Application of rapamycin combined vascular endothelial growth factor A in preparation of medicine for improving ovarian tissue function

Abstract

The invention discloses application of rapamycin combined with a vascular endothelial growth factor A in improvement of an ovarian tissue function. The improvement of the ovarian tissue function comprises any one or more than two of the following aspects: increasing the survival rate of follicles, prolonging the functional life of the ovarian tissue and improving the quality of oocytes. The effective concentration of the rapamycin is 500nM, and the effective concentration of the vascular endothelial growth factor A is 5ng / mL. The follicle loss dynamic after OTCT is tracked through a mouse model, it is found that rapid follicle loss is related to tip cell damage and primordial follicle activation, then a rapamycin and vascular endothelial growth factor A combination strategy is provided, follicle loss can be remarkably reduced, the reproductive function period can be prolonged, and the oocyte quality can be improved; according to the invention, a systematic solution is provided for OTCT technology optimization.

Description

Technical Field

[0001] The present invention relates to the application of rapamycin combined with vascular endothelial growth factor A in preparing a medicine for improving ovarian tissue function, and belongs to the technical field of specific therapeutic activity of pharmaceutical preparations. Background Art

[0002] Ovarian follicles are the core units of female reproductive function, but they are susceptible to damage from chemotherapy, leading to loss of fertility in cancer patients. Most patients still require fertility preservation after treatment, highlighting the crucial importance of fertility preservation technologies. Ovarian tissue cryopreservation and transplantation (OTCT) is a cutting-edge technology for fertility preservation, attracting considerable attention due to its ability to simultaneously preserve follicles and supporting stromal cells (maintaining endocrine function). Clinical studies have shown that OTCT can restore endocrine function in nearly 100% of patients, with a live birth rate of 41%. However, the survival of transplanted tissue is limited (only extending the survival period by an average of 2 to 5 years), primarily due to rapid depletion of the follicular reserve in the early post-transplant period. Ischemic injury and insufficient vascularization are important contributors to ovarian reserve depletion, and existing strategies are unable to effectively simultaneously inhibit overactivation of primordial follicles and repair the vascular network.

[0003] Vascular integrity is crucial for graft survival. Factors such as vascular endothelial growth factor A (VEGFA), secreted by ovarian follicles, are key regulators of angiogenesis. Combined administration of VEGFA and basic fibroblast growth factor (bFGF) can significantly increase vascular density in frozen-thawed human ovarian tissue following xenotransplantation. Recent studies have shown that co-transplantation of adipose-derived mesenchymal stem cells can promote vascular reconstruction in cryopreserved ovaries through VEGFA secretion, suggesting that promoting angiogenesis can improve ischemia and nutrient supply. However, how to integrate vascular support with follicle activation inhibition strategies remains to be explored.

[0004] Rapamycin (RAPA) is an mTORC1 inhibitor. It is a white solid crystal with a melting point of 183-185°C. It is lipophilic and soluble in organic solvents such as methanol, ethanol, acetone, and chloroform. It is very slightly soluble in water and almost insoluble in ether. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention provides a strategy for improving ovarian tissue function - vascular endothelial growth factor A-rapamycin combination strategy (VEGFA-Rapamycin Combination Strategy, VRCS).

[0006] The present invention is achieved through the following technical solutions:

[0007] Application of rapamycin combined with vascular endothelial growth factor A in the preparation of a drug for improving ovarian tissue function.

[0008] Furthermore, the improvement of ovarian tissue function includes any one or more of the following aspects: increasing follicle survival rate, extending the functional life of ovarian tissue, and improving oocyte quality.

[0009] Furthermore, the effective concentration of rapamycin is 500 nM, and the effective concentration of vascular endothelial growth factor A is 5 ng / mL.

[0010] Furthermore, the specific application method is: rapamycin is used for treatment during the collection, cryopreservation and recovery stages of ovarian tissue; and rapamycin and vascular endothelial growth factor A are used in combination for treatment during the in vitro culture stage of frozen ovarian tissue (i.e., the stage after recovery and before transplantation).

[0011] Furthermore, the specific application method is: in the collection stage, phosphate buffer containing 500nM rapamycin is used for washing; in the cryopreservation stage, 500nM rapamycin is added to the cryopreservation solution; in the resuscitation stage, 500nM rapamycin is added to the resuscitation solution; in the in vitro culture stage, 500nM rapamycin and 5ng / mL vascular endothelial growth factor A are added to the culture medium.

[0012] Furthermore, the freezing preservation fluid includes vitrification fluid I and vitrification fluid II, wherein vitrification fluid I is composed of 20% fetal bovine serum, 7.5% dimethyl sulfoxide, 7.5% ethylene glycol and the remainder DMEM / F12 culture medium, calculated by volume percentage; vitrification fluid II is composed of 20% fetal bovine serum, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.25M sucrose and the remainder DMEM / F12 culture medium, calculated by volume percentage.

[0013] Furthermore, the resuscitation fluid is a DMEM / F12 culture medium containing fetal bovine serum and decreasing concentrations of sucrose, wherein the fetal bovine serum accounts for 20% (volume percentage) and the sucrose concentrations are 0.5M, 0.25M, 0.125M and 0M respectively.

[0014] Furthermore, during the in vitro culture stage, the revived ovarian tissue was placed in DMEM / F12 culture medium containing 10% fetal bovine serum (volume percentage), 500 nM rapamycin and 5 ng / mL vascular endothelial growth factor A, and pre-cultured at 37°C, 5% CO2 and saturated humidity for 6 hours.

[0015] The present invention studies used vitrification to freeze wild-type and Tek-CreER T2The dynamics of follicle loss after OTCT in mTmG mouse ovarian tissue were analyzed using histology, proteomics, and high-resolution imaging. Experimental ovaries were pretreated with 500 nM rapamycin (to inhibit primordial follicle activation), 5 ng / mL vascular endothelial growth factor A (VEGF-A) (to promote angiogenesis), or a combination of both (cryo+VRCS). DMSO or H2O served as controls. Grafts were harvested 3, 7, 14, and 120 days after transplantation to assess vascular density, tip cell density, follicle activation, and residual follicles. Oocyte quality was assessed by in vitro fertilization, and graft survival was assessed by estrous cycle monitoring. Results showed that in this mouse model, follicle number decreased rapidly after transplantation. Proteomics and three-dimensional imaging revealed that this process was closely related to damage to key angiogenic cells (tip cells) and excessive activation of primordial follicles. Tip cell damage led to impaired vascular remodeling and ischemia, while mechanical stress induced by tissue dissection and cryopreservation triggered abnormal follicle activation. The combined use of RAPA to inhibit follicle activation and VEGFA to promote angiogenesis before transplantation significantly increases follicle survival, prolongs ovarian tissue functional lifespan, and effectively improves oocyte quality. This study, through a dual-drug strategy combining VEGFA and rapamycin, simultaneously targets vascular integrity and follicular homeostasis, providing a viable solution for improving the efficiency of fertility preservation based on ovarian tissue cryopreservation.

[0016] The present invention uses a mouse model to track the dynamics of follicle loss after OTCT and finds that rapid follicle loss is related to tip cell damage and primordial follicle activation. It then proposes a strategy of combining rapamycin (inhibiting activation) with VEGFA (promoting angiogenesis). This approach can significantly reduce follicle loss, prolong the reproductive function period, and improve oocyte quality, providing a systematic solution for the optimization of OTCT technology.

[0017] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic diagram of hematoxylin staining results.

[0019] Figure 2 : Photos of frozen ovarian-derived MⅡ oocytes, 2-cell embryos, and blastocysts.

[0020] Figure 3 : Schematic diagram of DDX4 immunostaining results, showing that the number of frozen ovarian follicles was less than that in the fresh control group 3, 7, and 14 days after transplantation. DDX4 immunostaining is shown in green, and the cell nuclei were counterstained with Hoechst (HOE, blue).

[0021] Figure 4: Schematic diagram of the changes in the number of frozen and fresh transplanted follicles 3, 7, and 14 days after transplantation. It can be seen that the decrease is most significant in the first 3 days; n≥4 per group at each time point.

[0022] Figure 5 : Schematic diagram of the follicle loss rate of frozen and fresh transplants 3, 7, and 14 days after transplantation, where n=3 per group at each time point.

[0023] Figure 6 : Volcano plot of protein expression in frozen and fresh ovarian tissues.

[0024] Figure 7 : Cluster analysis heat map of proteins expressed in frozen and fresh ovarian tissues.

[0025] Figure 8 : Schematic diagram of KEGG enrichment analysis of differentially expressed proteins, where fold change > 1.5, n = 2 per group, P < 0.05, angiogenesis-related pathways (blue) and primordial follicle activation pathways (red) are marked.

[0026] Figure 9 :Tek-CreER T2 ; mTmG ovary with mGFP labeled blood vessels, three-dimensional image acquired by confocal microscopy, where the scale bar in the lower left corner is 100 μm; the scale bar in the lower right corner is 20 μm.

[0027] Figure 10 : Schematic diagram of the vascular network of fresh and frozen ovaries.

[0028] Figure 11 : Schematic diagram comparing blood vessel density of fresh and frozen ovaries, where n=4 in the fresh group and n=3 in the frozen group.

[0029] Figure 12 : Schematic diagram of tip cells in fresh and frozen ovaries, where the red arrows indicate tip cells and the scale bar is 20μm.

[0030] Figure 13 : Schematic diagram comparing the number of tip cells in fresh and frozen ovaries, where n=4 in the fresh group and n=3 in the frozen group.

[0031] Figure 14 : Schematic diagram of tip cells in fresh and frozen grafts 3 days after transplantation, where the red arrows indicate tip cells, and the scale bar is 20 μm.

[0032] Figure 15 : Schematic diagram comparing the number of tip cells in fresh and frozen grafts 3 days after transplantation, where n=3 for the fresh group and n=6 for the frozen group.

[0033] Figure 16: Schematic diagram of the vascular network of fresh and frozen grafts 3 days after transplantation, where the scale bar is 100 μm.

[0034] Figure 17 : Schematic diagram comparing the vascular density of fresh and frozen grafts 3 days after transplantation, wherein, fresh group n = 6; frozen group n = 8.

[0035] Figure 18 : Schematic diagram of FOXO3A (green) and DDX4 (red) immunostaining results in normal in vivo ovaries, freshly isolated ovaries, and frozen ovaries. The white arrow indicates the cytoplasmic localization of FOXO3A, indicating oocyte activation; scale bar 100 μm.

[0036] Figure 19 : Schematic diagram comparing the FOXO3A ratios in normal ovaries, freshly isolated ovaries, and frozen ovaries, with n=3 in each group.

[0037] Figure 20 : Schematic diagram of the vascular network of frozen ovaries treated with VEGFA, where the scale bar is 100 μm.

[0038] Figure 21 : Schematic diagram of the comparison of vascular density in frozen ovaries treated with VEGFA, where n=3 in the Cryo+H2O group and n=4 in the Cryo+VEGFA group.

[0039] Figure 22 : Schematic diagram of tip cells in frozen ovaries treated with VEGFA, where the red arrows indicate tip cells, and the scale bar is 20 μm.

[0040] Figure 23 : Schematic diagram of the comparison of the number of tip cells in frozen ovaries treated with VEGFA, where n=4 in each group.

[0041] Figure 24 : Schematic diagram of the vascular network of a frozen ovary treated with VEGFA 3 days after transplantation. Scale bar: 100 μm.

[0042] Figure 25 : Schematic diagram comparing the vascular density of frozen ovaries treated with VEGFA 3 days after transplantation, where n=8 in the Cryo+H2O group and n=7 in the Cryo+VEGFA group.

[0043] Figure 26 : Schematic diagram of tip cells in frozen ovaries treated with VEGFA 3 days after transplantation, where the red arrows indicate tip cells, and the scale bar is 20 μm.

[0044] Figure 27: Schematic diagram of the comparison of the number of tip cells in frozen ovaries treated with VEGFA 3 days after transplantation, where n=5 in the Cryo+H2O group and n=6 in the Cryo+VEGFA group.

[0045] Figure 28 : Schematic diagram of the immunostaining results of FOXO3A (green) and DDX4 (red) in the rapamycin-treated group, where the white arrow indicates the cytoplasmic localization of FOXO3A, showing reduced cytoplasmic translocation, suggesting that oocyte activation is inhibited; scale bar 100 μm.

[0046] Figure 29 : Schematic diagram of the proportion of FOXO3A-positive oocytes in the rapamycin-treated group, wherein n=3 in each group.

[0047] Figure 30 : Schematic diagram of the number of surviving follicles in the rapamycin group 3 days after transplantation, where n=4 in each group.

[0048] Figure 31 : Schematic diagram of the follicle loss rate in the rapamycin group 3 days after transplantation, where n=4 in each group.

[0049] Figure 32 : Schematic diagram of the histological examination results of the rapamycin group 14 days after transplantation, where the red arrows indicate primordial follicles, and the scale bar is 20 μm.

[0050] Figure 33 : Schematic diagram of the number of primordial follicles in the rapamycin group 14 days after transplantation, where n=4 in each group.

[0051] Figure 34 : Schematic diagram comparing the number of follicles in the VRCS group and the rapamycin group 3 days after transplantation, where n=4 in each group.

[0052] Figure 35 : Schematic diagram comparing the follicle loss rate (percentage of daily loss) in the VRCS group and the rapamycin group 3 days after transplantation, wherein n=4 in each group.

[0053] Figure 36 : Schematic diagram of the histological examination results of the VRCS group and the rapamycin group 14 days after transplantation, where the red arrows indicate primordial follicles, and the scale bar is 20 μm.

[0054] Figure 37 : Schematic diagram comparing the number of follicles in the VRCS group and the rapamycin group 14 days after transplantation, where n=4 in each group.

[0055] Figure 38 : Schematic diagram of oocytes recovered 14 days after transplantation, 2-cell embryos formed by in vitro fertilization, and blastocysts. Scale bar: 100 μm.

[0056] Figure 39 : Schematic diagram comparing the number of oocytes retrieved by superovulation in the VRCS group and the rapamycin group 14 days after transplantation, wherein n=4 in each group.

[0057] Figure 40 : Schematic diagram of the proportion of oocytes developing into 2-cell embryos and blastocysts in the VRCS group and the rapamycin group 14 days after transplantation, where n=4 in each group.

[0058] Figure 41 : Schematic diagram of estrous cycle changes in the untreated, rapamycin-treated, and VRCS-treated groups after frozen ovary transplantation.

[0059] Figure 42 : Schematic diagram of estrous cycle analysis of the untreated, rapamycin-treated, and VRCS-treated groups after frozen ovary transplantation.

[0060] Figure 43 : Schematic diagram of the results of histological examination (hematoxylin staining) of ovarian grafts 4 months after transplantation, where the red arrows indicate the residual follicles, and the scale bar is 100 μm.

[0061] Figure 44 : Schematic diagram comparing the total number of residual follicles in each group 4 months after transplantation, where n=4 in each group.

[0062] Figure 45 : Schematic diagram comparing the number of follicles preserved at each stage in each group 4 months after transplantation, where n=4 in each group. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0064] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0065] Study on the mechanism and improvement strategy of follicle loss after experimental OTCT

[0066] (1) Materials and methods

[0067] (1) Experimental animals

[0068] Adult (6-8 weeks old) and adolescent (postnatal day 21, PD21) female C57BL / 6N mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0069] Tek-CreER T2 Mice were purchased from an experimental research institute in Taiwan Province, China. The CreERT2 recombinase regulated by the tek promoter can be inducibly expressed in the vascular endothelial cells of this strain of mice.

[0070] The method for constructing mTmG mice (strain 007576, Jackson Laboratory) is prior art.

[0071] Through male Tek-CreER T2 Mice were mated with female mTmG mice to obtain offspring Tek-CreER T2 mTmG double transgenic mice, after induction with tamoxifen (40 mg / kg body weight, product number 10540-29-1, Sigma-Aldrich), their vascular endothelial cells can be labeled with green fluorescence.

[0072] All mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center, College of Life Sciences, China Agricultural University. Environmental conditions were strictly controlled with a 12-h light / dark cycle, a constant temperature of 26°C, and a humidity of 40%–70%. They had free access to food and water. All animal experiments were approved by the Animal Experimentation Ethics Committee of China Agricultural University (No. AW11104202-3-1).

[0073] (2) Ovarian tissue collection

[0074] Pubertal female C57BL / 6N mice on PD21 were sacrificed by cervical dislocation, and ovaries were removed from the abdominal cavity. Under a stereomicroscope (Stemi 305, Zeiss), the ovaries were cleaned with sterile, pre-chilled phosphate-buffered saline (PBS, pH 7.4), and the ovarian bursa, adipose tissue, and connective tissue attached to the ovaries were removed using a microdissecting needle. Depending on the experimental design, some procedures were performed in PBS supplemented with dimethyl sulfoxide (DMSO, Product No. 196055, MP) or 500 nM rapamycin (Rapa) (Product No. AY-22989, Selleck).

[0075] (3) Ovarian tissue cryopreservation and recovery

[0076] Ovarian tissue cryopreservation (Cryo) and thawing methods are based on existing technologies and are briefly described as follows: The entire ovary is dissected into two halves and then balanced in two steps:

[0077] 1) Equilibrate in vitrification solution I (VSI) for 10 minutes. The composition of vitrification solution I is: 20% fetal bovine serum (FBS, Catalog No. A5670701, Thermo Fisher), 7.5% DMSO, 7.5% ethylene glycol (EG, Catalog No. 324558, Sigma-Aldrich), and the remainder is Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12, Catalog No. 1320033, Invitrogen). All percentages are by volume.

[0078] 2) Equilibrate in Vitrification Solution II (VSII) for 5 minutes. After equilibration, the ovarian tissue was immediately placed in liquid nitrogen and stored for two weeks. The composition of Vitrification Solution II is: 20% FBS, 15% DMSO, 15% EG, 0.25M sucrose (Cat. No. S818046, Macklin), with the balance being DMEM / F12.

[0079] 3) During thawing, tissues were sequentially transferred to DMEM / F12 (containing 20% FBS) containing decreasing concentrations of sucrose (0.5M, 0.25M, 0.125M, and 0M), soaking for 5 minutes each time. Finally, the tissues were transferred to sucrose-free medium to remove residual cryoprotectant and sucrose. Depending on the experimental design, DMSO or rapamycin was optionally added to some vitrification and thawing solutions.

[0080] (3) Ovarian tissue culture in vitro

[0081] Fresh or resuscitated ovarian tissue was cultured using an in vitro organ culture system. The specific method was as follows: ovarian tissue was placed on a Millicell cell culture insert (Cat. No. PICM0RG50, Millipore) in a 24-well cell culture plate (Cat. No. 702001, NEST), 400 μL of basal medium (DMEM / F12 supplemented with 10% FBS) was added, and pre-cultured at 37°C, 5% CO2, and saturated humidity for 6 hours.

[0082] Based on a dose-gradient experiment, ovarian tissues in the experimental groups were treated with culture medium containing 500 nM rapamycin (Cryo+Rapa), 5 ng / mL vascular endothelial growth factor A (VEGFA, Catalog No. V4512, Sigma-Aldrich) (Cryo+VEGFA), or a combination of the two (Cryo+VRCS) (500 nM rapamycin + 5 ng / mL VEGFA), respectively. The control group was treated with DMSO or H2O.

[0083] (4) Ovarian tissue transplantation

[0084] Cultured ovarian tissue was transplanted beneath the renal capsule of adult (6-8 weeks old) female C57BL / 6N mice. The procedure was as follows: After the mice were anesthetized with tribromoethanol (300 mg / kg, Catalog No. T48402, Sigma-Aldrich), a small incision was made in the renal capsule, and the ovarian tissue was implanted beneath the capsule. Recipient mice were sacrificed 3, 7, and 14 days after transplantation. The transplanted tissues were fixed with 4% paraformaldehyde (PFA, Catalog No. 30525-89-4, Santa Cruz) and used for follicle counting and developmental assessment.

[0085] (5) Estrous cycle detection

[0086] The estrous cycle stage of recipient mice was assessed daily by morphological examination of vaginal epithelial cells. The following procedure was performed daily between 3:00 PM and 5:00 PM: vaginal smears were collected using a medical cotton swab dipped in phosphate-buffered saline (PBS); the specimens were stained with hematoxylin; and cellular morphology was observed under a microscope to determine estrous stage. The estrous cycle is divided into four phases: proestrus (P) (characterized by predominantly nucleated epithelial cells), estrus (E) (predominantly anucleated keratinized epithelial cells), metestrus (M) (with the appearance of leukocytes), and diestrus (D) (predominantly neutrophils).

[0087] (6) Histological staining and follicle counting

[0088] Harvested ovarian tissue was fixed with 4% paraformaldehyde overnight at 4°C, then dehydrated and embedded in paraffin. Embedded tissue was serially sectioned (8 μm thickness) using a microtome and mounted on slides. Sections were stained with hematoxylin, and follicles were staged and counted based on granulosa cell morphology and surrounding oocyte structures: primordial follicles (oocytes surrounded by a single layer of flat granulosa cells); primary follicles (oocytes surrounded by a mixed layer of flat and cuboidal granulosa cells or a single cuboidal granulosa cell layer); secondary follicles (oocytes surrounded by multiple layers of granulosa cells); and antral follicles (granulosa cells with pyknotic nuclei and large lacunae between granulosa cells).

[0089] Method for counting primordial and primary follicles: Count every fifth section of each ovary, and multiply the total by five to estimate the total number. Secondary and antral follicles were counted in full on each section (only follicles containing visible oocytes with clear nuclei stained with hematoxylin were counted to avoid double counting). The total number of follicles per ovary was the sum of the number of follicles at all developmental stages, while the total number of follicles in the intact ovary was the sum of the number of follicles in both halves of the ovary. The follicle depletion rate was the ratio of the number of follicles consumed per day to the number of follicles initially transferred (PD 21 + day 0).

[0090] (7) Immunofluorescence and imaging

[0091] Ovarian tissue sections were deparaffinized in xylene and rehydrated with graded ethanol. Antigen retrieval was performed using microwave-assisted citrate buffer (pH 6.0). Sections were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated overnight at 4°C with the following primary antibodies: FOXO3A (dilution 1:300, Cat. No. 12829, rabbit, CellSignaling Technologies) and DEAD-box helicase 4 (DDX4) (dilution 1:300, Cat. No. ab27591, mouse, Abcam). After washing three times with PBS, sections were incubated with secondary antibodies: Alexa Fluor 555-conjugated donkey anti-mouse antibody or Alexa Fluor 488-conjugated donkey anti-rabbit antibody (dilution 1:100, Life Technologies). Nuclear staining was performed with Hoechst 33342 (dilution 1:100, Cat. No. C1022, Beyotime). Tissue imaging was performed using a Leica inverted microscope (DMi8) and an Andor Dragonfly spinning disk confocal microscope, and parameters such as primordial follicle activation were analyzed using ImageJ software. Immunofluorescence and follicle counting sections were obtained from different ovaries but processed using the same process.

[0092] (8) Ovarian tissue transparency and high-resolution three-dimensional imaging

[0093] The tissue clearing protocol is a slightly modified version of the existing technology. The specific steps are as follows: molten N-methylacetamide (Product No. M26305, Sigma-Aldrich) is diluted with phosphate-buffered saline (PBS) to a 40% (volume percentage) stock solution; Histodenz (Product No. D2158, Sigma-Aldrich) is dissolved in the stock solution to a final concentration of 86% (mass-volume ratio, unit: g / mL); Triton X-100 (0.1%, volume percentage) and 1-thioglycerol (Product No. M1753, Sigma-Aldrich) (0.5%, volume percentage) are added to prepare the final clearing solution.

[0094] Ovarian vascular network visualization process: Ovarian tissue or explants were washed in PBS containing 0.2% Triton X-100 and 0.5% 1-thioglycerol for 24 hours (at room temperature in the dark to remove residual blood cells). Subsequently, the tissue was immersed in clearing solution at a ratio of 1:50 (volume percentage) and incubated with rotation at room temperature in the dark for 48 hours. The cleared tissue was placed in a 35mm glass-bottomed dish (Cat. No. D35-14-1-N, Cellvis) containing fresh clearing solution for imaging.

[0095] High-resolution 3D imaging was performed using an Andor Dragonfly spinning disk confocal microscope (equipped with a ×40 objective, an Andor Zyla 4.2 scientific-grade CMOS camera, and an Andor integrated laser engine system) with excitation wavelengths of 405 nm (Hoechst), 488 nm, and 568 nm (mT). A spinning disk confocal scanner (Andor Dragonfly 500) enhanced imaging efficiency and quality, and image acquisition was performed using Fusion 2.1 software (https: / / andor.oxinst.com / products / dragonfly#fusion).

[0096] Image post-processing was performed using ImageJ software (http: / / rsbweb.nih.gov / ij / ): All z-stack projections were merged to generate a three-dimensional model of the vascular network. Vascular density was calculated as the percentage of green fluorescent vessel area to the total ovarian tissue area. To highlight the filopodia at the tip of the cells, the mG (488 nm) channel images were inverted to black and white using ImageJ.

[0097] (9) Quantitative analysis of the number of tip cells

[0098] Quantitative analysis of tip cells was based on reconstructed three-dimensional imaging data of the ovary. Images were analyzed using Imaris software (https: / / imaris.oxinst.com / ) in 3D view and slice mode. The overall imaging data were segmented into optical stacks approximately 50 μm thick along the z-axis. In randomly selected optical stacks, GFP-labeled blood vessels were manually inspected, and filopodia were identified using the filamentous structure tracking function of Imaris. Tip cell density was calculated by counting the number of tip cells in randomly selected areas of ovarian tissue or grafts (volume 500 μm × 500 μm × 50 μm), with 5 to 10 areas evaluated per ovary (n ≥ 3 ovaries per group). Tip cell density = total number of tip cells in the selected area / corresponding tissue volume.

[0099] (10) In vitro fertilization

[0100] Fourteen days after transplantation, recipient mice were intraperitoneally injected with 250 IU / kg pregnant mare serum gonadotropin (PMSG, Sansheng Biotechnology), followed 46-48 hours later by a booster injection of human chorionic gonadotropin (hCG, Sansheng Biotechnology). Thirteen hours after hCG injection, cumulus-ovarian complexes (COCs) were recovered from the subrenal capsule, and the number of MII oocytes was counted. MII oocytes were collected and placed in HTF medium (Cat. No. M1130, Abi Biotechnology). Epididymal sperm from male mice were incubated in HTF medium for 30 minutes to achieve capacitation. For in vitro fertilization, capacitated sperm were mixed with MII oocytes in HTF fertilization medium. Four hours later, zygotes were transferred to a drop of KSOM medium (Cat. No. M1430, Abi Biotechnology) covered with mineral oil (Cat. No. M8410, Sigma-Aldrich) for continued culture. Approximately 16 hours after fertilization, the number of two-cell embryos was counted, and the number of blastocysts was subsequently counted.

[0101] (11) Total protein extraction and purification

[0102] Fresh or frozen ovarian tissue was homogenized in RIPA buffer (Cat. No. 89900, ThermoFisher) in a 1.5 mL centrifuge tube on ice. Samples were sonicated at 70% power for 40 minutes (20 seconds of sonication / 40 seconds interval), followed by centrifugation at 14,000 rpm for 10 minutes. The supernatant was collected as a total protein extract. Protein concentration was determined using a BCA assay kit (Cat. No. 5000201, Bio-Rad) and standardized. Equal volumes of samples were washed three times with 8 M urea to remove residual detergents (particularly SDS). Trypsin was then added at a protein:enzyme ratio of 50:1 and digested overnight at 37°C. Protein expression profiles were analyzed using a Q Exactive mass spectrometer.

[0103] (12) Protein mass spectrometry analysis

[0104] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using a Waters nanoAcquity system (Waters, USA) coupled to a Q-Exactive high-resolution mass spectrometer (Thermo Scientific, USA). The peptides were enriched on a trapping column (Acclaim PepMap100, 75 μm × 2 mm, Thermo Scientific) and eluted onto a 20 cm column filled with a C18 stationary phase (Aqua C18, 3 μm, A fused silica capillary column (Phenomenex, USA) was used. The elution gradient was 125 minutes, with mobile phases A (0.1% formic acid in water) and B (acetonitrile) at a flow rate of 400 nL / min. The nanoelectrospray voltage was set at 2.0 kV. The full-scan mass spectrometry range was m / z 300–1800, with a resolution of 70,000 (at m / z 200). The secondary mass spectrometry (MS / MS) was performed at a resolution of 17,500, targeting the top 10 most abundant peptide signals, with a dynamic exclusion time of 20 seconds.

[0105] (13) Bioinformatics analysis

[0106] The significance of protein expression differences was assessed using the Student's t-test (Prism 9 for macOS, version 9.2.0), with a significance threshold of p < 0.05. Data are presented as mean ± standard deviation (SD). Differentially expressed proteins (DEPs) were identified based on a fold change > 1.5 and p < 0.05 and visualized using cluster heatmaps. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) enrichment analysis of DEPs were performed using the Metascape platform. Volcano plots, cluster heatmaps, and enrichment visualizations were generated using the Bioinformatics Online tool (https: / / www.bioinformatics.com.cn).

[0107] (14) Experimental design and statistical basis

[0108] This study aimed to evaluate protein expression differences between fresh and cryopreserved ovarian tissue from 21-day-old (PD21) mice using label-free mass spectrometry. Each group included at least two biological replicates (n = 2 ovaries per sample). Sample size was determined based on statistical power analysis. Fresh ovarian tissue served as a control to assess baseline protein expression levels and control for experimental variability. Biological replicates were defined as independent samples from different individuals. Both biological and technical replicates were included to ensure robustness of the results and assess inter- and intra-sample variability. Protein identification and quantification were performed using the PEAKS search engine (PEAKS studio 11, Bioinformatics Solutions Inc., Canada), including peak alignment, peak extraction, and protein quantification. Database search parameters were as follows: 1) Specific enzyme: trypsin, with a maximum of three missed cleavage sites allowed; 2) Fixed modification: carboxymethylation of cysteine residues (C); 3) Variable modification: oxidation of methionine (M); 4) Precursor ion mass tolerance: 10 ppm; 5) Fragment ion mass tolerance: 0.02 Da. Data were matched to the Uniprot human database (45,182 sequences, June 2018 version, https: / / www.uniprot.org / ). Points with mass spectrometry signals below the threshold or sample preparation anomalies were excluded, and all exclusion criteria were applied uniformly across the dataset. The Student's t-test assumed that the data were normally distributed; if normality was not satisfied, nonparametric tests were considered. Peptide identifications were filtered using a 1% false discovery rate (FDR) (FDR estimated using a decoy database), and only peptides with a confidence score >31 were selected for subsequent analysis. The raw mass spectrometry data have been submitted to ProteomeXchange under the accession number PXD061025 (https: / / www.iprox.cn / page / project.html?id=IPX0011049000).

[0109] (15) Statistical analysis

[0110] All experiments included at least three biological replicates. Data were analyzed using GraphPad Prism (version 9.2.0) and presented as mean ± standard deviation (SD). Intergroup comparisons were performed using the Student's t-test, with a significance threshold of P < 0.05. Significance levels are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no significant difference (P ≥ 0.05).

[0111] (2) Results

[0112] (1) Impaired angiogenesis and abnormal activation of primordial follicles lead to rapid loss of follicles in frozen ovarian tissue

[0113] To investigate the mechanism of follicle loss during ovarian tissue cryopreservation and transplantation (OTCT), this study used adolescent female mouse ovaries to establish a mouse model. The ovaries were dissected to simulate clinical injury, frozen, revived, and transplanted under the renal capsule. Within 14 days after transplantation, the frozen ovarian tissue developed into antral follicles (e.g., Figure 1 As shown, the red arrow indicates the antral follicle) and releases mature (MⅡ stage) oocytes (as shown Figure 2 These oocytes can be successfully fertilized in vitro and develop to the blastocyst stage (as shown on the left) through superovulation. Figure 2 This indicates that the experimental protocol effectively simulates the clinical OTCT process.

[0114] To quantify the degree of follicle depletion, ovarian grafts were collected 3, 7, and 14 days after transplantation. DDX4 staining confirmed the presence of oocytes and follicles at each time point (e.g. Figure 3 Quantitative analysis showed that the number of follicles in both frozen and fresh ovarian transplants decreased rapidly and significantly within the first 3 days after transplantation (as shown in Figure 2). Figure 4 As shown in the figure, 0 day: 2756±128 vs 2790±117; 3 days: 713±94 vs 1429±113), among which fresh grafts lost about half of the follicles, while frozen grafts were more depleted, losing more than two-thirds of the follicles. It is worth noting that the rate of follicle loss in frozen ovaries was significantly higher than that in fresh ovaries 0 to 3 days after transplantation (such as Figure 5 As shown in the results of the present study, the follicle loss rate was 24.71±1.13% vs 17.58±1.35% / day, 681±31 vs 490±38 / day. After that, the follicle loss rate slowed down (3-7 days: 2.10±0.98% vs 1.21±0.52% / day, 33±14 vs 59±27 / day; 7-14 days: 1.70±0.53% vs 1.37±0.53% / day, 38±15 vs 49±16 / day). These results indicate that the critical period of follicle loss is concentrated in the early stage after transplantation and that cryopreservation exacerbates follicle depletion.

[0115] To understand the mechanism of rapid loss, proteomic analysis was performed by mass spectrometry. A total of 610 differentially expressed proteins (DEPs) were identified between frozen and fresh ovarian tissues, of which 242 were upregulated and 368 were downregulated in the frozen group (e.g. Figure 6 Cluster analysis showed that the protein expression profiles of the two groups were significantly separated (as shown in Figure 7 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that DEPs were significantly enriched in angiogenesis-related pathways, including the “HIF-1 signaling pathway” and the “VEGF signaling pathway” (as shown in Figure 2). Figure 8Gene Ontology (GO) enrichment analysis further revealed vascular network development-related terms such as “endothelial cell development,” “negative regulation of angiogenesis,” “negative regulation of vascular morphogenesis,” and “vascular diameter maintenance,” suggesting that angiogenesis disorders may drive rapid follicle loss.

[0116] In addition to vascular-related changes, KEGG pathway analysis also found significant changes in pathways related to primordial follicle activation in frozen ovaries, such as the "PI3K-Akt signaling pathway" and the "FoxO signaling pathway" that regulate the activation of resting oocytes (such as Figure 8 In addition, the activation state of pregranular cells and their communication with surrounding tissues, such as the Hippo signaling pathway and the mTOR signaling pathway, also changed (e.g. Figure 8 (as indicated by the red mark in the figure).

[0117] The above results indicate that the mechanism of accelerated loss of frozen ovarian follicles may be due to the synergistic effect of vascular regeneration disorder and abnormal activation of primordial follicles.

[0118] (2) Cryoinjury-induced tip cell destruction and overactivation of primordial follicles jointly drive rapid follicle loss in OTCT

[0119] To understand the mechanism of angiogenesis disorder in frozen ovarian tissue, this study used Tek-CreER T2 mTmG endogenous fluorescence reporter mouse model. This model uses membrane-localized GFP to label the ovarian tissue vascular network, enabling subcellular resolution observation of vascular structures under whole-tissue transparent imaging. The results showed that both frozen and fresh ovarian tissues contain tip cells that guide angiogenesis (such as Figure 9 Quantitative analysis showed that the overall vascular density of frozen ovaries was significantly lower than that of fresh ovaries (as shown in Figure 10 、 Figure 11 As shown in the figure, 61.66±4.34% vs 74.57±3.22%), suggesting that the freezing process caused damage to the vascular network structure.

[0120] High-resolution imaging further revealed that the number of cells with typical filopodia tips per unit volume of frozen ovaries (e.g. Figure 12 The red arrow in the figure shows a sharp decrease compared to the fresh group (as shown in Figure 13 As shown, 1286.62±267.59 vs 5376.16±1941.72 cells / mm3). It is worth noting that compared with the fresh group, the number of cells in the frozen ovarian tip decreased by 76.08% (according to Figure 13 Calculated), while mature blood vessels only decreased by 17.31% (based on Figure 11This significant difference suggests that tip cells, responsible for guiding angiogenesis, are more susceptible to freezing damage than mature endothelial cells. The increased fragility of tip cells may be a key factor contributing to impaired vascular remodeling after transplantation.

[0121] To verify the effect of vascular injury on the vascular regeneration capacity of frozen ovarian transplants, whole-tissue high-resolution imaging analysis was performed 3 days after transplantation. Figure 14 The number of the 3D-type 247 cells in the fresh transplant group (shown by the red arrows in the figure) recovered somewhat, but was still significantly lower than that of the fresh transplant group (as shown in the figure Figure 15 As shown in Figure 5, 5028.03±997.49 vs 7792.87±2061.45 cells / mm3). The density of vascular reconstruction in frozen grafts was also significantly lower than that in the fresh group (e.g. Figure 16 、 17 These results suggest that cryotherapy-induced tip cell damage impairs the ability of ovarian tissue to rebuild its vascular network, which is an important mechanism leading to the rapid depletion of ovarian reserve after transplantation.

[0122] In addition to vascular damage, this study further explored the effect of freezing on the over-activation of primordial follicles. Proteomics suggested that primordial follicle activation was enhanced (e.g. Figure 8 (as shown in red), for this purpose, the present invention detected the localization changes of forkhead box protein O3 (FOXO3A), a key marker of follicle activation. FOXO3A is enriched in the nucleus of resting oocytes and translocated to the cytoplasm after activation (22,38,39). Comparison of normal ovaries, freshly isolated ovaries and frozen ovaries in vivo revealed that the proportion of FOXO3A cytoplasmic localization in primordial follicles in the freshly isolated group and the frozen group (as shown in red) was significantly higher than that in the frozen group. Figure 18 The white arrows in the figure) were significantly higher than those in the control group ( Figure 19 As shown in the results, the fresh isolation group had 22.53±1.19%, the frozen group had 49.18±1.93% vs the in vivo control group had 4.98±1.66%), confirming that tissue processing (mechanical manipulation and freezing) can induce abnormal activation of primordial follicles.

[0123] In summary, cryoinjury and mechanical stress have a dual destructive effect on ovarian tissue: on the one hand, cryotherapy specifically damages tip cells, leading to impaired angiogenesis; on the other hand, tissue handling and freezing synergistically trigger overactivation of primordial follicles. Therefore, optimizing OTCT strategies requires inhibiting follicle activation from the tissue isolation stage while simultaneously promoting angiogenesis.

[0124] (3) Combined use of VEGFA and rapamycin to alleviate vascular damage and inhibit excessive activation of primordial follicles

[0125] This study found that the rapid loss of frozen ovarian follicles is driven by vascular damage and overactivation of primordial follicles. T2 mTmG ovaries were treated with VEGFA (5 ng / mL) for 6 hours (the control group was treated with H2O). Whole-tissue high-resolution imaging showed that the vascular density in the VEGFA-treated group was significantly increased compared with the control group (e.g. Figure 19 、 20 As shown in Figure 2, 78.25±6.12% vs 51.02±3.97%), indicating that VEGFA can effectively repair the integrity of vascular structure. In addition, the cell density at the tip of the VEGFA-treated group was moderately increased compared with the control group (as shown in Figure 2). Figure 21 、 22 As shown in the figure, 3274.55±864.32 vs 1594.13±417.97 cells / mm3), suggesting that short-term VEGFA exposure enhances angiogenesis by promoting the recruitment of tip cells.

[0126] To evaluate the long-term effect of VEGFA on vascular development after transplantation, the vascular network of frozen ovarian grafts was examined 3 days after transplantation. The vascular density of the VEGFA pretreatment group was significantly higher than that of the control group (e.g. Figure 24 、 25 As shown, 49.50±7.13% vs 32.97±13.85%), the number of tip cells per unit volume also increased significantly (as shown in Figure 26 、 27 As shown, 8994.02±4895.86 vs 4996.05±2640.24 cells / cubic mm), confirming that VEGFA pretreatment can effectively alleviate freezing vascular injury and support vascular network reconstruction.

[0127] To inhibit the overactivation of primordial follicles, this study added the mTORC1 inhibitor rapamycin during the entire ovarian tissue processing process (including isolation, freezing, thawing and pre-culture). The proportion of cytoplasmic FOXO3A in oocytes in the rapamycin-treated group was significantly lower than that in the control group (e.g. Figure 28 As shown by the white arrow in Figure 29 As shown in Figure 2, 10.12±1.26% vs 48.19±4.78%), indicating that the activation of primordial follicles was inhibited. Three days after transplantation, the number of surviving follicles in the rapamycin-treated group was significantly higher than that in the untreated group (as shown in Figure 2). Figure 30 As shown, 965.50±129.58 vs 711.5±114.10), the rate of follicle loss also slowed down (as shown in Figure 31 As shown, 21.44±1.60% vs 24.86±1.36% / day).

[0128] Further monitoring showed that 14 days after transplantation, the rapamycin-treated group still retained more primordial follicles (e.g. Figure 32As shown by the red arrow in Figure 33 as shown, 298.5±67.09 vs 154±86.47).

[0129] These results suggest that rapamycin significantly improves follicle survival and maintains ovarian reserve by inhibiting overactivation of primordial follicles. The combined use of VEGFA (angiogenic) and rapamycin (activation inhibitor) provides a feasible approach to mitigate ovarian cryopreservation damage.

[0130] (4) VEGFA-rapamycin combination strategy improves ovarian reserve protection efficiency

[0131] Given the complementary protective effects of VEGFA and rapamycin on ovarian reserve, this study developed a VEGFA-Rapamycin Combination Strategy (VRCS) to improve follicle survival in cryopreserved ovarian tissue. In this strategy, rapamycin is administered throughout the entire process from tissue collection to freezing, thawing, and pre-culture, while VEGFA is added after tissue thawing and before transplantation to enhance follicle survival.

[0132] To evaluate the effectiveness of VRCS, the survival and development of follicles after frozen ovarian transplantation were compared with rapamycin alone (Cryo+Rapa) as a control. Three days after transplantation, the number of follicles in the VRCS group was significantly higher than that in the control group (e.g. Figure 34 As shown, 1197±124.11vs916±104.45), the follicle consumption rate was significantly reduced (as shown in Figure 35 This protective effect lasted until 14 days after transplantation, and the number of surviving primordial follicles in the VRCS group was still higher than that in the single-drug group (as shown in Figure 2). Figure 36 、 37 As shown, 329.4±36.12 vs 263.5±40.56), indicating that VRCS can provide better short-term protection for frozen ovarian reserves.

[0133] To analyze the effect of VRCS on oocyte yield and developmental potential, recipient mice were superovulated 14 days after transplantation and transplanted oocytes were collected (e.g. Figure 38 The number of oocytes retrieved per ovary in the VRCS group was significantly higher than that in the single-drug group (as shown on the left). Figure 39 In addition, the developmental potential of oocytes in the VRCS group was significantly enhanced: the formation of 2-cell embryos (such as Figure 28 As shown by the red arrow, Figure 40 As shown, VRCS: 72.93 ± 8.17% vs Rapa alone: 25.50 ± 10.52%) and blastocysts (as Figure 28 As shown by the right blue arrow, Figure 40 As shown in the results, the proportions of VRCS: 46.25±4.15% vs Rapa monotherapy: 12.5±9.01% were significantly increased, confirming that VRCS is superior to monotherapy in maintaining follicle survival and oocyte development ability.

[0134] In summary, the VEGFA-rapamycin combination strategy can more effectively maintain frozen ovarian tissue reserves and improve oocyte quality, providing an innovative solution for clinical fertility preservation.

[0135] (5) VEGFA-rapamycin combination strategy prolongs the duration of female reproductive function after OTCT

[0136] Based on the short-term protective effect of VRCS on frozen ovarian tissue, this study further evaluated its long-term protective effect on ovarian reserve. Frozen ovarian tissues with different treatments (untreated, rapamycin alone, VRCS) were transplanted under the renal capsule of recipient mice and the estrous cycle changes (such as Figure 41 The mice in the untreated group maintained regular estrous cycles (4-6 days / cycle) for up to 11 weeks, and the longest duration was 13 weeks (as shown). Figure 42 As shown, estrous cycle analysis showed that 50% of the mice in the untreated group maintained regular cycles until 11 weeks, and the remaining 50% maintained them until 13 weeks; 50% of the mice in the rapamycin group maintained them until 14 weeks, and the remaining until 16 weeks; while all mice in the VRCS group maintained regular cycles throughout the monitoring period); indicating that VRCS significantly prolongs the functional period of transplanted ovaries compared to single-drug treatment.

[0137] To evaluate the long-term preservation of ovarian reserve, grafts were collected four months later and the number of residual follicles was analyzed histologically. The number of residual follicles in the rapamycin group was higher than that in the untreated group, while the number of residual follicles in the VRCS group was the highest (e.g. Figure 43 The quantitative results showed that the total number of residual follicles in the untreated group was 74.5±34.77 (primordial follicles 1.75±2.49, primary follicles 27.75±18.16, secondary follicles 33.75±13.55, antral follicles 11.25±3.19) (as shown in the red arrow in the figure). Figure 44 、 45 The ovarian follicle count in the rapamycin group was 151.5±29.06 (primordial follicles 18.75±9.68, primary follicles 42.75±9.63, secondary follicles 67±10.05, antral follicles 23±8.15); the ovarian follicle count in the VRCS group was 283±71.93 (primordial follicles 92.75±58.46, primary follicles 73±12.02, secondary follicles 91.25±12.6, antral follicles 26±4.95) (as shown in Figure 2). Figure 44 、 45These results indicate that the protective effect of VRCS on ovarian reserve can last up to four months after transplantation and is significantly better than monotherapy.

[0138] In summary, VRCS significantly improves the long-term preservation and functional activity of frozen ovarian tissue. While rapamycin alone effectively inhibits overactivation of primordial follicles, it may limit oocyte developmental potential. Combining VRCS with VEGFA promotes angiogenesis and reconstruction after transplantation, reduces rapid follicle loss, and improves oocyte quality. The VEGFA-rapamycin combination strategy offers a superior approach for maintaining frozen ovarian function.

[0139] (3) Discussion

[0140] Ovarian tissue cryopreservation and transplantation (OTCT) has become a core clinical approach for fertility preservation and has resulted in hundreds of successful live births. However, its application still faces a significant challenge: ischemia-reperfusion injury after transplantation leads to a rapid loss of ovarian reserve. The graft is exposed to a hypoxic environment for 3 to 5 days after surgery, relying solely on diffusion from surrounding tissue for nutrients and oxygen. This critical "window period" leads to a significant depletion of primordial follicles. This study mapped an ovarian reserve depletion curve using a mouse model, clearly demonstrating that the first 3 days after transplantation represent a period of rapid follicle loss. Understanding the mechanisms underlying this period is crucial for elucidating the mechanisms of follicle loss in OTCT.

[0141] This study found that damage to tip cells, key cells for angiogenesis, is a significant driver of post-transplant follicle loss. Tip cells are crucial for vascular regeneration, and their number decreases significantly after cryotransplantation, severely impairing the ability to regenerate the vascular network. Using an endogenous fluorescent reporter mouse model combined with advanced imaging techniques, the researchers demonstrated that the cryothaw process disrupts the inherent structure of blood vessels, leading to a decrease in tip cell density and reduced angiogenesis after transplantation. This suggests that supporting vascular regeneration is key to preventing ovarian reserve depletion.

[0142] In addition to vascular damage, this study highlights the important role of overactivation of primordial follicles in ovarian reserve depletion during OTCT. This overactivation is not only due to cryoinjury but is also exacerbated by mechanical stress during early tissue manipulation. Results suggest that continuous application of rapamycin throughout tissue isolation, freezing, thawing, and pre-culture effectively inhibits follicle activation, potentially offering a protective advantage over intermittent application.

[0143] However, although rapamycin inhibits excessive follicular activation, it may have a negative impact on ovulation and oocyte developmental potential. In this study, the number of superovulated oocytes retrieved in the rapamycin-only treatment group was reduced, and the formation rate of 2-cell embryos and blastocysts was reduced, which may be related to rapamycin inhibiting the PI3K pathway, which is continuously active in developing follicles. In addition, studies have shown that rapamycin inhibits angiogenesis by downregulating VEGFA expression and its downstream VEGFR signaling pathway, which may aggravate post-transplant ischemia-reperfusion injury, increase reactive oxygen species (ROS) levels, and impair oocyte quality. Interestingly, the combined use of VEGFA can improve the above-mentioned negative effects by promoting ovulation and fertilization, but its specific mechanism still needs to be further analyzed.

[0144] (IV) Conclusion

[0145] The VEGFA-rapamycin combination strategy (VRCS) demonstrates significant advantages over monotherapy: by promoting angiogenesis, enhancing vascular remodeling, and continuously inhibiting primordial follicle activation, VRCS provides more effective protection of the ovarian reserve after transplantation. VRCS is expected to significantly improve OTCT outcomes and provide a new fertility preservation option for patients receiving gonadotoxic therapies (such as chemotherapy). This strategy not only helps restore fertility in cancer survivors but also has significant translational value in protecting endocrine function and improving patients' long-term quality of life.

[0146] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of rapamycin combined with vascular endothelial growth factor A in the preparation of drugs for improving ovarian tissue function.

2. The use according to claim 1, characterized in that The improvement of ovarian tissue function includes any one or more of the following aspects: increasing follicle survival rate, extending ovarian tissue function lifespan, and improving oocyte quality.

3. The use according to claim 1, characterized in that: The effective concentration of the rapamycin is 500 nM, and the effective concentration of the vascular endothelial growth factor A is 5 ng / mL.

4. The use according to claim 1, 2 or 3, characterized in that: The specific application method is: rapamycin is used to treat ovarian tissue during the collection, cryopreservation and recovery stages; during the in vitro culture stage of frozen ovarian tissue, rapamycin and vascular endothelial growth factor A are used in combination for treatment.

5. The use according to claim 4, characterized in that: During the collection phase, cells were washed with phosphate buffered saline containing 500 nM rapamycin; during the cryopreservation phase, 500 nM rapamycin was added to the cryopreservation solution; During the resuscitation phase, 500 nM rapamycin was added to the resuscitation solution; During the in vitro culture stage, 500 nM rapamycin and 5 ng / mL vascular endothelial growth factor A were added to the culture medium.

6. The use according to claim 5, characterized in that: The freezing preservation fluid includes vitrification fluid I and vitrification fluid II, wherein vitrification fluid I is composed of 20% fetal bovine serum, 7.5% dimethyl sulfoxide, 7.5% ethylene glycol and the remainder DMEM / F12 culture medium, calculated by volume percentage; vitrification fluid II is composed of 20% fetal bovine serum, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.25M sucrose and the remainder DMEM / F12 culture medium, calculated by volume percentage.

7. The use according to claim 5, characterized in that: The resuscitation fluid is a DMEM / F12 culture medium containing fetal bovine serum and decreasing concentrations of sucrose, wherein the fetal bovine serum accounts for 20%, and the sucrose concentrations are 0.5M, 0.25M, 0.125M and 0M respectively.

8. The use according to claim 5, characterized in that: During the in vitro culture stage, the revived ovarian tissue was placed in DMEM / F12 culture medium containing 10% fetal bovine serum, 500 nM rapamycin and 5 ng / mL vascular endothelial growth factor A, and pre-cultured at 37°C, 5% CO2 and saturated humidity for 6 hours.

Images