ovarian tissue or the isolated follicles. What is known already Ovarian tissue cryopreservation (OTC) is used for fertility preservation (the only possible approach applicable for prepubertal girls). To date, more than 200 babies have been born from cryopreserved ovarian tissue. However, a slow-freezing protocol has been used most of the time, and only four babies have been born using the vitrification protocol. This suggests that current vitrification protocols may have an unknown effect on ovarian tissue and the developing follicles. Identifying the effects that vitrification may have on ovarian tissue will make vitrification for OTC more available to everyone while reducing high costs and new equipment requirements. Study design, size, duration We obtained 19 bovine ovaries from the local slaughterhouse and processed further by separating the medulla from the cortex. Hematoxylin and Eosin stain (H&amp;E) was then used to calculate the follicle pool. Ovarian tissue pieces, where follicles were not identified, were excluded from the study. Participants/materials, setting, methods Ovarian cortical tissue pieces were fixed in Bouin’s solution and 4% paraformaldehyde and were used for morphological analysis using H&amp;E, Ki67 immunofluorescence and TUNEL assay. Moreover, bulk RNA sequencing was used, and ovarian pieces with RIN&amp;gt;7 were selected for library preparation. Finally, fresh and vitrified ovarian tissue pieces were used for follicle isolation. The isolated follicles were cultured for up to 6 days, followed by a viability test performed after every two days. Main results and the role of chance Vitrification and subsequent warming of the ovarian tissue showed no significant effect on the total population of follicles compared with the fresh tissue. However, from bulk RNA sequencing, we observed 165 differentially expressed genes (DEGs) in vitrified tissue (VT) compared with the fresh tissue (FT). Vitrified cultured (VTC) pieces showed a significant decrease in monolayered and antral follicles. Moreover, a significant increase was observed in the population of atretic follicles in the fresh cultured (FTC) and VTC pieces compared with their non-cultured tissue pieces. Also, culturing fresh and vitrified tissue pieces for six days increased the proliferation of ovarian stromal cells and showed the presence of DNA damage in the stromal cells of both study groups. The RNA sequencing analysis yielded 1,042 and 1,191 DEGs in FTC and VTC pieces, respectively, compared with their uncultured control groups. Furthermore, approximately 30% of the isolated follicles (152) from vitrified tissue were viable after isolation compared with the number of viable follicles isolated from the fresh tissue (525). The isolated follicles showed no changes in their viability rate for up to 4 days. We used a Two-way ANOVA with multiple comparisons to analyse the collected data. Limitations, reasons for caution The study's main limitation was the lack of possibility to use the slow freezing protocol to compare the effect between the slow freezing and vitrification methods. Wider implications of the findings The vitrification and warming of bovine ovarian tissue did not show significant changes in ovarian tissue morphology. The follicle isolation process and subsequent culturing indicate that the vitrification method could be a way to preserve ovarian tissue and its follicles. However, culturing the tissue pieces affects their transcriptomic profile. Trial registration number N/A