PVA / Fe3O4 / GO nanocomposite material, preparation method, and oocyte cryopreservation method based on synergistic anti-icing effect.

Abstract

This invention discloses a PVA / Fe3O4 / GO nanocomposite material, its preparation method, and a method for cryopreservation of oocytes based on a synergistic anti-icing effect. This composite nanomaterial combines polyvinyl alcohol, iron(III) oxide, and graphene oxide through hydrogen bonding and electrostatic interactions. It can reduce the supercooling of the solution during freezing, inhibiting the formation of sharp ice crystals. During the rewarming stage, it can inhibit recrystallization of ice crystals. Under the influence of external laser and alternating magnetic field, it can rapidly heat the solution, reducing internal thermal stress and anti-vitrification during the rewarming stage. This invention utilizes this nanocomposite material and a single, low-concentration permeable cryopreservative to achieve mass cryopreservation of oocytes. Oocytes cryopreserved using this nanocomposite-assisted method have higher survival rates after thawing and fewer gene mutations compared to traditional methods. Oocytes thawed using this cryopreservation method can achieve normal fertilization, development, and in vivo transplantation to produce healthy mouse offspring.

Description

Technical Field

[0001] This invention relates to the fields of nanocomposite materials, fertility preservation, and regenerative medicine, specifically to PVA / Fe3O4 / GO nanocomposite materials, their preparation methods, and a method for cryopreservation of oocytes based on a synergistic anti-icing effect. Background Technology

[0002] In recent years, female fertility loss due to various diseases or personal reasons has become increasingly common. This is particularly true given the rising incidence of cancer among younger women. After undergoing treatments such as radiotherapy, chemotherapy, and surgery, female patients are highly susceptible to ovarian dysfunction and loss of reproductive capacity. Therefore, fertility preservation before these treatments are crucial. Furthermore, many women today tend to postpone childbearing due to personal or social factors, creating a widespread demand for effective fertility preservation in advance among this group.

[0003] Currently, cryopreservation of female embryos and mature oocytes is the only method approved by the American Society for Reproductive Medicine. Among these methods, oocyte retrieval is widely used clinically for female fertility preservation due to its relative ease of acquisition and low risk of ethical issues. Vitrification is currently the primary method for oocyte cryopreservation. Vitrification prevents ice crystal formation during freezing, reducing ice crystal damage to oocytes. However, achieving this requires adding a high concentration of CPA and directly freezing individual oocytes. Current traditional methods use 4.3M CPA and a Cryotope device, which can only freeze one oocyte at a time.

[0004] Therefore, traditional vitrification not only causes severe chemical toxicity damage to oocytes due to high concentrations of CPA, but also severely limits the throughput of single cryopreservations. Thus, achieving efficient mass cryopreservation of oocytes at low CPA concentrations is a pressing scientific challenge in the field of female fertility preservation. Summary of the Invention

[0005] In view of the above problems, this invention proposes a PVA / Fe3O4 / GO nanocomposite material, its preparation method, and a method for cryopreservation of oocytes based on a synergistic anti-icing effect. The nanocomposite material possesses an anti-icing effect during both freezing and thawing stages. This composite material can simultaneously inhibit the formation of sharp ice crystal morphology, ice crystal recrystallization, and antivitrification damage. With its assistance, batch cryopreservation of oocytes under low-concentration CPA can be achieved, providing a novel approach for the efficient preservation of female fertility.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution.

[0007] A method for preparing a PVA / Fe3O4 / GO nanocomposite material involves first encapsulating polyvinyl alcohol (PVA) around Fe3O4 via hydrogen bonding to form PVA / Fe3O4 nanospheres; then, the PVA / Fe3O4 nanospheres are electrostatically adsorbed onto the surface of graphene oxide (GO), ultimately forming a PVA / Fe3O4 / GO nanocomposite material with an average particle size of hundreds of nanometers (300-700 nm). This PVA / Fe3O4 / GO nanocomposite material exhibits anti-icing effects during both freezing and rewarming stages. Preferably, the average particle size of the PVA / Fe3O4 nanospheres is 10–50 nm. For example, the average particle size of the PVA / Fe3O4 nanospheres can be 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 26 nm, 28 nm, 30 nm, 35 nm, 40 nm, 45 nm, or 50 nm.

[0008] Furthermore, the method includes the following steps:

[0009] (1) Preparation of Fe3O4;

[0010] (2) Polyvinyl alcohol and Fe3O4 were mixed and ultrasonically mixed at a water bath temperature of 60-90℃, followed by centrifugal washing to obtain Fe3O4 / PVA composite material.

[0011] (3) The Fe3O4 / PVA composite material, graphene oxide and deionized water were mixed, washed by centrifugation and dried under vacuum to obtain Fe3O4 / PVA / GO.

[0012] Further, in step (1), the average particle size of Fe3O4 is 10-30 nm; for example, the average particle size of Fe3O4 is 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 26 nm, 28 nm or 30 nm.

[0013] Preferably, in step (2), the mass ratio of polyvinyl alcohol to Fe3O4 is 100–250:80–120; for example, the mass ratios of polyvinyl alcohol to Fe3O4 are 100:80–120, 102:80–120, 105:80–120, 110:80–120, 115:80–120, 120:80–120, 125:80–120, 130:80–120, 135… 80~120, 140:80~120, 145:80~120, 150:80~120, 160:80~120, 170:80~120, 180:80~120, 190:80~120, 200:80~120, 210:80~120, 220:80~120, 230:80~120, 240:80~120 or 250:80~120.

[0014] Preferably, in step (3), the mass ratio of Fe3O4 / PVA composite material to graphene oxide is 100–200:200–400. For example, the mass ratio of Fe3O4 / PVA composite material to graphene oxide is 100:200–400, 105:200–400, 110:200–400, 120:200–400, 130:200–400, 140:200–400, 150:200–400, 160:200–400, 170:200–400, 180:200–400, 190:200–400, or 200:200–400.

[0015] Preferably, in step (3), the ratio of graphene oxide to deionized water is 200-500 mg:40 ml; for example, the ratio of graphene oxide to deionized water is 200 mg:40 ml, 250 mg:40 ml, 300 mg:40 ml, 350 mg:40 ml, 400 mg:40 ml, 450 mg:40 ml or 500 mg:40 ml.

[0016] The PVA / Fe3O4 / GO nanocomposite material prepared by the method described in any of the preceding methods.

[0017] Furthermore, the PVA / Fe3O4 / GO nanocomposite material is used in combination with ethylene glycol and trehalose when used for cryopreservation of oocytes.

[0018] This invention also provides a method for cryopreservation of oocytes based on a synergistic anti-icing effect, utilizing any of the PVA / Fe3O4 / GO nanocomposite materials described above, comprising the following steps:

[0019] (1) PVA / Fe3O4 / GO nanocomposite material is added to ethylene glycol to obtain a mixture; preferably, the concentration of PVA / Fe3O4 / GO nanocomposite material in the mixture is 1.5 to 2.5 mg / mL; preferably, the mixture also contains trehalose;

[0020] (2) Following the oocyte cryopreservation procedure, mouse MII oocytes were cryopreserved using the mixture prepared in step (1).

[0021] (3) After cryopreservation, the frozen oocytes are rewarmed using laser and alternating magnetic field;

[0022] (4) Gene sequencing of revived oocytes was performed by transcriptome sequencing to assess the impact of freezing on their genome.

[0023] Specifically, a method for cryopreservation of oocytes based on a synergistic anti-icing effect, using the PVA / Fe3O4 / GO nanocomposite material as described above, includes the following steps:

[0024] 1. Add 2 mg / mL of PVA / Fe3O4 / GO nanocomposite material to CPA, where CPA is a 2.5 M EG and 1 M trehalose solution;

[0025] 2. Following the oocyte cryopreservation procedure, mouse oocytes at the MII stage were cryopreserved using a PVA / Fe3O4 / GO nanocomposite material.

[0026] 3. After 24 hours of cryopreservation, the frozen oocytes are thawed using laser and alternating magnetic field.

[0027] 4. Transcriptome sequencing was performed on the revived oocytes to assess the impact of freezing on their genome. Furthermore, the revived oocytes were subjected to in vitro fertilization, development, and transplantation into surrogate mice for offspring reproduction to evaluate the biological function of the revived oocytes.

[0028] Specifically, this invention relates to a PVA / Fe3O4 / GO nanocomposite material prepared by hydrogen bonding and electrostatic interaction. For this nanocomposite material, PVA is first wrapped around Fe3O4 through hydrogen bonding to form PVA / Fe3O4 nanospheres with a size of 20 nm; then the PVA / Fe3O4 nanospheres are connected to the GO surface through electrostatic adsorption to finally form a PVA / Fe3O4 / GO nanocomposite material with a size of hundreds of nanometers.

[0029] Furthermore, by adding the PVA / Fe3O4 / GO nanocomposite material prepared above to a trehalose solution (2 mg / mL), the supercooling of the solution can be reduced at different freezing rates. At a rapid cooling rate (100 °C / min), the sharp ice crystal morphology formed in the solution during the cooling process can be suppressed.

[0030] Furthermore, an ice crystal recrystallization experiment was conducted using the PVA / Fe3O4 / GO nanocomposite material prepared above. Specifically, a trehalose solution containing the nanocomposite material was dropped from a height onto a glass slide cooled to -80°C, then reheated to -8°C at a rate of 15°C / min and maintained at this temperature for 30 min. The ice crystal size was observed using a cryogenic stage microscope. Quantitative analysis of the ice crystal size showed that, compared to the control group, the nanocomposite material effectively inhibited ice crystal recrystallization.

[0031] Furthermore, this nanocomposite material can be heated under dual physical fields of an external alternating magnetic field and a laser (magnetic field coil current 5-30A, laser control power 1-10W / cm²). 2 It can achieve uniform and rapid heating from the inside out of low-concentration penetrant (CPA) solutions, thus effectively avoiding the hazards caused by thermal stress and devitrification.

[0032] Furthermore, by utilizing the synergistic ice-suppressing effect of this nanocomposite material during the cooling and rewarming stages, it can be added to a low concentration of permeable CPA (2.5M EG) to achieve efficient cryopreservation of 10 oocytes in a single batch. Compared with traditional methods, the revived oocytes exhibit fewer genetic changes and have normal fertilization, development, and progeny production functions.

[0033] The present invention discloses a nanocomposite material with a synergistic anti-icing effect, and its application in the cryopreservation and thawing process of oocytes. Its main advantages are as follows:

[0034] 1. The nanocomposite material synthesized in this invention can achieve multiple ice suppression simultaneously during the cooling and reheating stages. Compared with the potential toxicity risks of previous chemical CPA methods for suppressing ice crystals, this method is entirely physical. Furthermore, the external alternating magnetic field and laser have the advantage of spatiotemporal controllability, enabling precise regulation during the ice crystal melting stage.

[0035] 2. Based on the nanocomposite material of the present invention, it is possible to achieve efficient and large-scale cryopreservation of mouse oocytes with a single, low-concentration (2.5M EG) CPA, and the survival rate after thawing reaches 98.6%.

[0036] 3. Oocytes cryopreserved with the assistance of the nanocomposite material of the present invention showed 85 gene changes after thawing compared to fresh oocytes, and 1396 gene changes compared to traditional freezing methods. This indicates that the nanocomposite material of the present invention can have a smaller impact on oocytes at the gene level.

[0037] 4. Oocytes revived with the assistance of nanocomposite materials can achieve normal in vitro fertilization, development, and in vivo transplantation, ultimately producing normal offspring mice. Attached Figure Description

[0038] Figure 1 Transmission electron microscopy (TEM) images of PVA / Fe3O4 and PVA / Fe3O4 / GO nanocomposites;

[0039] Figure 2 Nanocomposite materials reduce solution supercooling;

[0040] Figure 3 Nanocomposite materials suppress sharp ice crystal morphology during freezing;

[0041] Figure 4 Nanocomposite materials inhibit ice crystal recrystallization;

[0042] Figure 5 : Retemperature curve of CPA solution under the action of optical-magnetic physical field;

[0043] Figure 6 Nanocomposite materials inhibit the devitrification of CPA under the action of a light-magnetic physical field;

[0044] Figure 7 Steps for freezing and thawing mouse oocytes with the assistance of nanocomposite materials;

[0045] Figure 8 Comparison of oocyte survival rates under different freezing methods;

[0046] Figure 9 Comparison of the number of gene changes in oocytes frozen using different methods;

[0047] Figure 10 Development rate of oocytes frozen using different methods;

[0048] Figure 11 The birth rate of fertilized eggs transferred into the body after freezing oocytes using different methods. Detailed Implementation

[0049] To make the above-mentioned objectives, features and advantages of the invention more apparent and understandable, the specific embodiments of the invention will be described in detail below with reference to the accompanying drawings.

[0050] The present invention will be further described in detail below with reference to embodiments. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments. Specific operations are as follows:

[0051] In the following embodiments of the present invention, M2 culture medium (Sigma-Aldrich, Germany) is used.

[0052] Implementation Cases

[0053] Preparation of a PVA / Fe3O4 / GO nanocomposite material with synergistic anti-icing effect and its application in the freezing and thawing of oocytes.

[0054] The specific operation is as follows: Preparation of PVA / Fe3O4 / GO nanocomposite material: (1) Dissolve 0.85g FeCl3 and 0.3g FeCl2 in 200mL deionized water, add 20mL ammonia water (1mg / mL), stir rapidly under nitrogen atmosphere, then adjust pH=8, stir for 2h to prepare Fe3O4. (2) Prepare 10ml of 20mg / mL PVA solution and stir at 80℃, then ultrasonically disperse 80mg Fe3O4 in 5mL of deionized water to obtain Fe3O4 dispersion, slowly add Fe3O4 dispersion to polyvinyl alcohol solution (PVA), ultrasonically mix evenly, centrifuge and wash at 85℃ hot water to prepare Fe3O4 / PVA composite material. (3) Disperse 200mg Fe3O4 / PVA in 40mL of 10mg / mL graphene oxide (GO) solution, ultrasonically for 6h, centrifuge and wash, vacuum dry to obtain Fe3O4 / PVA / GO. PVA model is 1788, specification is 120 mesh. Transmission electron microscopy of Fe3O4 / PVA and PVA / Fe3O4 / GO nanocomposites, as shown in... Figure 1 As shown, the average particle size of Fe3O4 / PVA is approximately 15 nm, and the average particle size of PVA / Fe3O4 / GO nanocomposite is approximately 600 nm.

[0055] 1. Nanocomposite materials reduce supercooling: 2 mg of PVA / Fe3O4 / GO nanocomposite material was added to 2 mL of 1 mg / mL trehalose solution. The supercooling of the solution at different cooling rates was tested using a cryo-stage microscope. 20 μL of the solution was added dropwise to the cryo-stage and cooled from room temperature at rates of 10, 30, 50, and 100 °C / min. The freezing and thawing temperatures of the solution were quantitatively observed, and the supercooling was calculated. Quantitative analysis showed that the supercooling of the solution was significantly reduced under the action of the nanocomposite material (reduced by 5 °C at different cooling rates). The results are as follows: Figure 2As shown, the light-colored bars represent 1 mg / mL trehalose solution without the addition of nanocomposite materials; the dark-colored bars represent 1 mg / mL trehalose solution with the addition of 2 mg / mL nanocomposite materials.

[0056] 2. Suppressing sharp ice crystal morphology with nanocomposite materials: 2 mg of PVA / Fe3O4 / GO nanocomposite material was added to 2 mL of 1 mg / mL trehalose solution. First, 20 μL of the prepared nanocomposite material solution was dropped onto a cryogenic microscope. Then, the solution was cooled at a rate of 100 °C / min from room temperature to -80 °C using a cryogenic stage equipped with a high-speed camera. The results are as follows: Figure 3 As shown, compared to the trehalose solution without nanocomposite materials, the ice crystal morphology changed from sharp to rounded, indicating that the addition of nanocomposite materials can inhibit the damage caused by sharp ice crystal morphology.

[0057] 3. Inhibition of Ice Crystal Recrystallization by Nanocomposite Materials: 2 mg of PVA / Fe3O4 / GO nanocomposite material was added to 2 mL of a 1 mg / mL trehalose solution. 20 μL of the prepared nanocomposite solution was dropped from a height of 1.5 m onto a glass slide cooled to -80°C. The slide was then placed on a cryogenic stage and reheated to -8°C at a heating rate of 15°C / min, held at this temperature for 30 min. Optical imaging of the recrystallized ice crystals was performed using a cryogenic stage microscope, and the ice crystal size was recorded using a CCD high-speed camera. Image-J software was then used for quantitative statistical analysis of the ice crystal size. The results showed that the addition of the nanocomposite material effectively inhibited recrystallization and reduced the ice crystal size in the solution. Figure 4 As shown.

[0058] 4. Rapid rewarming under a photomagnetic physical field to suppress anti-glass transition: A 2 mg / mL PVA / Fe3O4 / GO nanocomposite material was prepared using 100 μL of 2.5 M ethylene glycol (EG) as a preservative. A fiber optic temperature sensor was placed in the center of a wheat tube containing the nanocomposite material and CPA. The tube was then placed in liquid nitrogen at -196°C. After complete freezing, the wheat tube was quickly removed and placed in a 50 mL centrifuge tube containing 37°C water. The electromagnetic coil (current control 5-30 A) and laser (power control 1-10 W / cm²) were then activated. 2 Nanoscale rewarming was performed on the wheat straw. Different methods were used to rewarm the CPA (CPA of 2.5 M EG). First, the wheat straw was completely immersed in liquid nitrogen at -196℃. Then, the wheat straw was placed under a separate laser (2W / cm²). 2 ), a single magnetic field (10A), with laser and magnetic field simultaneously activated (2W / cm). 2 ，The wheat tube was reheated using a 10A water bath at 37°C. The temperature of the wheat tube was recorded using optical fiber throughout the process, and the internal temperature curve of the wheat tube is shown below. Figure 5 As shown, the dual physical field method enables rapid rewarming (the curve illustrates how dual physical field heating allows the frozen wheat straw solution to be rewarmed to room temperature at a faster rate). Compared to traditional water bath rewarming (which involves immersing the wheat straw in a 37°C water bath), rapid rewarming suppresses the CPA anti-vitrification phenomenon, such as... Figure 6 As shown, Figure 6 Traditional water bath images show visible devitrification, as indicated by the arrow; however, reheating using dual physical field heating requires a shorter melting time and does not result in visible devitrification.

[0059] 5. Cryopreservation and thawing of mouse oocytes assisted by PVA / Fe3O4 / GO nanocomposite materials: Following the instructions... Figure 7As shown in the steps, transfer 8-10 fresh oocytes to a four-well culture dish (Thermo Fisher Scientific, USA) and add 20 μL of equilibration buffer (7.5% (v / v) EG). After incubating for 3 min, add 40 μL of equilibration buffer (equilibration buffer refers to 7.5% (v / v) EG). After incubating for 3 min, add another 260 μL of equilibration buffer (equilibration buffer 7.5% (v / v) EG). After incubating for 9 min, use a micropipette (Thermo Fisher Scientific, USA) to transfer the oocytes to 300 μL of cryoprotectant containing nanocomposite materials (the concentration of each component in the cryoprotectant containing nanocomposite materials is: 17.5% (v / v) EG + 1M trehalose + 2 mg / mL nanocomposite materials), and then place it in a four-well culture dish containing nanocomposite materials. After three aspiration and respiration cycles, oocytes were rapidly transferred within 1 minute to a plastic pipette (Thermo Fisher Scientific, USA) containing 20 μL of the above mixture (the above mixture refers to the cryoprotectant), and sealed with wax oil. The plastic pipette was then immersed in liquid nitrogen to vitrify the solution. After 24 hours, the pipette was transferred to a 50 mL centrifuge tube, and water at 37°C was added. Simultaneously, the centrifuge tube was placed in an environment with laser and alternating magnetic field. After heating, the sample was transferred to a 6 mm culture dish containing 300 μL of resuscitation medium (0.5 M trehalose) for 1 minute, and the nanocomposite material was removed using a magnet. Then, the sample was transferred to 300 μL of dilution medium (0.25 M trehalose) and incubated for 3 minutes. Subsequently, the oocytes were transferred to 300 μL of washing medium (the washing medium contained 20% (v / v) FBS + M2 medium) and incubated for 5 minutes. The same incubation procedure was repeated in the washing solution (the same incubation procedure was to transfer oocytes to 300 μL of washing solution and incubate for 5 min). Finally, the oocytes were transferred to M2 medium (Sigma-Aldrich, Germany) and cultured in an incubator (5% CO2, 37℃) for subsequent experiments. The equilibration solution was 7.5% (v / v) EG; the freezing solution was 2.5 M EG + 1 M trehalose; the thawing solution was 0.5 M trehalose; the dilution solution was 0.25 M trehalose; and the washing solution was M2 medium (Sigma-Aldrich, Germany) + 20% (v / v) FBS (based on the total volume of washing solution). 2 mg / mL (based on the total volume of washing solution) of PVA / Fe3O4 / GO nanocomposite material was added to the freezing solution.

[0060] 6. Performance evaluation of mouse oocytes after thawing: Recovered oocytes were stained using an acridine orange / ethidium bromide (AO / EB) staining kit (KGA502, KeyGen Biotech, China). The stained oocytes were then imaged using a fluorescence microscope (Eclipse Ti-U, Nikon, Japan). Green represents viable oocytes, and red represents dead oocytes. 40–50 oocytes were analyzed per experiment. The viability of thawed oocytes was defined as the ratio of green oocytes to the total number of oocytes. Figure 8 , 10 The "fresh" group in -11 refers to oocytes retrieved from mice (without cryopreservation or other treatments). After thawing, the oocytes were stained for viability; the survival rate results are as follows: Figure 8 As shown, oocytes cryopreserved using nanocomposite materials achieve a higher survival rate compared to traditional methods. Furthermore, we performed transcriptomic analysis (smart-seq2 sequencing technology) on oocytes thawed from both cryopreservation methods, such as... Figure 9 As shown, the cryopreservation method using nanocomposite materials can achieve minimal genetic changes. Furthermore, the in vitro development and fertilization capacity of thawed mouse oocytes were evaluated, revealing that oocytes cryopreserved using nanocomposite materials maintained normal fertilization and developmental potential after thawing and could be transplanted into the body to produce normal offspring. Figure 10 and 11 The conventional freezing method uses 4.3M osmotic CPA (composed of 2.14M EG + 2.16M DMSO) + 0.5M sucrose to assist in the cryopreservation and thawing of mouse oocytes. All other conditions are the same as those in the embodiments of this application.

[0061] Finally, it should be noted that the purpose of disclosing the embodiments is to help further understand the present invention. However, those skilled in the art will understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing a PVA / Fe3O4 / GO nanocomposite material, characterized in that, First, polyvinyl alcohol (PVA) is encapsulated around Fe3O4 via hydrogen bonding to form PVA / Fe3O4 nanospheres. Then, the PVA / Fe3O4 nanospheres are electrostatically adsorbed onto the surface of graphene oxide (GO), resulting in a PVA / Fe3O4 / GO nanocomposite material with an average particle size of 300–700 nm. This PVA / Fe3O4 / GO nanocomposite material exhibits anti-icing effects during both freezing and rewarming stages. The average particle size of the PVA / Fe3O4 nanospheres is 10–50 nm. The method includes the following steps: (1) Preparation of Fe3O4: FeCl3, FeCl2 and deionized water are mixed, ammonia water is added dropwise, and the mixture is stirred under a nitrogen atmosphere. Then the pH is adjusted to 7.5~8.5 and stirred to obtain Fe3O4. (2) Polyvinyl alcohol and Fe3O4 are mixed and ultrasonically mixed at a water bath temperature of 60~90℃, and then centrifuged and washed to obtain Fe3O4 / PVA composite material; wherein the mass ratio of polyvinyl alcohol to Fe3O4 is 100~250:80~120. (3) Mix Fe3O4 / PVA composite material, graphene oxide and deionized water, centrifuge and wash, and vacuum dry to obtain Fe3O4 / PVA / GO; wherein the mass ratio of Fe3O4 / PVA composite material and graphene oxide is 105~200:200~400.

2. The method according to claim 1, characterized in that, In step (1), the average particle size of Fe3O4 is 10~30nm; in step (3), the ratio of graphene oxide to deionized water is 200~500mg:40ml.

3. The PVA / Fe3O4 / GO nanocomposite material prepared by the method according to any one of claims 1-2.

4. A method for cryopreservation of oocytes based on a synergistic anti-icing effect, characterized in that, The method utilizes the PVA / Fe3O4 / GO nanocomposite material as described in claim 3, comprising the following steps: (1) PVA / Fe3O4 / GO nanocomposite material is added to ethylene glycol to obtain a mixture; in the mixture, the concentration of PVA / Fe3O4 / GO nanocomposite material is 1.5~2.5 mg / mL; the mixture also contains trehalose; (2) Following the oocyte cryopreservation procedure, mouse oocytes at the MII stage were cryopreserved using the mixture prepared in step (1). (3) After cryopreservation, the frozen oocytes are rewarmed using laser and alternating magnetic field; (4) Gene sequencing of revived oocytes was performed by transcriptome sequencing to assess the impact of freezing on their genome.

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