A method for in vitro activation of AOI oocytes and preservation of fertility

Abstract

The present invention relates to the technical field of in vitro oocytes, and specifically to a method for in vitro activation of AOI oocytes and preservation of fertility, comprising the following steps: S1, isolation and initial feature analysis of oocytes; S2, acquisition and typing of somatic cells; S3, construction of chimeric follicles; S4, optimization of 3D culture system; S5, design of functionalized biomaterials; S6, evaluation of follicle activity; S7, development of engineered cell systems; S8, development of non-invasive monitoring systems. The present invention uses animal and cell models and molecular biological methods to explain the interaction between somatic cells, oocytes and extracellular matrix and their specific mechanism of action, develop an optimized human follicle 3D culture system, and standardize the specific parameters of chimeric follicles and oocyte recovery, and further improve the quality and recovery potential of oocytes by designing an in vitro ovarian biological system.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro oocytes, and in particular to a method for in vitro activation of AOI oocytes and preservation of fertility. Background Art

[0002] With the development of modern society, economy and education, people's views on marriage and childbearing have changed greatly, and more and more women are delaying the age of first childbearing. At the same time, with the full opening of the two-child policy, many older women will choose to have children again. In this case, women generally need to face the problem of decreased fertility caused by late pregnancy.

[0003] Regarding this issue, the academic community generally believes that women's fertility will gradually decline with age until it is almost zero. The reason is that human fertility is determined by reproductive tissues, and its main functional unit is the follicle, which is contained in the ovary. The follicle is composed of eggs or oocytes and normal somatic cells. Among them, all cells are closely connected to the extracellular matrix, whether in the development process or in the state of homeostasis. The development of ovarian follicles depends on capillaries to provide nutrients and hormones. In addition, the interaction between follicles is conducive to the survival of follicles. These reveal the importance of the microenvironment to follicular development. Somatic cells nourish and help the developing eggs mature before ovulation. The mature oocytes are released by ovulation and then fertilized to become embryos. With age, ovarian tissue and extracellular matrix will change and harden. This tissue hardening is associated with the increase of age-related diseases and the loss of fertility. At the same time, the metabolites of the microenvironment around the follicles will change. Studies have shown that the main causes of ovarian aging are the following: a decrease in the number of follicles and a decrease in the quality of follicles; the ovarian microenvironment is also crucial to the healthy development of follicles. The decrease in the number of follicles is caused by an increase in the atresia rate of follicles in the primordial follicle pool, while the decrease in follicle quality is mainly due to endocrine-induced follicular dysplasia, which leads to granulosa cell dysfunction and decreased oocyte quality.

[0004] To solve the above problems, we provide a method for in vitro activation of AOI oocytes and preservation of fertility. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the first object of the present invention is to provide a method for in vitro activation and fertility preservation of AOI oocytes to solve the problems in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for in vitro activation of AOI oocytes and preservation of fertility, comprising the following steps:

[0008] S1, Oocyte isolation and initial characterization;

[0009] S2. Acquisition and typing of somatic cells;

[0010] S3, chimeric follicle construction;

[0011] S4. Optimization of 3D culture system;

[0012] S5. Functional biomaterial design;

[0013] S6, follicle activity assessment;

[0014] S7, Engineered cell system development;

[0015] S8. Development of non-invasive monitoring system.

[0016] The present invention is further configured as follows: in the step S1, separation and initial feature analysis of oocytes:

[0017] S1.1. Isolate oocytes from surgically obtained human ovarian tissue under sterile conditions;

[0018] S1.2, the tissue was first enzymatically digested with 0.25% collagenase I and 0.05% trypsin at 37°C for 15 min, and then filtered through a 40 μm cell sieve to remove larger tissue pieces;

[0019] S1.3, the isolated oocytes were temporarily stored in DMEM / F12 medium with HEPES buffer;

[0020] S1.4. Use a Nikon Ti-E fluorescence microscope to detect the integrity of the zona pellucida and label mitochondria with MitoTracker dye to observe their distribution and aggregation at an excitation wavelength of 488 nm.

[0021] S1.5, use PiumaNanoIndenter dynamic micro-pressure probe to evaluate the mechanical properties of the oocyte surface (hardness range is usually 1–10 kPa);

[0022] S1.6. Use qRT-PCR to perform preliminary screening of gene expression in oocytes.

[0023] The present invention is further configured as follows: in the step S2, obtaining and typing somatic cells:

[0024] S2.1, Granulosa cells and cumulus cells were isolated from ovarian tissue, high-quality cells were isolated from the gradient range of 30%-60% by Percoll density gradient centrifugation, and dead cells were excluded by Trypan Blue;

[0025] S2.2. Use the 10×Genomics single-cell transcriptome sequencing platform to accurately genotype the gene expression of these cells, with special attention to the expression of marker genes such as FOXL2 and CYP19A1;

[0026] S2.3. Within 24 hours after isolation, cells were cultured in a 2D tile format in DMEM medium containing 10% FBS and 1% insulin-transferrin-selenium, with the oxygen concentration controlled at 5%;

[0027] S2.4. GC-MAF and other secretory proteins were detected by Western Blot to evaluate their endocrine functions, combined with immunofluorescence to detect key pathway molecules.

[0028] The present invention is further configured as follows: in the step S3, constructing the chimera follicle:

[0029] S3.1. Granulosa cells and oocytes from subjects of different ages were assembled into chimeric follicles using an Eppendorf CellTram microinjection device in a CO2 incubator at 37°C and 5% CO2 concentration;

[0030] S3.2, the cell assembly was fixed by a three-dimensional collagen scaffold (concentration 1.5 mg / mL) and embedded in a hydrogel modified by natural hyaluronic acid and gelatin to maintain its structure;

[0031] S3.3. Real-time live cell imaging was used to observe cell-cell adhesion and interaction using a Leica SP8 laser confocal microscope, while SIRT1 repressor genes were knocked out in aged granulosa cells using CRISPR-Cas9;

[0032] S3.4, the constructed follicles are dynamically cultured in a low shear force microfluidic culture system. The present invention is further configured as follows: in the optimization of the 3D culture system in step S4:

[0033] S4.1. A dynamic 3D culture system was constructed using a PDMS microfluidic chip with a channel width of 100 μm, and a mixed ECM scaffold of collagen I and IV with a ratio of 3:1 to simulate ovarian tissue was added;

[0034] S4.2, hardness was controlled between 300–800 Pa by AFM atomic force microscopy to simulate the ovarian environment at different ages;

[0035] S4.3, AMH at a concentration of 10 ng / mL and VEGF at a concentration of 20 ng / mL were added to the culture medium, and the culture medium was renewed every 48 hours;

[0036] S4.4. Monitor the mechanical properties of the culture system in real time using a Brillouin microscope, and adjust the pH to 7.4 and the oxygen partial pressure to 5% according to cell growth conditions.

[0037] The present invention is further configured as follows: in the step S5, functionalized biomaterial design:

[0038] S5.1: Based on natural collagen and synthetic polyethylene glycol diacrylate at a concentration of 10% w / v, combined with glutaraldehyde crosslinker to generate scaffold material;

[0039] S5.2: Mechanical properties were tested by Instron 5940 tensile tester, and the Young's modulus was maintained between 2–5 kPa to adapt to folliculogenesis;

[0040] S5.3: The biomaterial is embedded with TGF-β and IGF-1 signaling molecules by solution polymerization, and the sustained release function is achieved by microencapsulation technology with a diameter of about 100 μm;

[0041] S5.4: Co-culture the material with ovarian follicles to verify its ability to promote oocyte growth.

[0042] The present invention is further configured as follows: in the step S6, follicle activity assessment:

[0043] S6.1: After follicles are extracted from the culture system, spindle morphology is examined using immunofluorescence staining, where antibody markers include α-tubulin;

[0044] S6.2: Extract oocyte RNA, perform transcriptome sequencing using the Illumina NovaSeq platform, and combine metabolomics detection to target energy metabolites ATP and NADH;

[0045] S6.3: 1×10 spermatozoa from healthy males were used in the in vitro fertilization experiment. 6 / mL to evaluate the fertilization rate, and observe the development of the blastocyst under a DAPI staining microscope;

[0046] S6.4: Use AI image processing technology to analyze follicle quality and maturity.

[0047] The present invention is further configured as follows: in the step S7, developing the engineered cell system:

[0048] S7.1: CRISPR-Cas9 gene editing was performed on granulosa cells to knock in the PGC1α gene;

[0049] S7.2: The edited cells were expanded in a rotating environment at RPM 20 / min in medium containing 5% FBS.

[0050] S7.3: These engineered granulosa cells were co-cultured with oocytes, 10 μM glutathione was added to enhance antioxidant capacity, and the concentration of FGF2 growth factor secreted by them was measured by LC-MS;

[0051] S7.4: Single-cell transcriptome sequencing was used to confirm the molecular stability of engineered cells cultured for a long time.

[0052] The present invention is further configured as follows: in the step S8, in the development of the non-invasive monitoring system:

[0053] S8.1: Design a real-time imaging platform based on optical coherence tomography with a resolution of 1 μm to monitor changes in follicle morphology;

[0054] S8.2: A responsive nanosensor with a gold nanorod diameter of 10 nm was embedded in the culture medium to detect the concentration of the metabolite lactate by changes in fluorescence intensity;

[0055] S8.3: Use nonlinear optical techniques in conjunction with reflectance spectroscopy to detect zona pellucida thickness;

[0056] S8.4: The AI ​​algorithm is written in Python and integrates multimodal data analysis to automatically generate dynamic reports on follicle growth and maturation, ensuring real-time regulation of system parameters.

[0057] Beneficial Effects

[0058] Compared with the known public technology, the technical solution provided by the present invention has the following beneficial effects:

[0059] The present invention uses animal and cell models and molecular biological methods to explain the interaction between somatic cells, oocytes and extracellular matrix and their specific mechanisms of action, develops an optimized human follicle 3D culture system, and standardizes the specific parameters of chimeric follicle and oocyte recovery. By designing an in vitro ovarian biological system, the quality and recovery potential of oocytes can be further improved. DETAILED DESCRIPTION

[0060] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the described embodiments are only part of the embodiments of the present application, rather than all the embodiments, and the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of this application.

[0061] It should be further explained that the embodiments of the present invention mainly describe the concept of the present invention. On the basis of this concept, the specific forms and settings of some connection relationships, positional relationships, power mechanisms, power supply systems, hydraulic systems and control systems may not be fully described. However, on the premise that those skilled in the art understand the concept of the present invention, those skilled in the art can implement the above-mentioned specific forms and settings in a well-known manner.

[0062] When an element is referred to as being "fixed to" or "disposed on" another element, it may be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it may be directly connected to the other element or indirectly connected to the other element.

[0063] The directional words "inside and outside" refer to the inside and outside relative to the outline of each component itself. The directions indicated by the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0064] For ease of description, spatially relative terms such as "on", "above", "on the upper surface of", "above", etc. may be used herein to describe the spatial positional relationship of a device or feature with other devices or features as shown in the figures. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figures. Thus, the exemplary term "above" may include both "above" and "below". The device may also be positioned in other different ways, and the spatially relative descriptions used herein are interpreted accordingly.

[0065] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "multiple" means two or more, and "several" means one or more, unless otherwise clearly and specifically defined.

[0066] The method for in vitro activation of AOI oocytes and preservation of fertility provided by the present invention is now described.

[0067] Example 1

[0068] The present invention provides a technical solution: a method for in vitro activation of AOI oocytes and preservation of fertility, wherein in step S1, separation and initial characteristic analysis of oocytes:

[0069] S1.1. Isolate oocytes from surgically obtained human ovarian tissue under sterile conditions;

[0070] S1.2, the tissue was first enzymatically digested with 0.25% collagenase I and 0.05% trypsin at 37°C for 15 min, and then filtered through a 40 μm cell sieve to remove larger tissue pieces;

[0071] S1.3, the isolated oocytes were temporarily stored in DMEM / F12 medium with HEPES buffer;

[0072] S1.4. Use a Nikon Ti-E fluorescence microscope to detect the integrity of the zona pellucida and label mitochondria with MitoTracker dye to observe their distribution and aggregation at an excitation wavelength of 488 nm.

[0073] S1.5, use Piuma NanoIndenter dynamic micro-pressure probe to evaluate the mechanical properties of the oocyte surface (stiffness range is usually 1–10 kPa);

[0074] S1.6, preliminary screening of gene expression in oocytes using qRT-PCR;

[0075] In the step S2, obtaining and typing somatic cells:

[0076] S2.1, Granulosa cells and cumulus cells were isolated from ovarian tissue, high-quality cells were isolated from the gradient range of 30%-60% by Percoll density gradient centrifugation, and dead cells were excluded by Trypan Blue;

[0077] S2.2. Use the 10×Genomics single-cell transcriptome sequencing platform to accurately genotype the gene expression of these cells, with special attention to the expression of marker genes such as FOXL2 and CYP19A1;

[0078] S2.3. Within 24 hours after isolation, cells were cultured in a 2D tile format in DMEM medium containing 10% FBS and 1% insulin-transferrin-selenium, with the oxygen concentration controlled at 5%;

[0079] S2.4, GC-MAF and other secreted proteins were detected by Western Blot to evaluate their endocrine function, combined with immunofluorescence to detect key pathway molecules;

[0080] In the step S3, constructing chimeric follicles:

[0081] S3.1. Granulosa cells and oocytes from subjects of different ages were assembled into chimeric follicles using an Eppendorf CellTram microinjection device in a CO2 incubator at 37°C and 5% CO2 concentration;

[0082] S3.2, the cell assembly was fixed by a three-dimensional collagen scaffold (concentration 1.5 mg / mL) and embedded in a hydrogel modified by natural hyaluronic acid and gelatin to maintain its structure;

[0083] S3.3. Real-time live cell imaging was used to observe cell-cell adhesion and interaction using a Leica SP8 laser confocal microscope, while SIRT1 repressor genes were knocked out in aged granulosa cells using CRISPR-Cas9;

[0084] S3.4. The constructed follicles are dynamically cultured in a low shear force microfluidic culture system.

[0085] In this embodiment, oocytes and granulosa cells are separated from ovarian tissue, and preliminary quality screening and genotyping are performed respectively. The cell health state is preliminarily evaluated by microscopic imaging and molecular biology techniques (transcriptomics, mitochondrial activity assessment). Subsequently, high-quality somatic cells and oocytes are recombined into chimera follicles with an optimized 3D scaffold system, and a stable culture environment is established by dynamic microfluidics. In this process, CRISPR-Cas9 technology is further combined to optimize somatic cell function, providing a high-quality chimera model for subsequent culture.

[0086] Example 2

[0087] The present invention provides a technical solution: a method for in vitro activation of AOI oocytes and preservation of fertility, wherein in step S4, in the optimization of the 3D culture system:

[0088] S4.1. A dynamic 3D culture system was constructed using a PDMS microfluidic chip with a channel width of 100 μm, and a mixed ECM scaffold of collagen I and IV with a ratio of 3:1 to simulate ovarian tissue was added;

[0089] S4.2, hardness was controlled between 300–800 Pa by AFM atomic force microscopy to simulate the ovarian environment at different ages;

[0090] S4.3, AMH at a concentration of 10 ng / mL and VEGF at a concentration of 20 ng / mL were added to the culture medium, and the culture medium was renewed every 48 hours;

[0091] S4.4, monitor the mechanical properties of the culture system in real time by Brillouin microscopy, and adjust the pH to 7.4 and the oxygen partial pressure to 5% according to the cell growth;

[0092] In the step S5, functionalized biomaterial design:

[0093] S5.1: Based on natural collagen and synthetic polyethylene glycol diacrylate at a concentration of 10% w / v, combined with glutaraldehyde crosslinker to generate scaffold material;

[0094] S5.2: Mechanical properties were tested by Instron 5940 tensile tester, and the Young's modulus was maintained between 2–5 kPa to adapt to folliculogenesis;

[0095] S5.3: The biomaterial is embedded with TGF-β and IGF-1 signaling molecules by solution polymerization, and the sustained release function is achieved by microencapsulation technology with a diameter of about 100 μm;

[0096] S5.4: Co-culture the material with ovarian follicles to verify its ability to promote oocyte growth;

[0097] In the step S7, developing the engineered cell system:

[0098] S7.1: CRISPR-Cas9 gene editing was performed on granulosa cells to knock in the PGC1α gene;

[0099] S7.2: The edited cells were expanded in a rotating environment at RPM 20 / min in medium containing 5% FBS.

[0100] S7.3: These engineered granulosa cells were co-cultured with oocytes, 10 μM glutathione was added to enhance antioxidant capacity, and the concentration of FGF2 growth factor secreted by them was measured by LC-MS;

[0101] S7.4: Single-cell transcriptome sequencing was used to confirm the molecular stability of engineered cells cultured for a long period of time.

[0102] In this embodiment, a dynamic 3D culture system is constructed, and oocytes and chimeric follicles are placed in a bionic scaffold for culture. Combined with microfluidics and ECM optimization methods, hardness, oxygen partial pressure and growth factor concentration are dynamically adjusted to simulate the young ovarian environment to the greatest extent. In addition, functionalized biomaterials (hydrogel scaffolds combined with signal molecules) are designed to provide long-term support. Engineered granulosa cells are developed simultaneously, and their support capacity is enhanced through gene editing and metabolic enhancement technology, combined with a dynamically adjusted 3D system, to further optimize the development environment of oocytes.

[0103] Example 3

[0104] The present invention provides a technical solution: a method for in vitro activation of AOI oocytes and preservation of fertility, wherein in step S6, follicle activity assessment:

[0105] S6.1: After follicles are extracted from the culture system, spindle morphology is examined using immunofluorescence staining, where antibody markers include α-tubulin;

[0106] S6.2: Extract oocyte RNA, perform transcriptome sequencing using the Illumina NovaSeq platform, and combine metabolomics detection to target energy metabolites ATP and NADH;

[0107] S6.3: 1×10 spermatozoa from healthy males were used in the in vitro fertilization experiment. 6 / mL to evaluate the fertilization rate, and observe the development of the blastocyst under a DAPI staining microscope;

[0108] S6.4: Analyze follicle quality and maturity using AI image processing technology;

[0109] In the step S8, the non-invasive monitoring system is being developed:

[0110] S8.1: Design a real-time imaging platform based on optical coherence tomography with a resolution of 1 μm to monitor changes in follicle morphology;

[0111] S8.2: A responsive nanosensor with a gold nanorod diameter of 10 nm was embedded in the culture medium to detect the concentration of the metabolite lactate by changes in fluorescence intensity;

[0112] S8.3: Use nonlinear optical techniques in conjunction with reflectance spectroscopy to detect zona pellucida thickness;

[0113] S8.4: The AI ​​algorithm is written in Python and integrates multimodal data analysis to automatically generate dynamic reports on follicle growth and maturation, ensuring real-time regulation of system parameters.

[0114] In this embodiment, the development process of follicles and oocytes is dynamically monitored in a 3D culture system, and real-time data is collected using OCT and Brillouin microscope optical imaging and nanoparticle embedded sensors. Based on these data, follicle quality, metabolic state and developmental potential are analyzed by machine learning algorithms. Immunofluorescence staining, transcriptome sequencing and metabolomics detection are combined to further verify the culture effect, and on this basis, the maturity of oocytes and the potential for early embryonic development are evaluated.

[0115] The present invention,

[0116] Oocytes and somatic cells (including granulosa cells and cumulus cells) were extracted from ovarian tissue under sterile conditions, and after removing tissue blocks by enzymatic hydrolysis with collagenase and trypsin combined with cell sieve filtration, they were stored in HEPES-buffered DMEM / F12 medium. The integrity of the zona pellucida of the oocytes was detected under a microscope, and mitochondrial activity and cell mechanical properties were evaluated using MitoTracker and micropressure probes, and their molecular characteristics were further analyzed by qRT-PCR and single-cell sequencing. Subsequently, the isolated high-quality granulosa cells and oocytes were fixed with a three-dimensional collagen scaffold, assembled into chimeric follicles in a CO2 incubator using microinjection technology, and the metabolic function of granulosa cells was optimized by CRISPR-Cas9 technology to provide a high-quality model for further research.

[0117] A dynamic culture system was established using a PDMS microfluidic chip, and the ovarian microenvironment was simulated by a 3:1 mixed scaffold of collagen I and IV. The scaffold hardness (300–800Pa) was detected by AFM to adapt to the mechanical properties of the ovaries of different ages. During the culture process, AMH and VEGF were added to support follicle growth, and the mechanical properties and cell behavior of the culture system were monitored by real-time microscopy. In addition, a functional scaffold material with sustained release function was designed by combining natural collagen and polyethylene glycol diacrylate, and the mechanical properties were tested by a tensile tester to ensure that it was adapted to the needs of follicle development. At the same time, engineered granulosa cells were constructed, and metabolic enhancement genes were knocked in through gene editing, combined with the antioxidant molecule glutathione to further support the growth and maturation of oocytes.

[0118] After extracting mature follicles from the culture system, immunofluorescence staining was used to detect spindle morphology, and transcriptome sequencing and metabolomics were used to analyze their molecular and metabolic characteristics. In vitro fertilization experiments were combined to evaluate the developmental potential of oocytes, and blastocyst formation and development were observed by microscopy. At the same time, an OCT-based optical imaging platform was designed to monitor follicle morphological changes in real time, embed responsive nanosensors to detect metabolite concentrations, and combine AI algorithms to conduct comprehensive analysis of multimodal data, automatically generate dynamic evaluation reports, and ensure optimal regulation of the culture system and accurate evaluation of oocyte quality.

[0119] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0120] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0121] Unless otherwise specifically stated, the relative arrangement of the parts and steps set forth in these embodiments, numerical expressions and numerical values ​​do not limit the scope of the application. The technology, method and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology, method and equipment should be considered as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

Claims

1. A method for in vitro activation of AOI oocytes and preservation of fertility, characterized in that: The following steps are involved: S1, Oocyte isolation and initial characterization; S2. Acquisition and typing of somatic cells; S3, chimeric follicle construction; S4. Optimization of 3D culture system; S5. Design of functional biomaterials; S6, follicle activity assessment; S7, Engineered cell system development; S8. Development of non-invasive monitoring system.

2. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S1, separation and initial feature analysis of oocytes: S1.

1. Isolate oocytes from surgically obtained human ovarian tissue under sterile conditions; S1.2, the tissue was first enzymatically digested with 0.25% collagenase I and 0.05% trypsin at 37°C for 15 min, and then filtered through a 40 μm cell sieve to remove larger tissue pieces; S1.3, the isolated oocytes were temporarily stored in DMEM / F12 medium with HEPES buffer; S1.

4. Use a Nikon Ti-E fluorescence microscope to detect the integrity of the zona pellucida and label mitochondria with MitoTracker dye to observe their distribution and aggregation at an excitation wavelength of 488 nm. S1.5, use Piuma NanoIndenter dynamic micro-pressure probe to evaluate the mechanical properties of the oocyte surface (stiffness range is usually 1–10 kPa); S1.

6. Use qRT-PCR to perform preliminary screening of gene expression in oocytes.

3. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S2, obtaining and typing somatic cells: S2.1, Granulosa cells and cumulus cells were isolated from ovarian tissue, high-quality cells were isolated from the gradient range of 30%-60% by Percoll density gradient centrifugation, and dead cells were excluded by Trypan Blue; S2.

2. Use the 10×Genomics single-cell transcriptome sequencing platform to accurately genotype the gene expression of these cells, with special attention to the expression of marker genes such as FOXL2 and CYP19A1; S2.

3. Within 24 hours after isolation, cells were cultured in a 2D tile format in DMEM medium containing 10% FBS and 1% insulin-transferrin-selenium, with the oxygen concentration controlled at 5%; S2.

4. GC-MAF and other secretory proteins were detected by Western Blot to evaluate their endocrine functions, combined with immunofluorescence to detect key pathway molecules.

4. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S3, constructing chimeric follicles: S3.

1. Granulosa cells and oocytes from subjects of different ages were assembled into chimeric follicles using an Eppendorf CellTram microinjection device in a CO2 incubator at 37°C and 5% CO2 concentration; S3.2, the cell assembly was fixed by a three-dimensional collagen scaffold (concentration 1.5 mg / mL) and embedded in a hydrogel modified by natural hyaluronic acid and gelatin to maintain its structure; S3.

3. Real-time live cell imaging was used to observe cell-cell adhesion and interaction using a Leica SP8 laser confocal microscope, while SIRT1 repressor genes were knocked out in aged granulosa cells using CRISPR-Cas9; S3.

4. The constructed follicles are dynamically cultured in a low shear force microfluidic culture system.

5. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S4, optimization of the 3D culture system: S4.

1. A dynamic 3D culture system was constructed using a PDMS microfluidic chip with a channel width of 100 μm, and a mixed ECM scaffold of collagen I and IV with a ratio of 3:1 to simulate ovarian tissue was added; S4.2, hardness was controlled between 300–800 Pa by AFM atomic force microscopy to simulate the ovarian environment at different ages; S4.3, AMH at a concentration of 10 ng / mL and VEGF at a concentration of 20 ng / mL were added to the culture medium, and the culture medium was renewed every 48 hours; S4.

4. Monitor the mechanical properties of the culture system in real time using a Brillouin microscope, and adjust the pH to 7.4 and the oxygen partial pressure to 5% according to cell growth conditions.

6. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S5, functionalized biomaterial design: S5.1: Based on natural collagen and synthetic polyethylene glycol diacrylate at a concentration of 10% w / v, combined with glutaraldehyde crosslinker to generate scaffold material; S5.2: Mechanical properties were tested by Instron 5940 tensile tester, and the Young's modulus was maintained between 2–5 kPa to adapt to folliculogenesis; S5.3: The biomaterial is embedded with TGF-β and IGF-1 signaling molecules by solution polymerization, and the sustained release function is achieved by microencapsulation technology with a diameter of about 100 μm; S5.4: Co-culture the material with ovarian follicles to verify its ability to promote oocyte growth.

7. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S6, follicle activity assessment: S6.1: After follicles are extracted from the culture system, spindle morphology is examined using immunofluorescence staining, where antibody markers include α-tubulin; S6.2: Extract oocyte RNA, perform transcriptome sequencing using the Illumina NovaSeq platform, and combine metabolomics detection to target energy metabolites ATP and NADH; S6.3: 1×10 spermatozoa from healthy males were used in the in vitro fertilization experiment. 6 / mL to evaluate the fertilization rate, and observe the development of the blastocyst under a DAPI staining microscope; S6.4: Use AI image processing technology to analyze follicle quality and maturity.

8. The method for in vitro activation of AOI oocytes and preservation of fertility according to claim 1, characterized in that: In the step S7, developing the engineered cell system: S7.1: CRISPR-Cas9 gene editing was performed on granulosa cells to knock in the PGC1α gene; S7.2: The edited cells were expanded in a rotating environment at RPM 20 / min in medium containing 5% FBS. S7.3: These engineered granulosa cells were co-cultured with oocytes, 10 μM glutathione was added to enhance antioxidant capacity, and the concentration of FGF2 growth factor secreted by them was measured by LC-MS; S7.4: Single-cell transcriptome sequencing was used to confirm the molecular stability of engineered cells cultured for a long time.

9. The method for in vitro activation and fertility preservation of AOI oocytes according to claim 1, characterized in that: In the step S8, the non-invasive monitoring system is being developed: S8.1: Design a real-time imaging platform based on optical coherence tomography with a resolution of 1 μm to monitor changes in follicle morphology; S8.2: A responsive nanosensor with a gold nanorod diameter of 10 nm was embedded in the culture medium to detect the concentration of the metabolite lactate by changes in fluorescence intensity; S8.3: Use nonlinear optical techniques in conjunction with reflectance spectroscopy to detect zona pellucida thickness; S8.4: The AI ​​algorithm is written in Python and integrates multimodal data analysis to automatically generate dynamic reports on follicle growth and maturation, ensuring real-time regulation of system parameters.