An agent for activating and increasing follicles and its application

Abstract

This invention relates to the field of ovarian tissue in vitro culture technology, and discloses a formulation for activating and increasing follicles and its application. The formulation consists of a PI3K / Akt / mTOR pathway activator and / or a PTEN inhibitor SF1670. The PI3K / Akt / mTOR pathway activator is a 5-15 μM statin, and the PTEN inhibitor is a 20-50 μM SF1670. Using this formulation can achieve the follicle activation and increase effect of existing technologies, contributing to the development and selection of in vitro follicle activators in clinical practice. Furthermore, the use of this formulation opens up a new technological application area for statins.

Description

Technical Field

[0001] This invention relates to the field of ovarian tissue in vitro culture technology, and in particular to a preparation for activating and increasing follicles and its application. Background Technology

[0002] In mammals, the growth and maturation of germ cells is a process finely regulated by hormones. These hormones include gonadotropins from the hypothalamus and hormones and growth factors produced by the reproductive organs themselves. In female mammals, the follicle is the basic functional unit of the ovary, consisting of an egg in the center and one or more layers of granulosa cells surrounding it. Based on the different growth and development stages of the oocyte within the follicle, they can be classified as primordial follicles, primary follicles, secondary follicles, cavitary follicles, and pre-ovulatory follicles. Primordial follicles are the resting follicle pool in the ovary; their number is fixed before or after birth in mammals, with approximately 400,000 primordial follicles in each human ovary. During the growth and development of the ovary, the primordial follicles are continuously activated, entering the growth phase through the primary and secondary follicle stages. At birth, starting with menarche, cyclical ovulation gradually depletes these follicles until only 1,000-2,000 remain. At this point, primordial follicles cease development, and the woman enters menopause. Since primordial follicles cease development after reaching a certain number, the absence of any follicles on an ultrasound scan does not necessarily mean that primordial follicles are absent from the ovary. As long as primordial follicles are present, in vitro primordial follicle activation technology can awaken them from their dormant state, allowing them to continue developing and ovulating.

[0003] Current in vitro follicle activation techniques for increasing follicle size primarily involve treating ovarian tissue with signaling pathway activators to activate primordial follicles, which is significant for treating patients with premature ovarian failure. Current follicle-increasing agents mainly include combinations of PI3K signaling pathway activators bpV and 740-YP, and mTOR signaling pathway activators phosphatidic acid and propranolol. However, due to the complexity of the activators involved in these patents, and the fact that some require 48 hours of in vitro treatment, patients often need to undergo two surgeries to complete the treatment in clinical practice. Therefore, finding new activation methods to shorten the activation time, enabling the treatment to be completed in a single surgery, reducing patient discomfort, and making the procedure convenient and quick, would represent a revolutionary leap forward for this technology. Summary of the Invention

[0004] The purpose of this invention is to provide a preparation for activating and increasing follicles and its application. The preparation is composed of a statin drug, an activator of the P13K / Akt / mTOR pathway, and / or a PTEN inhibitor SF1670. By using this preparation for activating and increasing follicles, the activation effect of existing technologies can be achieved. At the same time, the use of this preparation also opens up a new technical application field for statin drugs.

[0005] Technical solution: A preparation for activating and increasing follicles, comprising a PI3K / Akt / mTOR pathway activator and / or a PTEN inhibitor SF1670, wherein the PI3K / Akt / mTOR pathway activator is a 5-15 μM statin drug and the PTEN inhibitor is 20-50 μM SF1670.

[0006] The aforementioned statin drugs are one of lovastatin, pravastatin, and atorvastatin.

[0007] The application of the above-mentioned preparation in the preparation of human ovarian cortex culture medium.

[0008] The above-mentioned human ovarian cortex culture medium includes the following components: 1) statins, activators of the PI3K / Akt / mTOR pathway, and / or PTEN inhibitor SF1670; 2) basal culture medium.

[0009] The aforementioned human ovarian cortex culture medium, wherein the basal culture medium is MEMA cell culture medium, wherein 0.23 mM sodium pyruvate and 10 wt.% human serum albumin are added.

[0010] The above-mentioned human ovarian cortical culture medium has the following reagent composition: Solution I: basal culture medium; Reagent I: PI3K / Akt / mTOR pathway activator, which is a 5-15 μM statin drug, pre-amplified according to the dosage of 30 mL of basal culture medium; Reagent II: PTEN inhibitor, which is 20-50 μM SF1670, pre-amplified according to the dosage of 30 mL of basal culture medium.

[0011] The procedure for using human ovarian cortical culture medium for non-diagnostic and non-therapeutic purposes is as follows:

[0012] 1) Obtain human ovarian tissue in 10mL-15mL of cell culture medium, separate the ovarian cortex and cut it into 1mm pieces. 3 Small pieces;

[0013] 2) Take 1 mL of solution I and mix it with reagent I and / or reagent II to prepare 10 mL of tissue culture medium;

[0014] 3) Human ovarian cortex was cultured in a 24-well plate containing a suspension mesh. 400-3000 μL of tissue culture medium was added below the mesh and cultured for 2 hours at 37°C and 5% CO2.

[0015] 4) After 2 hours, the tissue culture is complete, and the cultured tissue can be used for subsequent operations.

[0016] The present invention has the following beneficial effects:

[0017] In summary, culturing mouse or human ovarian cortex in a culture medium containing statins (a P13K / Akt / mTOR pathway activator) and / or the PTEN inhibitor SF1670 can activate primordial follicles and promote follicular development within a short time (2 hours). The cultured ovarian cortex can then be surgically transplanted back into the mother to continue normal development until maturity. Therefore, this invention provides a simple and efficient primordial follicle activator, enabling more efficient, faster, and convenient application of this technology in the field of assisted reproductive technology. Attached Figure Description

[0018] Figure 1 This image shows the development of the mouse ovary after 2 hours of in vitro activation and culture of primordial follicles. Left (A) is the control, and right (B) is the activated ovary. Immunohistochemical results of AMH showed that, after 48 hours of culture, the activated ovary (B) had more follicles entering the secondary follicle stage compared to the control ovary (A).

[0019] Figure 2 After activating mouse ovarian tissue, mature oocytes were retrieved from the ovaries of the treated group and subjected to ICSI for embryonic development. A, ICSI operation on mature oocytes; B, Embryo at the 2-cell stage; C, Embryo at the 4-cell stage; D, Embryo at the 8-cell stage; E, Embryo at the morula stage; F, Embryo at the blastocyst stage. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0021] Example 1

[0022] A novel in vitro culture medium for human ovarian cortex, comprising the following components:

[0023] Solution I is prepared as follows:

[0024] Basic culture medium: MEMA (GIBCO) TM Minimum Essential Medium (MEM) Alpha Medium (1X) Liquid (Invitrogen) was used as the stock solution, with 10 wt.% human serum albumin and 0.23 mM sodium pyruvate added.

[0025] The preparation method for reagent I is as follows:

[0026] P13K / Akt / mTOR pathway activator, containing 15 μM atorvastatin, pre-amplified according to the dosage of 30 mL basic activated cell culture medium;

[0027] The above-mentioned activator is pre-packaged into small boxes, and then packaged together with Solution I into a larger box. When using, follow the ovarian cortex culture method described in the instructions.

[0028] The method for using the culture medium is as follows:

[0029] 1) Take mouse ovaries into 10 mL of cell culture medium and wash them repeatedly with the culture medium more than three times.

[0030] 2) Take 1 mL of solution I and reagent I. After reagent I is completely dissolved, use solution I to prepare 10 mL of tissue culture medium.

[0031] 3) Mouse ovaries were cultured in 24-well plates containing Millicell cell culture inserts (Millipores). 500 μL of cell activation medium was added below the Millipores and cultured for 2 hours at 37°C and 5% CO2.

[0032] 4) Two hours later, the activation process was completed. The treated mouse ovaries were transplanted under the kidney capsule of the recipient mouse, and the bilateral ovaries of the recipient mouse were removed at the same time.

[0033] 5) On the second day after the operation, the recipient mice were given an injection of follicle-stimulating hormone (FSH 1 IU / mouse) for 18 consecutive days.

[0034] 6) On the last day, recipient mice were treated with 10 IU of human chorionic gonadotropin (hCG). 24 hours later, the transplanted ovaries were collected, and mature oocytes were released by acupuncture.

[0035] 7) After in vitro fertilization of oocytes with sperm from male mice of the same strain, the resulting 2-cell embryos were transferred to pseudopregnant mice, and pregnancy was successfully established. Finally, the fertilization rate and litter size of mature oocytes obtained from the processed ovaries were compared with those obtained from mature oocytes obtained from normal adult ovaries after superovulation.

[0036] The experimental results are shown in Table 1. Table 1 compares the embryo development rate and embryo survival rate after in vitro fertilization of normal superovulated oocytes and in vitro activated oocytes.

[0037] Table 1

[0038] MII 2-cell embryo (％) 2-cell embryo Number of offspring (％) Treatment group eggs 120 86 71.67 60 15 25.00 Superovulation 81 69 85.18 60 22 36.67

[0039] Example 2

[0040] A novel in vitro culture medium for human ovarian cortex, comprising the following components:

[0041] Solution I is prepared as follows:

[0042] Basic culture medium: MEMA (GIBCO) TM Minimum Essential Medium (MEM) Alpha Medium (1X) Liquid (Invitrogen) was used as the stock solution, with 10 wt.% human serum albumin and 0.23 mM sodium pyruvate added.

[0043] The preparation method for reagent II is as follows:

[0044] PTEN inhibitor, containing 50 μM SF1670, pre-amplified at a dose of 30 mL of basic activated cell culture medium;

[0045] The above-mentioned activator is pre-packaged into small boxes, and then packaged together with Solution I into a larger box. When using, follow the ovarian cortex culture method described in the instructions.

[0046] The novel ovarian cortex in vitro culture medium described above was used in an experiment according to the method described in Example 1. The experimental results are shown in Table 2. Table 2 compares the embryo development rate and embryo survival rate after in vitro fertilization of normal superovulated oocytes and in vitro activated oocytes.

[0047] Table 2

[0048] MII 2-cell embryo (％) 2-cell embryo Number of offspring (％) Treatment group eggs 97 66 68.04 50 14 28.00 Superovulation 110 102 92.73 50 33 33.00

[0049] Example 3

[0050] A novel in vitro culture medium for human ovarian cortex, comprising the following components:

[0051] Solution I is prepared as follows:

[0052] Basic culture medium: MEMA (GIBCO) TM Minimum Essential Medium (MEM) Alpha Medium (1X) Liquid (Invitrogen) was used as the stock solution, with 10 wt.% human serum albumin and 0.23 mM sodium pyruvate added.

[0053] The preparation method for reagent I is as follows:

[0054] P13K / Akt / mTOR pathway activator, containing 5 μM pravastatin, pre-amplified according to the dosage of 30 mL basic activated cell culture medium;

[0055] The preparation method for reagent II is as follows:

[0056] PTEN inhibitor, containing 20 μM SF1670, pre-amplified according to a dose of 30 mL basic activated cell culture medium;

[0057] The above-mentioned activator is pre-packaged into small boxes, and then packaged together with Solution I into a larger box. When using, follow the ovarian cortex culture method described in the instructions.

[0058] The novel ovarian cortex in vitro culture medium described above was tested using the method described in Example 1. The experimental results are shown in Table 3, which compares the embryo development rate and embryo survival rate after in vitro fertilization of normal superovulated oocytes and in vitro activated oocytes.

[0059] Table 3

[0060] MII 2-cell embryo (％) 2-cell embryo Number of offspring (％) Treatment group eggs 95 70 73.68 65 18 27.69 Superovulation 83 75 90.36 65 23 35.38

[0061] The results in Tables 1, 2, and 3 show that the development rate of 2-cell embryos from mature oocytes in the treatment group after in vitro fertilization was lower than that from normal superovulation oocytes. However, compared with the activation protocols in existing patents, the development rate of 2-cell embryos was at a higher level. Although the development rate of 2-cell embryos in all activation treatment groups was lower than that in the normal superovulation oocyte group, the activation protocol involved in this patent significantly obtained more 2-cell embryos, indicating that the culture medium formed by this activation protocol can better promote follicle development and improve the quality of oocytes.

[0062] The above methods have been proven effective through testing, and the relevant test results are as follows:

[0063] Ovarian development in mice 2 hours after in vitro activation and culture of primordial follicles.

[0064] Mouse ovaries were activated with an in vitro primordial follicle activator and cultured for 48 hours. The ovaries were then fixed and subjected to immunohistochemical detection of marker molecules. Figure 1 The left side (A) is the control, and the right side (B) is the activated ovary. Immunohistochemical results of AMH showed that, after 48 hours of culture, the activated ovary (B) had more follicles entering the secondary follicle stage compared to the control ovary (A).

[0065] II. Follicles that develop normally under the renal capsule of mice after treatment with an in vitro activation agent for mouse ovarian tissue.

[0066] Mouse ovarian tissue was treated with an in vitro activating agent, then allowed to develop under the renal capsule in mice. After 12 hours of hCG treatment, the ovaries were collected, pre-ovulatory follicles were mechanically punctured, and mature oocytes were collected for ICSI embryonic development to evaluate early in vitro embryonic development. Figure 2 As shown, mature oocytes subjected to ICSI (A) develop normally into embryos at the blastocyst stage (F) after passing through the 2-cell (B), 4-cell (C), 8-cell (D), and morula (E) stages. This demonstrates that the follicles produced by activating and increasing follicles in mouse ovaries develop and function completely normally.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a P13K / Akt / mTOR pathway activator and a PTEN inhibitor SF1670 for the preparation of a preparation for activating an increased number of follicles, characterized in that: consisting of a P13K / Akt / mTOR pathway activator, 5 μM statin, and a PTEN inhibitor, 20-50 μM SF1670; the statin is pravastatin; the preparation can be used for preparing human ovarian cortex culture solution, which comprises the following components: 1) a P13K / Akt / mTOR pathway activator, statin, and a PTEN inhibitor, SF1670; 2) a basic culture solution; wherein the basic culture solution is MEMa cell culture solution, wherein sodium pyruvate 0.23 mM and human serum albumin 10 wt.% are added; the reagent composition is: solution I: the basic culture solution; reagent I: a P13K / Akt / mTOR pathway activator, 5 μM statin, which is pre-packaged according to the dosage of 30 mL basic culture solution; reagent II: a PTEN inhibitor, 20-50 μM SF1670, which is pre-packaged according to the dosage of 30 mL basic culture solution.

2. Use according to claim 1, wherein the use steps of the human ovarian cortex culture solution are as follows: 1) Take the ovarian tissue in 10-15 mL cell culture solution, separate the ovarian cortex and cut into 1 mm 3 small pieces; 2) 1 mL solution I is taken and mixed with reagent I and reagent II to prepare 10 mL tissue culture solution; 3) human ovarian cortex is cultured in a 24-well plate containing suspended small nets, 400-3000 μL tissue culture solution is added below the small nets, and the culture is carried out for 2 hours under the condition of 37℃ and 5% CO2; 4) after 2 hours, the tissue culture is completed, and the cultured tissue can be used for subsequent operation.

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