Follicle Growth Research Articles

Multiomic meta-analysis suggests a correlation between steroid hormone-related genes and litter size in goats.

Litter size is a key indicator of production performance in livestock. However, its genetic basis in goats remains poorly understood. In this work, a genome-wide selection sweep analysis (GWSA) on 100 published goat genomes with different litter rates was performed for the first time to identify candidate genes related to kidding rate. This analysis was combined with the public RNA-sequencing data of ovary tissues (follicular phase) from high- and low-yielding goats. A total of 2278 genes were identified by GWSA. Most of these genes were enriched in signaling pathways related to ovarian follicle development and hormone secretion. Moreover, 208 differentially expressed genes between groups were obtained from the ovaries of goats with different litter sizes. These genes were substantially enriched in the cholesterol and steroid synthesis signaling pathways. Meanwhile, the weighted gene co-expression network was used to perform modular analysis of differentially expressed genes. The results showed that seven modules were reconstructed, of which one module showed a very strong correlation with litter size (r = -0.51 and p-value <0.001). There were 51 genes in this module, and 39 hub genes were screened by Pearson's correlation coefficient between core genes > 0.4, correlation coefficient between module members > 0.80 and intra-module connectivity ≥5. Finally, based on the results of GWSA and hub gene Venn analysis, seven key genes (ACSS2, HECW2, KDR, LHCGR, NAMPT, PTGFR and TFPI) were found to be associated with steroid synthesis and follicle growth development. This work contributes to understanding of the genetic basis of goat litter size and provides theoretical support for goat molecular breeding.

Study of Sonographic Indicators of Ovarian Reserves in Women With WHO-Defined Anovulatory Disorders.

Depending on the age of the wife or female partner, infertility is defined as the inability of a couple to achieve conception after 12 months if the age is ≤35 years and six months if the age is ≥35 years. About half of female infertility is attributed to ovulatory dysfunction, and the rest is either due to tubal and pelvic pathology or problems such as thyroid disease or anatomic abnormalities. Ovulatory disorders are one of the major factors for infertility problems in couples. Ultrasound monitoring of ovarian follicle growth plays a pivotal role in infertility treatment. The total number of small antral follicles in both ovaries is an important predictive parameter of the ovarian induction cycle, whereas ovarian volume is the parameter most easily assessed by ultrasound. The purpose of this study is to analyze the sonographic indicators of infertility (antral follicle count and ovarian volume) in four World Health Organization (WHO)-defined anovulatory groups and compare the same with that of healthy fertile women.

Three-Dimensionally Printed Agarose Micromold Supports Scaffold-Free Mouse Ex Vivo Follicle Growth, Ovulation, and Luteinization.

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery.

Engineered Exosomes Biopotentiated Hydrogel Promote Hair Follicle Growth via Reprogramming the Perifollicular Microenvironment.

Androgenetic alopecia (AGA) is a highly prevalent condition in contemporary society. The conventional treatment of minoxidil tincture is hindered by issues such as skin irritation caused by ethanol, non-specific accumulation in hair follicles, and short retention due to its liquid form. Herein, we have developed a novel minoxidil-incorporated engineered exosomes biopotentiated hydrogel (Gel@MNs) that has the capability to modulate the perifollicular microenvironment for the treatment of AGA. Leveraging the exceptional skin penetration abilities of flexible liposomes and the targeting properties of exosomes, the encapsulated minoxidil can be effectively delivered to the hair follicles. In comparison to free minoxidil, Gel@MNs demonstrated accelerated hair regeneration in an AGA mouse model without causing significant skin irritation. This was evidenced by an increase in both the number and size of hair follicles within the dermal layer, enhanced capillary formation surrounding the follicles, and the regulation of the transition of hair follicle cells from the telogen phase to the anagen growth phase. Therefore, this safe and microenvironment-modifying hybrid exosome-embedded hydrogel shows promising potential for clinical treatment of AGA.

Quantitative label-free proteomic analysis of mouse ovarian antral follicles following oral exposure to a human-relevant mixture of three phthalates.

Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human-relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/d; PHT 32) and occupationally exposed (500 µg/kg/d; PHT 500) populations for 10 d. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; whereas those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women.

Shh Gene Regulates the Proliferation and Apoptosis of Dermal Papilla Cells to Affect Its Differential Expression in Secondary Hair Follicle Growth Cycle of Cashmere Goats.

Sonic hedgehog (Shh) is a component of the Hedgehog signaling pathway, playing an important role in regulating cell proliferation, differentiation, apoptosis, and the repair of damaged organisms. To further clarify the expression pattern of Shh gene in the secondary hair follicle growth cycle of cashmere goats and its mechanism of action on secondary hair follicle papilla cells, and improve cashmere quality, in this study, we took Inner Mongolia Albas white cashmere goats as the research objects and collected skin samples at different growth stages to obtain secondary hair follicles, detected Shh and its gene expression by RT-qPCR, Western blot, immunohistochemistry, and other techniques, while we also cultured DPCs in vitro. Shh gene overexpression and interference vectors were constructed, and the effects of Shh gene on the proliferation and apoptosis of DPCs were studied through cell transfection technology. The results showed that there are significant differences in Shh and its gene expression in the secondary hair follicle growth cycle skins of cashmere goats, with the highest expression level in anagen, followed by catagen, and the lowest expression level in telogen. Shh was mainly expressed in the inner root sheath, outer root sheath, and secondary hair follicle papilla. After the overexpression of Shh gene, the proliferation and vitality of the hair papilla cells were enhanced compared to the interference group. After Shh gene interference, the apoptosis rate of the cells increased, indicating that Shh gene can regulate downstream Ptch, Smo, and Gli2 gene expression to promote the proliferation of DPCs, and thus form its expression pattern in the secondary hair follicle growth cycle of cashmere goats.

Tiny messengers, big results: A review of exosome-mediated treatments and considerations in dermatology

Exosomes are small extracellular vesicles that play an important role in intercellular communication by transporting proteins, lipids, and nucleic acids between cells. They have emerged as relevant research areas due to their involvement in regulating various physiological and pathological processes. This review explores the potential applications of exosome-based therapies in dermatology and examines the safety aspects of these treatments. Past research has demonstrated that exosomes may effectively treat conditions such as alopecia and accelerate wound healing by stimulating hair follicle growth and enhancing tissue regeneration. Studies have shown that exosomes can promote the proliferation of dermal papilla cells and hair follicle growth in cases of alopecia. They also accelerate wound healing by modulating processes involved in inflammation, cell migration, and tissue remodeling. However, more research is needed to fully characterize the long-term safety profile of exosomes and establish standardized clinical protocols. Both human-derived and plant-derived exosomes appear to have favorable safety profiles based on current evidence, though plant sources may offer advantages in terms of production and biocompatibility. Continued exploration of exosomes&amp;rsquo; mechanisms and potential risks will optimize these innovations and offer safe, effective exosome treatments to patients. While further research is warranted, current findings provide valuable insights into the applications of exosome therapy for dermatological conditions and its emerging role in precision medicine.

P-380 Improvement of follicle growth and health during dynamic culture of bovine ovarian cortical tissue (BOCT) is associated to a higher remodeling of the extracellular matrix

Abstract Study question Is improvement of follicle growth and health during dynamic culture in Perifusion Bioreactor (PB) associated to a healthier condition and remodeling of extracellular matrix? Summary answer The dynamic culture in PB allows the extracellular matrix (ECM) network rearrangement improving follicle growth and viability compared to static culture What is known already Over the past two decades, in vitro folliculogenesis has achieved moderate success in large mammals, emerging as a distinctive and reliable method for obtaining secondary follicles. Recently, although dynamic culture in bioreactors has been demonstrated to enhance follicle progression, quality, and viability compared to static culture, less emphasis has been devoted to ECM remodeling. Ovarian tissue ECM plays a crucial role in cell–cell, and cell-ECM interactions involved in follicle activation and growth. The comprehension of ECM function deepens our understanding of follicular development and holds significant promise for future reproductive technologies Study design, size, duration Bovine ovaries (age 8-24 months) were collected at slaughterhouse. BOCT strips (1x1x0.5mm) from the same ovary were cultured in groups of 10 for 14 days in PB (dynamic culture) and conventional dishes (CD, static culture). At the end of culture, collagen thickness and packing density was assessed through PicroSirius red (PSR) staining, follicle stages and quality through histology, and follicle viability through live-dead far-red and propidium iodide labeling under confocal microscopy Participants/materials, setting, methods Neosynthesis and remodeling of collagen fibers was analyzed on PSR-stained samples under polarized light measuring the color of each pixel. A color threshold was set to isolate the four colors seen in PSR-stained samples under polarized light: green, thin fibers (neosynthesized); yellow, mid-sized fibers (low assembly); orange, mid-sized fibers (medium assembly); red, mature thick fibers (high assembly). The color threshold was set as follows: red 2–9, yellow 39-51, orange 10–38, green 52–128 Main results and the role of chance In fresh tissue, analysis of PSR-stained samples showed the following values: Red 1.35; Orange 4.93; Yellow 0.18; Green 0.15. At Day 14, compared to the static culture, culture in PB, exhibits a significantly higher production of green (PB 0.22 vs CD 0.02 P &amp;lt; 0.01) and yellow (PB 0.56 vs CD 0.26 P &amp;lt; 0.05) fibers indicating an improved neosynthesis and remodeling of collagen and ECM health condition. Such higher ECM remodeling after dynamic culture is associated to a significantly higher percentage of secondary follicles (PB 15.4– CD 4.1%, P &amp;lt; 0.01 ), superior follicle quality (grade I/II, PB 70,5 - CD 28.4%, P &amp;lt; 0.01) and viability (PB 70 – CD 41.1%, P &amp;lt; 0.01). PSR analysis suggests that the continuous nutrients and oxygen supply, catabolites removal, and biomechanical stimulation during dynamic PB culture, stimulates the deposition of newly synthesized collagen which in turn, promotes follicle growth Limitations, reasons for caution Although the bovine is a suitable reproductive model for human, such findings should be replicated on human ovarian tissue Wider implications of the findings The aim of ovarian tissue culture is to promote the growth of healthy secondary follicles that can be isolated for further culture to generate MII oocytes. The present findings suggest that adoption of dynamic ovarian tissue culture could enhance the outcome of folliculogenesis in vitro Trial registration number not applicable

P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with &amp;gt;1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p &amp;lt; 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p &amp;lt; 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable

P-700 Modelling individual follicle growth rates during ovarian stimulation in medically assisted reproduction cycles to enable prediction of final follicle profiles

Abstract Study question Can the growth of individual follicles during ovarian stimulation in medically assisted reproduction (MAR) cycles be modelled to enable prediction of final follicle profiles? Summary answer Mean follicle growth rate is 1.35 mm per day. Our model reliably forecasts follicle size profiles on the final scan within ±2mm with 75.1% accuracy. What is known already Follicle growth rates during ovarian stimulation are believed to be linear with various mean growth rates reported between 1 to 4 mm per day. Follicles are believed to grow faster during ovarian stimulation than in the natural menstrual cycle. The change in mean follicle size was 1.69 mm per day with ovarian stimulation in 131 MAR cycles, and 1.4 mm per day in 50 natural cycles. However, there are only very limited data using individual follicle sizes to model follicle growth to enable prediction of future follicle size profiles. Study design, size, duration We conducted a retrospective cohort study of 12,950 patients from 11 European clinics (2005-2023). First IVF/ICSI cycles with at least three follicles and two scans were analysed. Using 8,564 cycles with scans one or two days apart, 98,029 follicles were modelled to estimate follicle growth rates per day. Stratification by age, weight, total antral follicle count (AFC), initial FSH dose, and suppressant protocol was performed. Predictive modelling was conducted using 39,698 scans including 434,082 follicles. Participants/materials, setting, methods Assessment of Follicle Growth: Follicles were ranked and the difference in corresponding follicle sizes over consecutive scans estimated, and the impact of baseline characteristics evaluated. Prediction of follicle size profile on final scan: A kernel density estimator and random forest model were developed using the same training data. Both approaches estimated future follicle growth from the initial scan. The two predictions were combined using maximum-likelihood adjustments. Generalisation error was estimated using independent test data. Main results and the role of chance The mean growth rate of individual follicles was 1.350 mm per day (95% CI 1.346 – 1.353 mm per day), which was lower than the previously reported mean follicle growth rate during ovarian stimulation (1.7 mm per day). The large number of patient-cycles and follicles assessed enabled a precise estimate of follicle growth per day during ovarian stimulation. Age, total AFC, initial FSH dose, bodyweight, and suppressant protocol had no statistically significant effect on the estimated mean growth rate. Using only the follicle sizes from the initial scan during ovarian stimulation, our model had 75.1% accuracy for predicting follicle sizes on the final scan within ±2mm. Including follicle size data from the first two scans improved the accuracy of the model to predict the follicle sizes on the final scan to 80.4%. In a confirmatory analysis, we cross-validated our findings across each of 11 individual clinics, and similar estimates for the generalisation error as for the standard test/train split were maintained, suggesting suitable generalisability of these findings. Limitations, reasons for caution A key simplifying assumption in this study is that follicles demonstrate monotonic growth patterns. Specifically, the relative ordering of follicle sizes is preserved over the entire cycle. This assumption enables straightforward extraction of growth statistics from the observed data but assumes that follicles do not regress in size. Wider implications of the findings We have used a large high-quality dataset, allied to advanced modern machine learning techniques, to precisely estimate follicle growth rates during ovarian stimulation, and use this to reliably predict future follicle size profiles during ovarian stimulation. This supports the design, personalisation, streamlining and success of future assisted conception cycles. Trial registration number None

P-671 Modelling human follicle growth and development during ovarian stimulation: a physiological computational approach

Abstract Study question Can key elements of ovarian physiology be accurately modelled, thereby providing a tool to generate novel insights and guide development of new therapeutics? Summary answer A physiological computational model was developed to describe follicle growth and follicle size distributions observed in agonist and antagonist ovarian stimulation (OS) protocols. What is known already The outcome of OS remains unpredictable despite identification of several prognostic factors. The average first cycle pregnancy rate is 30-40% across fertility centres, highlighting the need for improved stimulation protocols. This is an active area of clinical research; however, clinical trials are costly, time-consuming, and frequently have outcomes that are unexpected or difficult to interpret. We integrated available physiological knowledge of human follicle growth and development and clinical trial data into a computational model to enable in silico simulations with the aim to optimise clinical trial design. Study design, size, duration Computational model building was performed using a mechanistic approach aimed at capturing well-understood physiology that is relevant in OS protocols. Model inputs included patient characteristics (such as anti-Müllerian hormone (AMH) level), OS regimen and pituitary downregulation with gonadotrophin releasing hormone (GnRH) agonist or antagonist. Key outputs of the model included follicle number and mean size, duration of stimulation and serum hormone concentrations (oestradiol, testosterone, inhibin B and androstenedione). Participants/materials, setting, methods Published physiological data on regulation of follicle development and hormone production informed model design. Following implementation of the model in the Python platform, data from two OS clinical trials were used to calibrate (partially censored) and test the model. The two trials provided data on the effects of the recombinant follicle stimulating hormone preparation follitropin delta over a range of fixed doses and compared the effects of follitropin delta in agonist and antagonist OS protocols. Main results and the role of chance The computational model describes the interplay between pituitary gonadotrophins ovarian steroid hormones and follicle growth and development. Processes spanning the cellular level (such as steroidogenesis and receptor dynamics in theca and granulosa cells), the ovaries (follicle number and size) and the organism (pharmacokinetics and hypothalamic-pituitary-ovarian axis) are included as a system of coupled differential equations. Approximately 70 compartments, 500+ parameters and 1700+ reactions constitute the model framework. Follicles are recruited in the model at variable times, starting from 7 days prior to the start of stimulation. Follicles are initialised with individual sensitivity to gonadotrophins and growth rates. The fate of each follicle (dominance or atresia) is governed by these properties, which determine whether the balance shifts towards proliferation or atresia under the influence of gonadotrophins. Model-simulated numbers of follicles, their average size and mean serum hormone concentrations match closely with observed values in the two clinical trials. For each of these variables, their dependence on time, the dose of follitropin delta, AMH level and downregulation protocol were described with good precision. The inter-patient variability in the simulated number of follicles and in serum hormone levels matched closely with the observed variability based on comparison of the interquartile range. Limitations, reasons for caution The computational model currently describes follicle development during treatment with follitropin delta. We envision extending the model to encompass other treatment outcomes and additional preparations of exogenous gonadotrophins. While the model incorporates our current understanding of mechanisms important for follicle development, ongoing research in the field will likely drive improvements. Wider implications of the findings The computational model is hypothesis-generating and can improve the design of future clinical trials through in silico trial simulation. For instance, the effects of changing subject inclusion/exclusion criteria or of novel dosing regimens can be investigated. Trial registration number NCT01426386, NCT03809429

P-803 Intra-ovarian injection of plasma rich in stem cell-secreted and platelet-enclosed factors to promote follicle rescue and reproductive outcomes in women with very low ovarian reserve

Abstract Study question Could intraovarian injection of plasma rich in stem-cell and platelet growth factors optimize ovarian reserve and IVF outcomes in women with very low ovarian reserve? Summary answer Intraovarian injection of G-CSF mobilized plasma rich in grown factors improved follicle count since first follow-up and led to better fecundation, implantation and pregnancy’ rates. What is known already Autologous stem cell ovarian transplantation (ASCOT) of bone marrow derived stem cells optimized ovarian reserve and reproductive outcomes in poor responders (POR) and this effect seems to be due to the growth factors secreted by the stem cells, acting through paracrine pathways. Similarly, platelet-rich Plasma (PRP) has been also offered to promote ovarian reactivation in patients with impaired ovarian reserve by providing positive growth factors contained in platelets. Indeed, a technique that combines both the non-cellular components of ASCOT and PRP (ASCOT-1) has been proposed to optimize follicle rescue and IVF outcomes in women with very low reproductive prognosis. Study design, size, duration Retrospective observational study with 159 women (≤ 45 years) diagnosed with POR and primary ovarian insufficiency (POI) according to ESHRE criteria, who underwent the ASCOT-1 technique after refusing oocyte donation, between June 2021-October 2023 at IVIRMA Alicante, Spain (IRB approval 2310-FIVI-188-SH). The ASCOT-1 consisted in the intraovarian injection of plasma rich in both stem cell-secreted and platelet-enclosed factors obtained after granulocyte colony stimulating factor (G-CSF) mobilization and PRP obtention (Endoret Kit BTI, Biotechnology institute). Participants/materials, setting, methods Medical records of patients who underwent the ASCOT-1 as an ovarian reactivation treatment were examined. Follow up of AFC and AMH was carried out for a maximum of 4 menstrual cycles to evaluate follicular activation. Moreover, when follicle growth was detected, patients underwent a controlled ovarian stimulation (COS). IVF and reproductive outcomes were compared with previous COS cycles started in our clinics, when available. T-test was used where appropriate. Main results and the role of chance Overall 159 POR/POI women (age 38.7±4.2years, AMH0 0.22±0.41ng/ml; AFC0 1.4±1.9) underwent the ASCOT-1 technique. Mean volume of plasma injected was 6.8±0.8ml, distributed equally in both ovaries. First follow-up was done in 131 women showing statistically significant improvements in the AFC during the entire follow-up compared to the basal levels measured before the treatment (AFC1 3.3±3.4, p &amp;lt; 0.001; AFC2 3.3±3.6, p &amp;lt; 0.001; AFC3 2.9±2.6, p &amp;lt; 0.001; AFC4 2.4±2.6, p = 0.07). After ASCOT-1, 98 COS were initiated in 48 women in our clinics, which were compared to their previous 66 cycles. Although age was significantly higher in their posterior cycles (38.0±2.9 vs40.0±3.6; p &amp;lt; 0.001), after ASCOT-1 oocyte retrieval was reached in a greater amount of cases (75.5% vs87.5%, p=NS). ASCOT-1 showed a positive, but non statistically significant effect in the number of MII-oocytes (2.8±4.1 vs3.2±3.9, p=NS) and blastocysts obtained (1.8±2.8 vs2.4±3.1, p=NS), but relevant improvements were observed in quality related parameters such as oocyte maturation (61.5%, 95%IC 45.8-77.3, vs86.1%, 95I%IC 69.9-85.5; p 0.03), fertilization (62.6%, 95%CI 53.0-71.3, vs76.5%, 95%CI 70.0-82.0; p &amp;lt; 0.001), implantation (25.0%, 95%CI 8.3-52.5 vs41.1%, 95%CI 19.4-66.5; p &amp;lt; 0.001) and clinical pregnancy rate (25.0%, 95%CI 8.3-52.5 vs46.6%, 95%CI 22.2-72.5; p &amp;lt; 0.001). Ultimately, 14 clinical pregnancies (7 after ET and 7 by natural conception) were obtained after ASCOT-1. Limitations, reasons for caution This retrospective study involves short periods of follow up and limited amount of IVF attempts. Thus, further prospective studies with increased sample size are required to validate our results, to establish the duration of the effects, and to identify which subgroups of patients could benefit the most from this treatment. Wider implications of the findings Growth factors contained in the G-CSF mobilized PRP could play a role in follicular development, providing a new tool to increase the reproductive potential of women with very low ovarian reserve where oocyte donation is the only practical option, allowing them to access to homologous IVF treatment. Trial registration number 2310-FIVI-188-SH

P-526 Identification of mouse cumulus cell-specific miRNAs and their role in the acquisition of the oocyte developmental competence

Abstract Study question Do cumulus cells (CCs), during mouse germinal vesicle (GV)-to-metaphase II (MII) transition, produce miRNAs with a role in the acquisition of the oocyte developmental competence? Summary answer We identified a group of 74 miRNAs with a known role in key processes of oocytes maturation and developmental competence. What is known already We recently developed a co-culture platform whereby CC-free, denuded, GV oocytes (DOs) were matured to MII upon a feeder layer (FL) of CCs derived either from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) oocytes. When cultured upon FL-SN-CCs, DOs displayed a developmental competence equal to that showed by control cumulus-enclosed oocytes (COCs). Instead, when cultured upon FL-NSN-CCs, all DOs arrested at the 2-cell stage. We found that CCs released into the medium large amounts of extracellular vesicles (EVs), whose content analysis by NGS led to the identification of candidate miRNAs. Study design, size, duration Five-eight-week-old CD1 mouse females (Charles River) were injected with 10 I.U. Pregnant Mare Serum Gonadotropin (PMSG). After 48 hr, the ovaries were isolated and their surface punctured with a thin sterile needle to collect fully-grown antral oocytes which were separated from the surrounding CCs. Eight-ten DOs were cultured upon FL-SN-CCs or FL-NSN-CCs for 15 hr in 50 µl exosome-free alpha-MEM Glutamax medium for EVs production. Experiments were performed in triplicates. Participants/materials, setting, methods EVs were collected and processed for EVs characterisation by Amnis-Imaging-flow-cytometer and transmission electron microscopy (TEM), or for RNA extraction (miRNeasy Micro Kit, Qiagen), libraries production (Small RNA-Seq Library Prep kit, Lexogen) and NGS (Illumina Genome Analyzer and the NextSeq 500/550 High Output v2.5). To identify miRNA’s target genes, we used “mirPath v.3” server of the DIANA tools website (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath). Then, STRING and KEGG were used to highlight the pathways in which these genes are involved. Main results and the role of chance Imaging flow cytometry recorded an amount of released EVs in both FL-SN-CCs or FL-NSN-CCs in the mean range of 5-5.9 x 10(8) particles/ml (p &amp;gt; 0.05), with &amp;gt;96% positive to CD9 exosome surface marker. TEM analysis showed an EVs size in diameter comprised between 18.5-54.9 nm. By comparing FL-SN-CCs vs. FL-NSN-CCs, NGS of EVs’ miRNAs content displayed 74 differentially expressed miRNAs, 43 up- and 31 down-regulated. Of them, 7 resulted involved in the regulation of 71 target genes with a known function in meiosis resumption (N = 24), follicle growth (N = 23), fertilisation (N = 1) and in the acquisition of the oocyte’s developmental competence (N = 23). Limitations, reasons for caution We used an in vitro platform to mimic the cumulus-oocyte-complex environment and, thus, the artificial set-up may differ from that of the natural condition. Also, our hypothesis on the role of these miRNAs, needs to be further confirmed by functional assays (e.g., miRNA inactivation). Wider implications of the findings Overall, our results indicate CC-derived miRNAs as candidates of the CC-to-oocyte bidirectional communication and suggest a group of miRNAs as potential regulative molecules in the acquisition of the oocyte developmental competence. Trial registration number NOT APPLICABLE

P-793 New approaches for treating diminished ovarian reserve: MSC-derived extracellular vesicles and autologous platelet rich plasma

Abstract Study question Does intraovarian injection of MSC-Derived Extracellular Vesicles (EVs) and autologous platelet rich plasma (PRP) improves the outcome of IVF in women with Diminished Ovarian reserve? Summary answer EVs and PRP therapies increse Antral follicle count (AFC), lower FSH level and result in higher amount of mature oocytes and blastocysts What is known already Diminished ovarian reserve (DOR) and premature ovarian insufficiency (POI) are significant impediments to the success of in vitro fertilization (IVF). Current strategies for ovarian stimulation primarily affects only the growth of antral follicles. Even in scenarios where ovarian ovulatory function is compromised, a reservoir of dormant follicles persists, which could potentially be activated by stem cells. Mesenchymal stem cells (MSCs), owing to their multipotent differentiation capabilities and paracrine signaling properties, have emerged as leading candidates for innovative therapeutic approaches. Recent scientific inquiries have highlighted the paracrine impact of EVs secreted by stem cells, underscoring their prospective therapeutic relevance. Study design, size, duration This prospective interventional study was conducted at the V.I. Kulakov Scientific Research Center for Obstetrics, Gynecology, and Perinatology in Russia. It involved 60 infertile female participants aged 20 to 38 years, each with DOR and a history of at least two unsuccessful IVF cycles. These participants were enrolled in the study during their subsequent IVF cycle. Prior to inclusion, all patients provided written informed consent. Participants/materials, setting, methods Patients in group 1 (n = 30) received treatment with exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-Exos) prior to initiating their in vitro fertilization (IVF) cycle. In contrast, participants in group 2 (n = 30) underwent intraovarian therapy with autologous platelet-rich plasma (PRP) in preparation for their IVF cycle. The comparative analysis encompassed antral follicle count (AFC), anti-Müllerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, ovarian stimulation outcomes, and the primary characteristics of oogenesis and embryogenesis. Main results and the role of chance After transplantation of EVs and PRP the ovarian function-related hormone levels and the AFC returned to nearly normal parameteres.Meanwhile, after exosomal treatment the improvement in reproductive outcomes was more significant. FSH concentration was significantly different before and 30 days after the procedure in both groups. In group 1 FSH values were less than the initial data In 95% of cases, in group 2 – in 72% of cases. AMH levels did not change significantly in both groups. The AFC increased in group 1 compared to group 2. All Patients underwent an IVF antagonist cycle after 3 months from the treatment. The number of obtained oocytes in group 1 ranged from 2 to 8, MII from 1 to 6; in group 2: from 0-5, MII – 0-5. The number of blastocysts in group 1 ranged from 1 to 5, in group 2 – from 0 to 3. In group 1, pregnancy occurred in 9 patients (30%): 1 women ( 3,3%) conceived spontaneously in the cycle of procedures, 8 (26,7%) attempted IVF, in group 2 pregnancy occurred in 4 patients (13,3%): 4 attempted IVF. EVs and PRP injections improve outcomes of IVF results in women with low ovarian reserve. Limitations, reasons for caution Limitations may include the fact that the study is single-center and has a rather small amount of patients enrolled in the study. Fundamental approaches to further research in this area are also required to better understand the obtained results. Wider implications of the findings EVs and PRP therapies appear to enhance ovarian reserve metrics and the success rates of IVF programs in patients with DOR. The clinical implementation of these interventions may offer improved reproductive prospects for patients whose sole option was previously oocyte donation. Trial registration number 121040600410-7

P-620 Oocyte competence in Vitro Fertilization: relation between serum anti-Müllerian hormone (AMH) concentrations and complement C4 protein expression in follicular fluid

Abstract Study question To investigate if serum AMH concentration variations are correlated with protein expression in follicular microenvironment affecting IVF outcomes. Summary answer Serum AMH concentrations were associated with altered levels of intrafollicular protein involvement of the complement pathway in immunity response in follicular fluid (FF). What is known already Anti-Müllerian hormone (AMH) plays a crucial role in regulating growth and development of ovarian follicles. The follicular fluid is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. AMH concentration in follicular fluid can be a marker of ovarian reserve status and oocyte competence. AMH elevated levels may indicate a higher ovarian reserve and are sometimes associated with a greater ovarian response to ovarian stimulation used in IVF. However, the exact relationship between AMH levels in follicular fluid and IVF outcomes may vary among women and depend on various factors. Study design, size, duration The study included FF collected from 75 patients underwent IVF from December 2018 to June 2019.All patients enrolled underwent GnRH antagonist stimulation protocol with r-FSH according to clinical/ovarian features.Patients were sorted into three groups according to serum AMH concentrations: AMH≤1.0 ng/ml, n = 25 (A group); AMH=1.1-2.0 ng/ml, n = 29 (B Group); AMH&amp;gt;2.0 ng/ml, n = 21 (C group).Cutoffs for defining low(A), average(B), and high(C) AMH serum concentrations corresponded to round values of 30th and 70th centiles of each measurement. Participants/materials, setting, methods Exclusion criteria: endometriosis, metabolic/endocrinology diseases and oligoasthenoteratozoospermia. FF aspirates from individual follicles (diameter ≥18 mm) were collected on the day of oocyte retrieval. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). Oocyte number and quality, fertilization rate are IVF outcome measures. Data were analyzed using the unpaired Student’s t-test by IBM SPSS-STATISTICS versions 20.0 and considered significant if p-value ≤ 0.05. Main results and the role of chance 180 protein spots were highlighted in three groups: 80% is represented in all the samples analyzed, whereas 19 protein spots out of 180 are group-specific. Five unique upregulated proteins were identified in the A group and five in C group respect to control group (B). These were identified IG, CO4, ALBU, TRFE, IGLC in A group, while A1AT, ANT3 A2HS, HPT and CO4 in C group. A and C group showed worse oocyte quality associated with a lower fertilization rate than B group. On the basis of the proteome analysis, the component of the complement cascade C4 was found more plentiful in patient with low and high AMH serum concentration compared to average AMH serum concentration ones. The presence of high levels of the unprocessed precursor C4 correlated to serum AMH fluctuations is an indication of non-activation of the complement pathways fundamental for follicle maturation and ovulation compromising IVF outcome. Limitations, reasons for caution Limited number of patients. Age and lifestyle factors (i.e. smoking, diet) are not considered. Wider implications of the findings Results showed an increase of CO4 in FF of patients with low and high AMH serum concentrations. CO4 FF high levels could be indicative of defective complement activation during ovulation with consequent impaired processes and lower standard oocyte competence. Thus, CO4 could be identify as biomarker to predict IVF success. Trial registration number Not Applicable

P-343 Assessing the effect of adipose tissue-derived stem cell-conditioned medium on follicles and stromal cells in cultured ovarian tissue

Abstract Study question Does addition of adipose tissue-derived stem cell-conditioned medium (ASC-CM) enhance follicle survival and development and stromal cell viability in ovarian tissue culture? Summary answer Adding ASC-CM to ovarian tissue in vitro culture (IVC) promotes follicle survival, development, estradiol (E2) production, and stromal cell viability. What is known already Despite extensive efforts to optimize ovarian tissue IVC conditions, outcomes of follicle in vitro development remain suboptimal, mainly showing poor follicle survival and growth. IVC conditions that best resemble the in vivo environment are likely to generate less cellular distress and yield better outcomes. ASCs are known for promoting cell proliferation and survival through secretion of growth factors and cytokines. CM obtained from ASC culture contains these beneficial biomolecules and could potentially be a valuable supplement for ovarian tissue IVC. Study design, size, duration This prospective experimental study used 8 fragments of bovine ovarian cortex, which were cultured for 8 days in two different groups: (i) standard ovarian tissue culture (OT Ctrl D8); and (ii) ovarian tissue cultured with ASC-CM supplementation (OT+CM D8). One fragment per sample served as a non-cultured control (D0). Half of the culture medium was replaced every other day, and medium from days 2 and 8 was stored for further analyses. Participants/materials, setting, methods E2 production was evaluated on days 2 and 8 by ELISA. Follicle classification was established using hematoxylin and eosin (H&amp;E) staining. Follicle and stromal cell apoptosis rates were assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) labeling served as a follicle quality marker. ASC-CM was analyzed to identify and measure soluble factors like vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-β1), associated with protective effects. Main results and the role of chance The OT+CM D8 group showed significantly lower primordial follicle rates (p = 0.03) and higher secondary follicle rates (p = 0.02) than the OT Ctrl D8 group. Concerning apoptosis, the OT+CM D8 group demonstrated significantly lower follicle (p = 0.014) and stromal cell (p = 0.001) rates than the OT Ctrl D8 group. Furthermore, follicles in the OT+CM D8 group exhibited a significant increase (p = 0.002) in GDF-9 expression compared to those in the OT Ctrl D8 group. E2 production was also significantly higher (p = 0.04) after 8 days of culture in the OT+CM D8 group. All studied paracrine factors (VEGF, IL-6 and TGF-b1) were found to be present in the ASC-CM. Limitations, reasons for caution This study focuses only on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue, and not on subsequent steps. Wider implications of the findings Our study demonstrates that adding ASC-CM to ovarian tissue IVC medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. Moreover, our data suggest that ASC-derived factors VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects. Trial registration number not applicable

P-693 Three-dimensional modeling of follicular microenvironment dynamics in the human adult ovary

Abstract Study question How does the ovarian microenvironment, involving neural fibers, vascular networks, and immune cells, interplay with follicular dynamics in the human adult ovary? Summary answer Neural fibers growing into the ovarian cortex and vascular network expansion were associated with primordial follicle activation and growth. Macrophages associated with antral follicular atresia. What is known already The ovarian follicles, responsible for preserving and supplying oocytes, play a crucial role in fertility, with their fate influenced by the ovarian microenvironment. However, the three-dimensional (3D), irregular, and dynamic structural changes of the ovarian microenvironment are difficult to interpret using two-dimensional analysis. 3D-imaging technologies have been developed to reveal intricate structural and functional relationships particularly in rodents, but these approaches have not been widely adopted for human ovarian research. Study design, size, duration Our study aims to explore the follicular microenvironment dynamics in the human adult ovary. In this work, we optimized whole-mount immunofluorescence using TUBB3, PODXL, PECAM1, ACTA2 and CD68, and conducted 3D imaging on intact adult ovarian tissue, offering a visualization of the 3D structures and dynamic changes within ovarian follicles, neural fibers, vascular networks, and immune cells in human adult ovary at the single-cell level. Participants/materials, setting, methods The human ovarian samples were collected from donors (N = 5) who underwent oophorectomy after testosterone treatment. Part of the intact ovary, including cortex and medulla, was fixed and used for whole-mount immunofluorescence using markers for neural fibers (TUBB3), blood vessels (PODXL, PECAM1, ACTA2), and immune cells (CD68). Subsequently, an enhanced iDiSCO method was employed for tissue clearance. Finally, 3D images were acquired using a lightsheet microscope, followed by image analysis and 3D reconstruction using Imaris software. Main results and the role of chance Our results showed that TUBB3-labeled nerve fibers predominantly distributed in the ovarian cortex, with few presented in the medullary region. Notably, their presence surrounding secondary and antral follicles was limited. We observed a rich distribution of PODXL-labeled capillaries surrounding primordial and primary follicles in the cortical layer of the adult ovary. As follicles progressed to the secondary stage, PECAM1-labeled vasculature contributed to the construction of a vascular network enveloping the secondary follicles and connecting extensively to larger-caliber arteries containing (ACTA2+) smooth muscle cells. In antral follicles, large larger-caliber arteries were also detected, maintaining proximity to the theca cell layer. Interestingly, in cases of follicular atresia, vascular invasion within the follicular cavity was observed. Additionally, clusters of CD68+ macrophages exhibited a scattered distribution around the follicles at various stages, with a significant accumulation in atretic small-antral follicles. Limitations, reasons for caution Our study was conducted on ovaries from a limited number of donors after gender-affirming surgery. Although we aimed to provide insights into the ovarian microenvironment, we were so far unable to obtain ovaries with large antral follicles, ovulatory follicles and the formation of the corpus luteum. Wider implications of the findings The views provided by 3D-image analysis of the human ovary will augment the current knowledge of the complex interactions between the follicles and the ovarian microenvironment. 3D ovarian mapping will also provide a roadmap for generating oocytes in vitro for fertility preservation and ovarian disease modelling. Trial registration number N/A.

O-076 Novel method to enhance preantral follicle growth by granulocyte colony-stimulating factor administration improves embryos and pregnancy rate in poor ovarian reserve: a randomized controlled trial

Abstract Study question Whether priming with granulocyte colony-stimulating factor (G-CSF) before ART improves embryo and pregnancy rate while increasing anti-Müllerian hormone (AMH) in patients with poor ovarian reserve. Summary answer G-CSF priming improved embryonic development and pregnancy rate in ART with poor ovarian reserve, simultaneously increasing serum AMH. Enhanced preantral follicle growth likely was responsible. What is known already Eleven years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering G-CSF with embryo transfers. No implantation succeeded directly, but serendipitously 3 of the 10 spontaneously became pregnant 2 months later. One of these pregnancies involved twins in a 45-year-old woman without ovulation induction. We hypothesized that G-CSF administration might have stimulated preantral follicle growth, with improved ovulation after 2 months. In animal models with diminished ovarian reserve, G-CSF administration consistently increased ovarian preantral follicles and AMH. Therefore, we designed this clinical trial. Study design, size, duration Patients were prospectively randomized to priming or control groups. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of IVF/ICSI and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Participants/materials, setting, methods Hundred ART patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. Main results and the role of chance Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by G-CSF priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts (53% with priming vs. 24% in controls). The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. The efficacy of G-CSF priming was not associated with genotypes for killer-cell immunoglobulin-like receptors. No adverse effects of priming were observed. Limitations, reasons for caution Enhancement of preantral follicle growth by G-CSF administration needs to be confirmed histologically as this clinical trial could not allow ovarian biopsies for its purpose of infertility treatments. Wider implications of the findings This study proposes a novel, simple, and safe treatment for poor ovarian reserve. In addition, enhancement of preantral follicle growth by G-CSF priming may also improve ovulatory dysfunctions in polycystic ovary syndrome with elevated AMH, reflecting pooling of retarded/arrested growing preantral follicles. Trial registration number UMIN000013956

P-370 The age-associated increase in ovarian stiffness compromises follicle development and induces changes in the follicles’ transcriptome, resulting in poor-quality oocytes

Abstract Study question Does the age-associated increase in ovarian stiffness trigger the decline in oocyte quantity and quality observed in women of advanced maternal age? Summary answer An encapsulated in vitro follicle culture mimicking ovarian biomechanics revealed that age-associated increase in stiffness impacts follicle growth, modulates follicles’ transcriptome and reduces oocyte quality. What is known already Female reproductive aging is characterized by a progressive decline in oocyte quality and quantity, resulting in sub-fertility. We recently discovered that with aging the mouse ovary assumes a pro-fibrotic milieu which induces ovarian stiffness. Moreover, human ovaries also become stiffer with aging; however, it is not known whether this age-associated increase in stiffness impacts ovarian function. The present study aims to examine whether stiffness is a novel mechanism that mediates the age-associated progressive decline in oocyte quality and how stiffness affects the transcriptome of growing follicles, resulting in poor-quality oocytes. Study design, size, duration We used an in vitro alginate-encapsulated follicle culture to mimic young and old environments in which follicles grow. Alginate hydrogel synthesis replicated the soft (0.5% - 1.79±0.08 kPa) and stiff (2% - 4.56±2.03 kPa) environments of young and aged ovaries, respectively. Mouse secondary follicles were cultured in 0.5 or 2% alginate and used for i) long-term culture (day 0-10) to evaluate follicle development and oocyte quality; ii) short-term culture (hour 0-24) to investigate follicles’ transcriptome. Participants/materials, setting, methods During the long-term culture, markers of follicle development were evaluated (n = 31 follicles), including: follicle size, estradiol production (ELISA), cell death (cleaved-caspase3,CC3; immunohistochemistry). At D10, oocyte quality was inspected on isolated oocytes. For the short-term culture experiment, 32 follicles were analyzed by RNAsequencing at 3h (baseline) and 24h. We compared 3h and 24h gene expression (DESeq2,R/Bioconductor) in 0.5% and 2%. Significant differences reported at padj&amp;lt;0.05 and log2FoldChange±2. Two mice strains (CB6F1;CD1) were used in all experiments. Main results and the role of chance Follicles cultured in stiff environment showed a significant reduction in follicle size compared to follicles cultured in soft environment (0.5% 226.9±17.4um, 2% 160.8±9.9um; p &amp;lt; 0.0001). These size differences suggest that granulosa cells are not proliferating in a stiff environment. Estradiol production was reduced in follicles cultured at 2% (0.5% 11.3±15.9ng/ml, 2% 0.3±0.5ng/ml, p = 0.296), while CC3 intensity was increased (CC3 staining intensity/area, 0.5% 20.4±5.8a.u., 2% 27.1±4.3a.u., p &amp;lt; 0.05), suggesting that the stiff environment impacts granulosa cell viability. Oocyte quality significantly declined in follicles cultured in 2%, with 68.9±16.8% of the oocytes degenerated, compared to 23.6±9.2% in 0.5% alginate. The use of two different mice strains yielded comparable results. Using RNAsequencing analysis, we found 1033 differentially expressed genes (DEGs) between 3h and 24h of culture in 2% alginate, mostly involved in apoptosis, inflammation and collagen processes, biological pathways typically associated with aging. The 64% of those DEGs were upregulated at 24h. Conversely, the 1295 DEGs (61% upregulated at 24h) detected comparing 3h and 24h in 0.5% alginate were relate to metabolism, cell cycle and development pathways, biological processes associated to the normal behavior of growing follicles. These differences in gene expression suggest an early-response in the transcriptome of follicles cultured in a stiff environment. Limitations, reasons for caution The stiffness of the alginate gels was based on whole ovary biomechanics; future investigations are needed to measure each follicle-stage-specific stiffness. The changes in follicles’ transcriptome in the stiff environment were evaluated only in short-term culture. This study utilizes in vitro approaches, in vivo modulation of stiffness is currently undergoing. Wider implications of the findings Increased ovarian stiffness is observed in conditions such as aging or ovaries exposed to cancer treatments. Studying how stiffness impacts oocyte quality will allow the development of suitable in vitro systems to support human folliculogenesis and in vivo approaches to modulate biomechanics of the ovary, preserving female fertility. Trial registration number not applicable

P-629 In vivo follicular activation (IVFA) through repeated ovarian punction: results of an experimental study in a premature ovarian insufficiency rodent model

Abstract Study question Can the new IVFA therapy effectively promote ovarian follicle development and increase oocyte number in a premature ovarian insufficiency (POI) animal model. Summary answer The IVFA technique reveals favourable reproductive outcomes in the animal model, demonstrating increased oocyte retrieval without compromising quality, making a promising advancement in fertility restoration. What is known already POI is characterized by the loss of the primordial follicular pool before the age of 40. Ovarian fragmentation for in vitro follicular activation (IVA) is a technique based on the disruption of the ovarian extracellular matrix, subsequently promoting actin polymerization and interfering with the evolutionarily conserved tumour suppressor Salvador-Hippo-Warts (SHW) signalling pathway. This has been proven to increase expression of downstream growth factors, thus promoting the primordial follicle growth and the generation of mature oocytes. Yet, this procedure is invasive and expensive. Consequently, a demand exists for less invasive in vivo therapies. Study design, size, duration Experimental animal study. Sixty 6-week-old C57BL/6 female mice were divided into control (CT) and POI groups. Each group, was further divided into three subsets (n = 10/group): without ovarian punctures (CT-0 and POI-0), 15 punctures (CT-15 and POI-15), and 22 punctures (CT-22 and POI-22). Twenty-one days after POI induction, the new IVFA technique was performed on the anaesthetized animals. Subsequently, controlled ovarian stimulation (COS) was initiated, followed by euthanasia for the oocyte retrieval and histological ovarian evaluation. Participants/materials, setting, methods POI condition was induced with intraperitoneally injections of 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan. The IVFA technique involved ovarian punctures using a 30-gauge needle. COS was induced with 10 IU of PMSG and 10 IU of hCG 48 hours later, leading to euthanasia for oocytes and ovaries collection. Ovaries (n = 6/group) were fixed for follicular counts and immunohistochemistry. Oocyte quality assessment included reactive oxygen species (ROS) levels, mitochondrial fluorescence (Mitotracker), and spindle analysis. Main results and the role of chance The control groups subjected to the IVFA technique (CT-15 and CT-22) demonstrated significantly higher mean total oocyte counts compared to the non-intervention group (CT-0) (30.0±9.8 and 27.7±7.9 vs 18.1±11.7, respectively; p-value&amp;lt;0.05 in both comparisons). Similarly, within the POI model, interventions (POI-15 and POI-22) showed significantly elevated mean total oocyte counts than the non-intervention group (POI-0) (8.1±4.4 and 7.2±2.8 vs 3.8±3.6, respectively; p-value&amp;lt;0.05). The intensity of ROS fluorescence, Mitotracker fluorescence, and spindle analysis in retrieved oocytes were not affected by the IVFA therapy, thereby preserving oocyte quality. Notably, the percentage of growing follicles was significantly increased in the intervention groups compared to the non-intervention groups in the control (60±5.7% and 58.1±4.7% vs 41.5±4.5%; p-value&amp;lt;0.001). In the POI model, statistically significant differences were observed for POI-15 vs POI-0 (57.8±9% vs 41.6±13.3%; p-value&amp;lt;0.05), but not for POI-22 vs POI-0 (51.5±14.2% vs 41.6±13.3%, p=n.s.) Moreover, the global Ki67 proliferation marker demonstrated a significant increase in the percentage of proliferative cells in the CT-15 intervention group compared to the CT-0 group (53±11.2% vs 46.1±15.1; p &amp;lt; 0.01). Finally, in the POI model, the percentage of Ki-67 positive cells was also significantly higher in both IVFA groups (39.4±18.5% and 38.1±17.3% vs 32±17.5%, respectively; p &amp;lt; 0.05). Limitations, reasons for caution Since this is an animal model of POI, further validation of the new therapy with human ovarian samples would be required. The magnitude of the effect could vary due to inter-species variability. Wider implications of the findings The positive results of the new therapy in promoting follicle development and increasing oocyte retrieval, without compromising quality, have significant implications for developing new fertility treatments. If validated in humans, this approach could offer a promising solution for women with POI. Trial registration number not applicable