Application of EGCG combined with CHK2 inhibitor in improving egg maturation or embryo development

Abstract

The present invention discloses the application of EGCG combined with CHK2 inhibitor in the preparation of a preparation for improving egg maturation or embryonic development, and belongs to the field of assisted reproductive technology of mammals. The present invention also discloses a preparation for improving egg maturation or embryonic development, the active ingredients of which are EGCG and CHK2 inhibitor. The present invention also discloses the application of CHK2 inhibitor in the preparation of a preparation for improving egg maturation or embryonic development. The present invention has found through research that a low concentration of CHK2 inhibitor BML-277 can significantly improve the development rate of aged oocytes, and can achieve better developmental outcomes in combination with an appropriate concentration of EGCG. This shows that the CHK2 inhibitor BML-277, as well as EGCG combined with CHK2 inhibitor, have great application potential in improving the fertilization ability and pregnancy outcomes of mammals, especially elderly mammals.

Description

Technical Field

[0001] The invention relates to application of EGCG combined with a CHK2 inhibitor in improving egg maturation or embryo development, and belongs to the technical field of assisted reproduction of mammals. Background Art

[0002] Reproductive health is crucial to maintaining the sustainability of the population. However, with the continuous changes in the natural and social environment in which humans live, fertility issues are becoming increasingly severe, and the global prevalence of infertility is increasing. In order to solve the fertility dilemma of infertile couples, assisted reproductive technology has emerged and developed rapidly, and has been widely used around the world. However, the current successful pregnancy rate of assisted reproductive technology is still low, and researchers are in urgent need of further improving the embryo culture system.

[0003] With the increase of age, the number and quality of oocytes in the ovaries decrease, accompanied by impaired ovarian endocrine function. This age-related decline in fertility is called reproductive aging. At present, with the relaxation of my country's fertility policy, the proportion of elderly childbearing has increased sharply, but the quality of oocytes in elderly women has declined, resulting in high infertility rates, high miscarriage rates, and difficulties in the prevention and control of birth defects. How to improve the quality of oocytes in elderly women is an important problem that needs to be solved urgently. It is known that oxidative stress, mitochondrial defects, meiotic chromosome segregation errors, genetic mutations, etc. are important factors that lead to the decline in oocyte quality in elderly women. Exploring targeted therapeutic drugs or intervention plans for these reasons will provide effective measures for assisted pregnancy in elderly women in the future.

[0004] In recent decades, the application of assisted reproductive technologies such as in vitro oocyte maturation culture (IVM) and in vitro fertilization-embryo transfer (IVF-ET) has brought hope to a large number of infertile patients. However, there are problems such as insufficient developmental potential of oocytes and embryos and low successful pregnancy rates. In addition, the effect of assisted pregnancy becomes worse as the age of assisted pregnancy increases. In-depth exploration of methods to improve assisted reproductive technologies and increase the success rate of assisted pregnancy will benefit a large number of infertility patients. At present, this field urgently needs methods and culture media that can enhance the maturation of oocytes and the developmental potential of embryos in the in vivo or in vitro cultivation of eggs and embryos, thereby improving the fertilization ability and pregnancy outcomes of mammals, especially elderly mammals.

[0005] Epigallocatechin gallate [(-)-Epigallocatechin gallate, EGCG] is the most abundant polyphenol compound in green tea, and has multiple biological effects such as antioxidant, anti-inflammatory, and anti-tumor. Many studies have shown that EGCG plays an important role in the prevention and treatment of chronic diseases such as tumors, diabetes, cardiovascular diseases, and nervous system diseases.

[0006] Gu Yaqing's master's thesis "Effects of Epigallocatechin Gallate (EGCG) on Mouse Oocyte In Vitro Maturation, Fertilization and Embryonic Development" studied the effects of EGCG on mouse oocyte in vitro maturation, fertilization and later embryonic development by adding different concentrations of EGCG. The results showed that in the HTF system, the addition of 20μM EGCG when culturing COCs can significantly increase the maturation rate of oocytes, as well as the later fertilized egg and embryo development rates. In the HTF system, the addition of EGCG has no obvious effect on DO (naked eggs), cannot increase the maturation rate of oocytes, and has little effect on the fertilized egg and embryo development rate. Excessive addition of EGCG can significantly reduce the maturation rate of oocytes, fertilized eggs and embryo development rates. However, the research object is young eggs, and it is still unclear how EGCG affects the maturation rate of old oocytes and the in vivo and in vitro development outcomes.

[0007] Checkpoint kinase 2 (CHK2) is an important signal transduction protein in the DNA damage repair pathway, which can repair damaged DNA and maintain chromosome stability. Enhancing the sensitivity of tumor cells to DNA damage by using CHK2 inhibitors has been widely studied in the field of tumor treatment. BML-277 is an ATP-competitive inhibitor of checkpoint kinase 2, which can protect human CD4(+) and CD8(+) T cells from apoptosis caused by ionizing radiation in a dose-dependent manner. A recent study reported ("CHK2 is essential for spindle assembly and DNA repair during the first cleavage of mouse embryos") that the application of 25μM BML-277 can seriously affect the development of early embryos, causing embryonic DNA damage and inducing oxidative stress.

[0008] A recent study reported (“Dominant mutations in CHK1 cause pronuclear fusion failure and zygote arrest that can be rescued by CHK1 inhibitor”) that mutations in the gene encoding cell cycle checkpoint kinase 1 (CHK1) can lead to cleavage disorders in human fertilized eggs. The mutation increases the kinase activity of CHK1, which causes cell cycle arrest of fertilized eggs by phosphorylating and inhibiting the downstream CDC25C / CDK1 pathway. According to this mechanism, the use of an appropriate concentration of CHK1 inhibitor to maintain the kinase activity of mutant CHK1 at the normal threshold can successfully enable the patient's fertilized eggs to develop into transplantable blastocysts. CHK1 is a protein kinase that plays an important role in the cell cycle checkpoint pathway, and together with the DNA damage response pathway, it constitutes a genome monitoring system. Although the blastocysts treated with CHK1 inhibitors have complete genomic ploidy and pluripotency, the long-term safety of inhibiting CHK1 to treat cleavage disorders is currently unknown, and a safer and more effective embryo culture program needs to be explored. Summary of the invention

[0009] In view of the above-mentioned prior art, the present invention has found through research that EGCG, CHK2 inhibitors and their combination have great application potential in improving egg maturation and embryonic development.

[0010] The present invention is achieved through the following technical solutions:

[0011] Application of EGCG combined with CHK2 inhibitor in the preparation of preparations for improving egg maturation or embryonic development.

[0012] The egg maturation specifically refers to the maturation rate of mammalian oocytes, especially aged eggs (clinically, it generally refers to eggs of patients older than 37 years old, and mouse eggs older than 9 months are often used as aged egg models in experiments).

[0013] The improvement of egg maturation is specifically manifested in one, some or all of the following effects: improving the maturation rate of oocytes, especially old eggs, reducing the proportion of oocyte death and fragmentation, reducing the reactive oxygen in the oocyte, improving the distribution of mitochondria in the oocyte, increasing the mitochondrial membrane potential in the oocyte, improving the spindle morphology in the oocyte, reducing the proportion of abnormal spindles in the oocyte, and increasing the ATP content in the oocyte.

[0014] The embryonic development specifically refers to the ability of a mammalian fertilized egg to develop into a blastocyst.

[0015] The improvement of embryonic development is specifically manifested in: increasing the success rate of fertilized eggs developing to the blastocyst stage.

[0016] Furthermore, the CHK2 inhibitor is selected from BML-277.

[0017] A preparation for improving egg maturation or embryonic development, wherein the active ingredients are EGCG and CHK2, wherein the effective concentration of EGCG is 1 to 100 μM, and the effective concentration of the CHK2 inhibitor is 0.01 to 100 nM. Preferably, the effective concentration of EGCG is 5 to 20 μM, more preferably 10 μM; the effective concentration of the CHK2 inhibitor is 5 to 20 nM, more preferably 10 nM.

[0018] Furthermore, the preparation for improving egg maturation or embryo development also contains a basic embryo culture medium. The basic embryo culture medium is selected from any suitable mammalian embryo culture medium known in the art, such as bicarbonate buffered medium, Hepes buffer or MOPS buffered medium or phosphate buffered saline, G1 / G1-Plus, G2 / G2-Plus, GMOPS, KSOM, M16, M2, PBS and other commonly used culture media.

[0019] Use of a CHK2 inhibitor in preparing a preparation for improving egg maturation or embryo development.

[0020] Furthermore, the CHK2 inhibitor is selected from BML-277.

[0021] Application of EGCG in the preparation of preparations for improving egg maturation or embryonic development.

[0022] The present invention has found through research that a low concentration of CHK2 inhibitor BML-277 can significantly improve the development rate of mouse old oocytes, and can achieve better developmental outcomes when used in combination with an appropriate concentration of EGCG. This is contrary to the previously reported results that high concentrations (25 μM) of BML-277 seriously affect early embryonic development. This may be because low concentrations of BML-277 can cause other DNA damage repair pathways to activate through compensatory mechanisms, thereby promoting the development of old eggs. This shows that the CHK2 inhibitor BML-277, as well as EGCG combined with CHK2 inhibitors, have great application potential in improving the fertilization ability and pregnancy outcomes of mammals, especially elderly mammals. The present invention is of great significance and value to the research and development of reproductive technology.

[0023] 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

[0024] Figure 1 :Typical images of cleavage of mouse mutant (p.F441fs*16) fertilized eggs treated with culture medium containing EGCG at different concentrations.

[0025] Figure 2 :Statistical graph of cleavage of mouse mutant (p.F441fs*16) fertilized eggs treated with culture medium containing EGCG at different concentrations.

[0026] Figure 3 :Typical pictures of blastocyst development after mouse mutant (p.F441fs*16) fertilized eggs were treated with culture medium containing different concentrations of EGCG.

[0027] Figure 4 :Statistical graph of blastocyst development after mouse mutant (p.F441fs*16) fertilized eggs were treated with culture medium containing different concentrations of EGCG.

[0028] Figure 5 :Expression of inner cell mass (OCT3 / 4) and trophoblast factor (Cdx2) in blastocysts obtained from wild-type (WT) and mutant (p.F441fs*16) mouse fertilized eggs treated with medium containing EGCG (10 μM)

[0029] Figure 6 : Statistical graph of the expression of inner cell mass factor (OCT3 / 4) in blastocysts obtained after wild-type (WT) and mutant (p.F441fs*16) mouse fertilized eggs were treated with culture medium containing EGCG (10 μM).

[0030] Figure 7 : Statistical graph of the expression of trophoblast factor (Cdx2) in blastocysts obtained after wild-type (WT) and mutant (p.F441fs*16) mouse fertilized eggs were treated with culture medium containing EGCG (10 μM).

[0031] Figure 8 : Typical images of mature eggs (MⅡ) obtained after young mouse oocytes were treated with medium containing EGCG (10 μM).

[0032] Fig. 9 : Statistical graph of mature eggs (MⅡ) obtained after young mouse oocytes were treated with culture medium containing EGCG (10μM).

[0033] Fig.10 : ROS expression in MⅡ eggs obtained after young mouse oocytes were treated with medium containing EGCG (10μM).

[0034] Fig.11 : Statistical graph of ROS fluorescence signals of MⅡ eggs obtained after young mouse oocytes were treated with medium containing EGCG (10μM).

[0035] Fig.12: Typical images of mature eggs (MⅡ) obtained after old mouse oocytes were treated with medium containing EGCG (10μM).

[0036] Fig.13 :Statistical graph of mature eggs (MⅡ) obtained after old mouse oocytes were treated with culture medium containing EGCG (10μM).

[0037] Fig.14 : ROS expression in MⅡ oocytes obtained after aged mouse oocytes were treated with medium containing EGCG (10μM).

[0038] Fig.15 : Mitochondrial distribution of MⅡ oocytes obtained after aged mouse oocytes were treated with medium containing EGCG (10μM).

[0039] Fig.16 :Detection of mitochondrial potential of MⅡ oocytes obtained after old mouse oocytes were treated with medium containing EGCG (10μM).

[0040] Fig.17 : Spindle morphology of MⅡ oocytes obtained after old mouse oocytes were treated with medium containing EGCG (10μM).

[0041] Fig.18 : Statistical graph of ROS fluorescence signals of MⅡ eggs obtained after aged mouse oocytes were treated with medium containing EGCG (10μM).

[0042] Fig.19 : Statistical graph of mitochondrial potential (JC-1; Red: Aggregator; Green: Monomer) of MⅡ oocytes obtained after aged mouse oocytes were treated with medium containing EGCG (10 μM).

[0043] Fig. 20 : Statistical graph of ATP content in MⅡ oocytes obtained after old mouse oocytes were treated with medium containing EGCG (10μM).

[0044] Fig.21 :MⅡ ovalbumin obtained after injection of different concentrations of EGCG in aged mice.

[0045] Fig. 22 : Schematic diagram of the number of eggs obtained after injecting different concentrations of EGCG into old mice.

[0046] Fig.23 : Schematic diagram of the percentage of mature eggs obtained after injecting different concentrations of EGCG into old mice (eggs containing PB1 are MⅡ eggs).

[0047] Fig.24: Schematic diagram of the percentage of dead or fragmented eggs obtained after injection of different concentrations of EGCG in aged mice.

[0048] Fig.25 : Spindle morphology of MⅡ eggs obtained after injection of different concentrations of EGCG in aged mice.

[0049] Fig.26 : Schematic diagram of the proportion of abnormal spindles in MⅡ eggs obtained after injection of different concentrations of EGCG in aged mice.

[0050] Fig. 27 :Image of mature oocytes obtained after old mouse oocytes were treated with EGCG / BML-277 / EGCG&BML-277.

[0051] Fig.28 : Schematic diagram of the GVBD (germinal vesicle breakdown) rate of aged mouse oocytes after treatment with EGCG / BML-277 / EGCG&BML-277.

[0052] Fig.29 :Schematic diagram of the MⅡ oocyte rate obtained after aged mouse oocytes were treated with EGCG / BML-277 / EGCG&BML-277.

[0053] Fig.30 :DNA damage (γH2AX) signal diagram of MⅡ oocytes obtained after old mouse oocytes were treated with EGCG / EGCG&BML-277.

[0054] Fig.31 :Statistical graph of DNA damage (γH2AX) signal of MⅡ oocytes obtained after aged mouse oocytes were treated with EGCG / EGCG&BML-277. DETAILED DESCRIPTION

[0055] 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. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0056] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0057] Experimental Effects of EGCG and CHK2 on Oocytes and Fertilized Eggs

[0058] (1) Conventional mouse oocyte in vitro fertilization

[0059] Female mice of appropriate age (5 weeks) were selected for superovulation: 10 IU pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally, 10 IU human chorionic gonadotropin (HCG) was injected 48 hours later, and the cumulus-oocyte complex in the ampulla of the oviduct was collected about 16 hours later.

[0060] Sperm was collected from the cauda epididymis of adult male mice and capacitated for 1 hour in a conventional capacitation medium (G-IVF plus medium from Vitrolife). The cumulus-oocyte complex and the capacitated sperm were then added to a conventional in vitro fertilization medium (G-IVF plus medium from Vitrolife) covered with mineral oil and cultured for 6 hours at 37°C and 5% CO2. The fertilized eggs were transferred to an embryo culture medium (G-1plus medium from Vitrolife) covered with mineral oil and cultured continuously at 37°C and 5% CO2. The development rate and quality of embryos at each stage (including 2-cell, 4-cell, 8-cell and blastocyst) were then counted.

[0061] (2) Conventional mouse oocyte in vitro maturation

[0062] Female mice of appropriate age (5 weeks) were selected and intraperitoneally injected with 10 IU PMSG. After 48 hours, the ovaries of the female mice were taken to separate the oocytes. At this time, the oocytes were germinal vesicle (GV) stage oocytes. They were cultured in vitro for 16 hours in the medium for preimplantation embryo in vitro culture (M16 from Sigma) at 37°C and 5% CO2. After the oocytes matured, they were metaphase (MⅡ) oocytes of the second meiotic division. Subsequently, the maturation rate of MⅡ oocytes, as well as the reactive oxygen species (ROS) level, mitochondrial distribution, mitochondrial potential, ATP level, spindle morphology and other indicators for evaluating oocyte quality were monitored.

[0063] (3) Effects of EGCG-containing culture medium on in vitro culture of oocytes and embryos

[0064] EGCG powder was diluted with DMSO to prepare a 1000X storage solution and stored at -20°C.

[0065] The EGCG-containing culture medium was prepared immediately when the eggs or embryos were cultured in vitro. The storage solution or DMSO containing EGCG was added to the basal culture medium to prepare 4 different EGCG concentrations (0, 1, 10, 100 μM) (the DMSO dose used in each concentration group was the same) (when the EGCG concentration was 0, DMSO was added to the basal culture medium at a ratio of 1:1000). The basal culture medium used for in vitro culture of oocytes was the M16 medium of Sigma. The basal culture medium used for in vitro culture of embryos was the G-1plus medium of Vitrolife.

[0066] In vitro culture of oocytes: The retrieved GV-stage oocytes were placed in the above-prepared culture medium containing EGCG and cultured in vitro for 16 h.

[0067] Embryo culture in vitro: The fertilized eggs obtained 6 hours after in vitro fertilization were placed in the above-prepared culture medium containing EGCG and cultured continuously until the blastocyst stage.

[0068] result:

[0069] Mouse fertilized eggs were overexpressed with wild-type (WT) and mutant (Mutated) CHK1 by microinjection, and then placed in a medium containing EGCG and cultured until the blastocyst stage. Most of the mouse fertilized eggs overexpressing mutant CHK1 failed to divide and could not successfully develop into blastocysts, which is similar to the patient's phenotype. However, after adding an appropriate concentration of EGCG to the culture medium, the cleavage rate and blastocyst development rate of the mutant fertilized eggs were significantly improved (e.g. Figures 1 to 4 As shown in Figure 2 ). Given that mutant fertilized eggs rarely develop to the blastocyst stage, we detected the blastocysts obtained from mutant fertilized eggs after the application of EGCG. The signal intensity of the inner cell mass and trophoblast (Oct3 / 4 and Cdx2) was not significantly different from that of wild-type fertilized eggs ( Figure 5 , 6 , 7).

[0070] Five-week-old young mouse oocytes were cultured in vitro (using a medium containing EGCG), and the proportion of oocytes developing to the MⅡ stage was significantly increased ( Figure 8 , 9 We detected the level of reactive oxygen species in MⅡ stage oocytes and found that adding appropriate concentrations of EGCG to the culture medium could significantly reduce the level of reactive oxygen species in MⅡ oocytes ( Fig.10 , 11 ), which may be related to the antioxidant effect of EGCG. (Note: These two figures are not included in the revised manuscript, and the fertilized eggs mentioned here are described in the previous paragraph)

[0071] (4) To explore the culture strategy to improve the development of eggs and embryos in older women, based on the above findings, we further used 10-month-old mice to observe the culture effect after adding EGCG to the culture medium.

[0072] The results are gratifying: after culturing aged oocytes with medium containing EGCG, the in vitro maturation rate of oocytes was significantly improved ( Fig.12 , 13 To further examine the quality of MⅡ oocytes after EGCG treatment, we then tested their reactive oxygen levels, mitochondrial distribution, mitochondrial membrane potential, spindle morphology, and ATP content. Figures 14 to 20The results showed that EGCG treatment could significantly reduce the reactive oxygen species in the in vitro matured oocytes of aged mice, improve the mitochondrial distribution of aged oocytes, increase the mitochondrial membrane potential, improve the spindle morphology, and increase the ATP content in aged oocytes.

[0073] Conclusion: EGCG can significantly improve the development rate of CHK1 mutant fertilized eggs. EGCG can improve the in vitro developmental outcomes of young eggs and embryos. EGCG can improve the development rate of aged oocytes and improve oocyte quality.

[0074] (5) Effect of EGCG on oocyte maturation in vivo

[0075] In order to further explore methods to improve the outcomes of egg and embryo development in older women, EGCG was injected intraperitoneally into aged mice to explore its effect on improving oocyte development in vivo.

[0076] 10-month-old female mice were intraperitoneally injected with 0.1-10 mg / kg EGCG for 6 consecutive days (a total of 5 groups of different doses were injected, the doses were: 0, 0.1, 1, 5, 10 mg / kg; EGCG powder was diluted with normal saline and injected) at the same time. On the 4th day, 10 IU PMSG was injected at the same time, and on the 6th day (within 48 hours after the 4th day), 10 IU HCG was injected at the same time. After 16 hours, the cumulus-oocyte complex was removed from the ampulla of the oviduct, and the granulosa cells of the cumulus-oocyte complex were digested with hyaluronidase to expose the MⅡ oocyte. The number of ovulations, the rate of first polar body (PB1) extrusion, and the rate of abnormal oocytes (death or fragmentation) were monitored, and immunofluorescence staining was performed to monitor the spindle morphology.

[0077] Results: After 6 consecutive days of intraperitoneal injection of EGCG, the mature oocytes obtained after superovulation were taken out, and the granulosa cells were digested with hyaluronidase to observe the maturation rate and quality of oocytes in each group. Figures 21 to 26 The results showed that the appropriate concentration of EGCG injected in vivo could significantly increase the in vivo maturation rate of oocytes and significantly reduce the proportion of oocyte death and fragmentation. The spindle morphology of each group was observed by immunofluorescence, and the results showed that the appropriate concentration of EGCG could also significantly reduce the proportion of abnormal spindles in mature oocytes.

[0078] Conclusion: EGCG can improve the developmental outcome of aged oocytes in vivo.

[0079] (6) Effects of BML-277, EGCG and their combined use on the oocyte maturation rate of aged mice

[0080] In summary, the above research results suggest that EGCG reduces the level of oxidative stress in oocytes and improves the developmental outcome of aged oocytes. On this basis, in order to further explore methods to improve the maturation rate of aged eggs, we tried to find a feasible solution from the perspective of DNA damage and investigate the effects of BML-277, EGCG and their combined application on the maturation rate of aged mouse oocytes.

[0081] Culture medium containing BML-277: BML-277 powder was diluted with DMSO to prepare 1000X stock solution and stored at -20°C. Prepared immediately before in vitro culture of oocytes, the basic culture medium used was M16 culture medium from Sigma. The concentration of BML-277 was 10 nM.

[0082] EGCG-containing culture medium: EGCG powder was diluted with DMSO to prepare a 1000X storage solution and stored at -20°C. Prepared immediately before in vitro culture, the concentration of EGCG was 10 μM. The basic culture medium used was M16 culture medium from Sigma.

[0083] Culture medium containing BML-277 and EGCG: The concentration of BML-277 is 10 nM, and the concentration of EGCG is 10 μM. The basic culture medium used is M16 culture medium from Sigma.

[0084] In vitro culture of oocytes: GV-stage oocytes taken from 10-month-old mice were placed in the above-prepared culture medium containing BML-277, culture medium containing EGCG, and culture medium containing BML-277 and EGCG, and cultured in vitro for 16 hours.

[0085] Results: Surprisingly, low concentrations of the CHK2 inhibitor BML-277 significantly increased the developmental rate of aged oocytes, and combined with appropriate concentrations of EGCG, a better developmental outcome was achieved (e.g. Fig. 27 , 28 , 29); and the combined use of BML-277 can significantly reduce DNA damage in aged eggs compared with the use of EGCG alone (e.g. Fig.30 , 31 This is contrary to the previous report that high concentrations (25 μM) of BML-277 seriously affect early embryonic development (generally speaking, the quality of oocytes is an important factor affecting embryonic development potential. If the quality of oocytes can be improved, embryonic development will also improve. Although the research subjects reported in this literature are embryos, BML-277 theoretically inhibits CHK2 by inhibiting DNA repair and causing DNA damage accumulation, thereby affecting cell development). This may be because low concentrations of BML-277 can promote the development of older eggs by activating other DNA damage repair pathways through compensatory mechanisms.

[0086] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended 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. The use of EGCG combined with a CHK2 inhibitor in the preparation of a preparation for improving egg maturation, characterized in that: The effective concentration of EGCG is 10 μM, and the effective concentration of CHK2 inhibitor is 10 nM; the CHK2 inhibitor is selected from BML-277; The preparation for improving egg maturation has one, some or all of the following effects: improving the maturation rate of oocytes, reducing the proportion of oocyte death and fragmentation, reducing the reactive oxygen in oocytes, improving the distribution of mitochondria in oocytes, increasing the mitochondrial membrane potential in oocytes, improving the spindle morphology in oocytes, reducing the proportion of abnormal spindles in oocytes, and increasing the ATP content in oocytes; The oocytes are selected from aged eggs.

2. A preparation for improving egg maturation, characterized in that: The active ingredients are EGCG and a CHK2 inhibitor, the effective concentration of EGCG is 10 μM, and the effective concentration of the CHK2 inhibitor is 10 nM; the CHK2 inhibitor is BML-277; The preparation for improving egg maturation has one, some or all of the following effects: improving the maturation rate of oocytes, reducing the proportion of oocyte death and fragmentation, reducing the reactive oxygen in oocytes, improving the distribution of mitochondria in oocytes, increasing the mitochondrial membrane potential in oocytes, improving the spindle morphology in oocytes, reducing the proportion of abnormal spindles in oocytes, and increasing the ATP content in oocytes; The oocytes are selected from aged eggs.

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