PRP concentrated storage liquid capable of improving embryo encapsulation rate, preparation method, embryo culture medium and application

Abstract

The invention provides a PRP concentrated storage solution capable of improving embryo encapsulation rate, a preparation method, an embryo culture medium and application, and belongs to the technical field of biology. According to the method, the PRP concentrated storage liquid after platelet separation is introduced into an early embryo in-vitro culture system, a novel culture system for simulating a maternal physiological microenvironment is successfully constructed, the biochemical microenvironment for embryonic development is remarkably optimized through multiple-effect growth factors such as PDGF and IGF-1 and a fibrous protein matrix contained in the PRP concentrated storage liquid, and the survival rate of embryonic development is improved. The method can obviously accelerate the proliferation speed of the blastocytes of mouse early embryo in-vitro culture, greatly increase the number of the blastocytes, enable the number of the blastocytes and the blastocyst rate to highly approach to the in-vivo development embryo level, effectively improve the blastocyst formation rate and the embryo quality of in-vitro embryonic development, and enhance the embryo implantation capability, is simple and convenient to operate, short in time consumption, and low in cost. And a solution with great potential is provided for improving the blastocyst formation efficiency of in-vitro culture of embryos in an auxiliary growth process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a PRP concentrated stock solution capable of improving the blastocyst formation rate of embryos, a preparation method, an embryo culture medium and applications thereof. Background Art

[0002] In vitro fertilization-embryo transfer (IVF-ET, commonly known as test-tube baby) technology is a core assisted reproductive method in which mature oocytes of the female and sperm of the male are fertilized in vitro, and then the embryos are cultured in vitro to a specific developmental stage (usually the cleavage-stage embryos on the 3rd day or the blastocyst-stage embryos on the 5th - 6th day) and transferred back to the mother's uterus. However, currently, the average clinical pregnancy rate of global IVF-ET is only about 50%, and the live birth rate is further reduced to 30% - 40%. Research shows that approximately 60% of the assisted reproductive failure cases are attributed to embryonic developmental arrest in vitro, that is, the fertilized eggs cannot break through the developmental bottleneck in the in vitro culture system to form high-quality embryos with transplantation value (such as cleavage-stage embryos with ≥8 cells and <15% fragmentation rate on the 3rd day, or blastocysts with a morphological score ≥3BB on the 5th day). This developmental arrest phenomenon mostly occurs at the key nodes of zygotic genome activation (ZGA, around the 3rd day) and blastocyst formation (on the 5th - 6th day) in human embryos, and its root cause is closely related to the inability of the in vitro culture microenvironment to accurately simulate the physiological conditions of the mother's body.

[0003] Consistent with the in vitro culture of human embryos, when mouse fertilized eggs are cultured in vitro in a commercial KSOM culture medium, the in vitro developmental rate of the embryos is significantly lower than that of in vivo embryonic development, and both the blastocyst rate and the number of blastocyst blastomeres are significantly lower than those of in vivo blastocysts.

[0004] The low developmental potential and developmental arrest phenomenon of embryos are inextricably linked to the in vitro developmental microenvironment. Although modern assisted reproductive technologies have been continuously innovated and the quality of the culture medium has been significantly improved, from basic nutritional components to the addition of special growth factors, all are trying to approximate the in vivo environment as much as possible, but so far there is still no culture medium that can completely replicate the physiological conditions of the female reproductive tract. The female reproductive tract not only provides nutrients for embryonic development but also constructs a dynamically and precisely regulated biochemical and physical environment, and this complexity far exceeds the simulation ability of the current in vitro culture system.

[0005] Numerous studies on different species such as humans, mice, and cattle have revealed many problems in in vitro embryonic development. At the cytoplasmic level, embryos produced in vitro often show excessive lipid accumulation, which may interfere with normal cell metabolism and division; in terms of cytogenetics, the abnormal rates of chromosomal aneuploidy, structural aberrations, etc. are significantly increased, affecting the developmental potential and genetic stability of embryos; at the epigenetic level, changes in regulatory patterns such as DNA methylation and histone modification may lead to gene expression disorders. In addition, morphological changes such as a decrease in the number of embryonic blastomeres and abnormal cell - cell connections are also very common. These abnormalities are intertwined, jointly constituting the potential risk of embryonic developmental arrest, highlighting the urgent need to optimize the in vitro developmental microenvironment and innovate the culture system. Platelet - rich plasma (PRP), a platelet concentrate extracted by centrifuging blood, contains various growth factors such as platelet - derived growth factor (PDGF), transforming growth factor, IGF - 1, fibroblast growth factor, vascular endothelial growth factor, and fibrin, and is rich in various growth factors that promote tissue regeneration and anti - inflammatory repair, playing an important role in facial anti - aging, scar removal, and wound repair. However, it has not been applied to in vitro embryo culture.

[0006] Currently, the components of in vitro embryo culture methods are relatively single, and there are still many active components such as growth factors in the in vivo oviduct fluid, such as epidermal growth factor (EGF), transforming growth factor (TGF), insulin - like growth factor 1 (IGF - 1), leukemia inhibitory factor (LIF), etc. The lack of active factors such as the microenvironment may be the key factor for embryonic developmental arrest during in vitro embryo culture. Therefore, developing new in vitro embryo culture technologies for improving the blastocyst formation rate of embryos is particularly important for scientific research based on mouse embryos and future clinical applications. Summary of the Invention

[0007] The purpose of the present invention is to provide a PRP concentrated stock solution, a preparation method, an embryo culture medium, and an application that can improve the blastocyst formation rate of embryos, which can effectively increase the blastocyst formation rate and embryo quality of in vitro embryonic development, enhance the embryo implantation ability, and have simple operation and short time - consumption.

[0008] In order to achieve the above - mentioned invention purpose, the present invention provides the following technical solutions:

[0009] The present invention provides a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos, and the PRP concentrated stock solution is obtained by filtering platelet - rich plasma.

[0010] The present invention also provides a method for preparing a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos, comprising the following steps:

[0011] Take platelet-rich plasma, adjust the concentration, activate it, and filter it to obtain the PRP concentrated stock solution.

[0012] Preferably, when adjusting the concentration, the final concentration of platelets is 1×10 8 cells / mL to 1×10 9 cells / mL.

[0013] According to the method for preparing a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos according to claim 2, characterized in that the activation method is:

[0014] Put the platelet-rich plasma with adjusted concentration into a -80°C refrigerator for 24 to 48 hours, take it out, water bath at 37°C for 1 to 2 hours, and then centrifuge at 1000 to 3000 g for 5 to 20 minutes.

[0015] Preferably, in step (2), the pore size of the filtration is 0.2 to 0.23 μm.

[0016] The present invention also provides the application of the PRP concentrated stock solution prepared by the method for preparing a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos in the preparation of embryo culture media.

[0017] The present invention also provides the application of the PRP concentrated stock solution prepared by the method for preparing a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos in increasing the proportion of blastocysts.

[0018] The present invention also provides an embryo culture medium, characterized in that the embryo culture medium includes the PRP concentrated stock solution prepared by the method for preparing a PRP concentrated stock solution that can improve the blastocyst formation rate of embryos.

[0019] Preferably, the concentration of the PRP concentrated stock solution in the embryo culture medium is 1 to 10×10 5 cells / mL in terms of platelet concentration.

[0020] The present invention also provides the application of an embryo culture medium in embryo culture.

[0021] The beneficial effects of the present invention compared with the prior art are:

[0022] The present invention introduces the PRP concentrated stock solution after separating platelets (with a concentration of 1×10 5 cells / mL to 1×10 6(at a density of cells / mL), a new culture system that simulates the physiological microenvironment of the mother body was successfully constructed. Through multiple growth factors such as PDGF and IGF-1 and fibrin matrix contained in the PRP concentrated stock solution, the biochemical microenvironment for embryonic development was significantly optimized, which could significantly accelerate the cleavage sphere proliferation rate of early mouse embryos in vitro culture, greatly increase the number of cleavage spheres, and make the number of cleavage spheres and blastocyst rate highly approach the level of embryos developed in vivo. This method can effectively improve the blastocyst formation rate and embryo quality of in vitro embryo development, enhance the embryo implantation ability, not only has simple operation and short time consumption, but also provides a highly potential solution for improving the efficiency of in vitro cultured embryos to form blastocysts during the assisted reproduction process, showing outstanding technical advantages and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 It is the preparation process of the PRP concentrated stock solution in Test Example 1 of the present invention;

[0025] Figure 2 It is the in vitro development situation and statistical chart of mouse embryos under different concentration gradients of the action of the PRP concentrated stock solution in Test Example 1 of the present invention. Among them, A is the picture of mouse embryos after culturing fertilized eggs obtained in vivo (hCG24) in KSOM medium supplemented with different concentrations of the PRP concentrated stock solution for hCG 96h; B is the statistical chart of the blastocyst rate of mouse embryos cultured in vitro under different concentration gradients of the action of the PRP concentrated stock solution;

[0026] Figure 3 It is the result chart of the promotion of PRP on the proliferation and cell number of early in vitro embryo culture in Test Example 2 of the present invention. Among them, Figure A is the in vitro control, the 0.05% PRP in vitro treatment group and the embryo picture obtained in vivo; Figure B is the statistical chart of the number of cleavage spheres of hCG 96h embryos;

[0027] Figure 4 It is the fluorescence staining and statistical chart of CDX2 and NANOG of mouse embryos in Test Example 2 of the present invention. Among them, A is the fluorescence staining picture of CDX2 of mouse embryos, B is the fluorescence staining picture of NANOG, and C is the Western blot of CDX2 and NANOG of mouse blastocysts. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0029] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0032] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0033] The present invention provides a method for preparing a PRP concentrated stock solution that can improve the embryonic cyst formation rate. The specific steps are as follows: Take platelet-rich plasma and adjust the platelet concentration to 1·10 9 cells / mL; Place the platelet-rich plasma with adjusted concentration in a -80°C refrigerator for 24 - 48 h, take it out and place it in a 37°C water bath for 1 - 2 h, then centrifuge at 1000 - 3000 g for 5 - 20 min for activation, and take the supernatant after centrifugation and filter it through a 0.2 - 0.23 μm filter membrane to obtain the PRP concentrated stock solution.

[0034] Example 1

[0035] Example 1 of the present invention provides a method for preparing a PRP concentrated stock solution that can improve the embryonic cyst formation rate. The specific steps are as follows:

[0036] 1) PRP Platelet Quantification: PRP is sourced from the routine preparation process in the hospital blood transfusion department. According to expert consensus, the platelet concentration in the recommended PRP is ≥ 4 times the patient's baseline platelet concentration. The platelet concentration in PRP collected by apheresis is greater than 1000·10 9 cells / L (i.e., greater than 1000·10 6 cells / mL). Platelets in PRP are counted using a hemocytometer, and PRP is diluted with normal saline to a platelet stock solution concentration of 1000·10 6 cells / mL.

[0037] 2) PRP Activation: The centrifuge tube containing the platelet stock solution from step (1) is placed in an -80°C refrigerator for 24 h, then in a 37°C water bath for 1 h, and then centrifuged at 2000g at room temperature for 10 min to activate the PRP.

[0038] 3) PRP Filtration: The supernatant of the activated PRP is taken and filtered using a 0.22 - μm sterile filter to remove cells, platelets, cell debris, etc., obtaining a concentrated PRP stock solution that is activated and can be added to the embryo culture medium.

[0039] The concentrated PRP stock solution of the present invention mainly includes growth factors released by platelets and no longer contains platelets. Since there are many growth factors and it is difficult to select one for quantification, and the number of platelets is proportional to the released growth factors, the platelet concentration is subsequently used to quantify the concentration of PRP release, that is, the concentration of the concentrated PRP stock solution is equivalent to 1000·10 6 platelets / mL before activation.

[0040] 4) Sub - packaging of Concentrated PRP Stock Solution: The concentrated PRP stock solution is sub - packaged, with 20 μL in each tube and sub - packaged into sterile 0.2 - mL PCR tubes, stored at -80°C for standby. Take out one tube each time for use to avoid repeated freezing and thawing.

[0041] Experimental Example 1

[0042] Experimental Example 1 of the present invention detected the effects of different concentrations of the concentrated PRP stock solution prepared in Example 1. The specific steps are as follows:

[0043] Forty - week - old mice were injected with 5 IU of PMSG (Ningbo Sansheng). After 48 h, 5 IU of hCG was injected, and then they were caged with wild - type male mice. The mice were sacrificed 24 h after hCG injection, and the oviduct ampulla was taken to obtain fertilized eggs. The concentrated PRP stock solution prepared in Example 1 was taken and diluted with KSOM culture medium. Using the concentration gradient method, it was diluted 10000 - fold, 2000 - fold, 1000 - fold, 500 - fold, 100 - fold, 50 - fold, and 20 - fold (as Figure 1As shown in the figure, PRP+KSOM culture drops with concentrations of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, and 5% were prepared respectively, with each drop being 40 μL. The retrieved fertilized eggs were evenly placed into the respective concentration drops and cultured for 96 h, and the blastocyst formation rate of in vitro embryos was observed (the results are as Figure 2 shown).

[0044] It can be seen from Figure 2 that the optimal proportion of the PRP concentrated stock solution for promoting embryo development is 0.01% to 0.1%, that is, the concentration of the PRP concentrated stock solution in the embryo culture medium is 1×10 5 cells / mL to 1×10 6 cells / mL of PRP is the best. All the subsequently retrieved fertilized eggs were cultured with 0.05% PRP (equivalent to a platelet concentration of 5×10 5 cells / mL in the culture medium) and the experiment was carried out.

[0045] Test Example 2

[0046] In Test Example 2 of the present invention, the promoting effect of the PRP concentrated stock solution prepared in Example 1 on the quality of blastocysts was detected, and the specific steps are as follows:

[0047] Take the blastocysts treated with the 0.05% PRP+KSOM culture drops in Test Example 1, and perform immunofluorescence staining and Western blot on the inner cell mass expressing the marker protein NANOG and the trophectoderm expressing the marker protein CDX2. The results are as Figure 3 , 4 shown.

[0048] It can be seen from Figure 3 that compared with the morphology of embryos cultured conventionally in vitro, after adding 0.05% PRP to the embryo culture medium, the proliferation ability of fertilized eggs developing into embryos at all levels is significantly improved, and the development speed is closer to the embryo development situation in the mouse body.

[0049] It can be seen from Figure 4 that compared with embryos cultured conventionally in vitro, after adding 0.05% PRP to the embryo culture medium, the differentiation potential of the inner cell mass and trophectoderm developing into blastocysts is improved, indicating that PRP promotes the quality of blastocysts cultured in vitro.

[0050] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A PRP concentrated storage solution that can improve the blastocyst formation rate of embryos, characterized in that, The PRP concentrated storage solution is obtained by filtering platelet-rich plasma.

2. A method for preparing the PRP concentrated stock solution capable of increasing the blastocyst formation rate of embryos according to claim 1, characterized in that, It includes the following steps: Take platelet-rich plasma, adjust its concentration, activate it, and filter it to obtain the PRP concentrated storage solution.

3. The preparation method of the PRP concentrated storage solution capable of improving the embryonic blastocyst formation rate according to claim 2, wherein, The final concentration of platelets when adjusting the concentration is 1·10 8 cells / mL to 1·10 9 cells / mL.

4. The preparation method of the PRP concentrated storage solution capable of improving the embryo blastocyst formation rate according to claim 2, characterized in that, The activation method is as follows: Put the platelet-rich plasma with adjusted concentration into a -80°C refrigerator for 24 to 48 hours, take it out and place it in a 37°C water bath for 1 to 2 hours, and then centrifuge it at 1000 to 3000 g for 5 to 20 minutes.

5. The preparation method of the PRP concentrated storage solution capable of improving the embryo blastocyst formation rate according to claim 2, characterized in that, In step (2), the pore size of the filtration is 0.2 to 0.23 μm.

6. Application of the PRP concentrated storage solution prepared by the preparation method of the PRP concentrated storage solution capable of increasing the blastocyst formation rate according to any one of claims 2 to 5 in the preparation of an embryo culture medium.

7. Application of the PRP concentrated storage solution prepared by the preparation method of the PRP concentrated storage solution capable of increasing the blastocyst formation rate according to any one of claims 2 to 5 in increasing the blastocyst ratio.

8. An embryo culture medium, characterized in that, The embryo culture medium includes the PRP concentrated storage solution prepared by the preparation method of the PRP concentrated storage solution capable of increasing the blastocyst formation rate according to any one of claims 2 to 5.

9. The embryo culture medium according to claim 8, wherein The concentration of the PRP concentrated solution in the embryo culture medium is 1 to 10·10 5 per mL in terms of platelet concentration.

10. Application of the embryo culture medium according to any one of claims 8 to 9 in embryo culture.

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