Application of NAD signaling pathway agonists in in vitro embryo culture

Abstract

The present invention provides a method for culturing embryonic cells in vitro. According to an embodiment of the present invention, the method comprises: (i) culturing fertilized egg cells or their derivatives in vitro in the presence of an NAD signaling pathway agonist. During the culturing of the fertilized egg cells or their derivatives, the quality of the in vitro cultured embryos, such as their developmental potential, can be effectively improved by adding the NAD signaling pathway agonist.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to the application of NAD signaling pathway agonists in in vitro embryo culture. Background Art

[0002] In human in vitro fertilization and embryo transfer (commonly known as "test tube baby"), early immature eggs, mature eggs, sperm, fertilized eggs, and early embryos all need to be incubated in a specific culture system. The current physical environment such as culture temperature, humidity, and oxygen content is basically perfect and constant, but although the egg in vitro maturation culture medium, fertilized egg culture medium, and early embryo culture medium are commercially produced and used, they are far from perfect and mature, and have not yet met medical expectations. The prominent problem caused is that 40%-60% of human embryos experience early developmental arrest, and most embryos stop developing in the early stages of development and then die, resulting in low efficiency of "test tube baby" treatment, especially for women over 35 years old and young women (under 35 years old) whose ovarian function declines prematurely.

[0003] Therefore, the means of culturing embryos in vitro still need to be improved. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems of the related art to a certain extent. In view of this, the present invention proposes a means for effectively culturing embryos in vitro.

[0005] The present invention is accomplished based on the following findings of the inventors:

[0006] The inventor of the present invention unexpectedly discovered during the in-depth study of "test tube babies" that in the culture process of eggs, sperm or embryos, by adding NAD signaling pathway agonists, especially NR, to the culture medium, the quality of eggs, sperm or embryos, such as developmental potential, can be effectively improved. Therefore, the present invention proposes a method that can improve the success rate of "test tube babies", thereby effectively promoting the development of the industry and making effective contributions to human "test tube baby" treatment.

[0007] Therefore, in one aspect of the present invention, a method for culturing embryonic cells in vitro is proposed. According to an embodiment of the present invention, the method comprises: (i) culturing fertilized egg cells or their derivatives in vitro in the presence of an NAD signaling pathway agonist.

[0008] As mentioned above, the inventors of the present invention have found that the quality of in vitro cultured embryos, such as developmental potential, can be effectively improved by adding NAD signaling pathway agonists during the culture process of fertilized oocytes or their derivatives.

[0009] The inventors of the present invention have found in their research for many years that human embryo arrest occurs mostly at the 6-8 cell stage during the in vitro process of fertilized eggs. After egg fertilization, maternal gene regulation develops into embryonic gene regulation. This transition usually requires zygotic genome activation (ZGA), from the degradation of maternal substances to the activation of the embryonic genome. This process is called maternal-to-embryo transition. During this transition, the transcription and expression of genes in the embryo undergo a series of huge changes, including post-fertilization reprogramming, genome activation, demethylation and remethylation, X chromosome inactivation, etc., which are easily affected by various factors and cause embryonic development disorders.

[0010] At present, some progress has been made in the phenomenon of early embryonic development block in mammals such as mice and pigs. In the in vitro development study of mammalian embryos such as mice, cattle and sheep, it was found that embryonic development block was related to the silencing of certain genes and mitochondrial abnormalities. In terms of treatment, the focus is currently on the field of antioxidant mechanisms. In addition, embryonic development block can also be improved by co-culturing oviduct cells and improving culture medium. Research on in vitro development block of human embryos is still in its initial stage. The causes of early embryonic development block are quite diverse, including abnormal oocyte metabolism, decreased ATP content, abnormal transcription, asynchronous nucleocytoplasmic development, polyploid egg formation and other changes that affect oocyte quality, as well as factors such as poor sperm quality.

[0011] Existing sperm, egg and embryo culture fluids mainly contain water, various electrolytes, balanced salts, energy substances such as sodium pyruvate, glucose, growth factors, etc. There are various culture fluids on the market, some of which are sequential cultures, that is, at different developmental stages, the nutritional components are slightly different. Some are one-step cultures, and various nutrients are added in a culture fluid to meet the needs of the entire different developmental stages. Although various culture fluids are different and have different formulas, none of them solve the problem of direct energy supply for early embryo development, which is the main cause of early embryo development block or dysplasia. At present, different energy substances (such as sodium pyruvate, glucose) and cytokines are added to various links such as in vitro maturation of eggs, egg culture, embryo culture, and sperm incubation stages. However, the addition of these substances does not significantly promote embryo development, but is far from helping the embryo to continue to develop, does not alleviate or solve the problem of embryo development block, and does not establish a perfect culture fluid for optimizing ideal egg, sperm, and embryo culture. Through research, the inventor unexpectedly discovered that the main reason is that eggs, sperm, and embryos lack the ability to directly utilize energy substances such as sodium pyruvate and glucose.

[0012] According to an embodiment of the present invention, the NAD signaling pathway agonist includes NR, NMN or a functional analogue thereof.

[0013] Of course, those skilled in the art will understand that the above-mentioned NAD signaling pathway agonists may also include the above-mentioned NR, NMN or their functionally similar precursor compounds or pharmaceutically acceptable salts and other derivatives. Nicotinamide riboside (NR) is a derivative of vitamin B3 and a precursor substrate of nicotinamide adenine dinucleotide (NAD). After ingestion, it can increase NAD levels, participate in more than 100 metabolic activities of cells, and significantly improve cell activity (sperm, eggs and early embryos).

[0014] According to an embodiment of the present invention, the NAD signaling pathway agonist is preferably NR. Nicotinamide Riboside (NR) is a derivative of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD). NR has the following compound structure:

[0015]

[0016] The inventors of the present invention were surprised to find that the effect of adding NR is significantly better than directly adding NMN. The reason may be that the physical and chemical properties (such as pH) of NR, as a precursor compound of NMN, are more suitable for in vitro fertilized egg cell derivatives (such as the growth of early embryos), and after NR is taken up by cells, it can be quickly converted into NAD, thereby playing the role of an agonist of the NAD signaling pathway, thereby promoting the development of early embryos.

[0017] According to an embodiment of the present invention, the fertilized egg cell derivative includes a dysplastic embryo. In particular, according to an embodiment of the present invention, the dysplastic embryo is in the one-cell to blastocyst stage (pre-implantation) embryo stage. The inventors of the present invention unexpectedly discovered that by contacting the dysplastic embryo with an NAD signaling pathway agonist, especially NR, the embryo before the blastocyst stage can effectively improve the quality of the in vitro cultured embryo, such as developmental potential, thereby saving precious embryo resources and improving the success rate of test-tube babies.

[0018] According to an embodiment of the present invention, in step (i), the in vitro culture is carried out by placing the fertilized egg cell or its derivative in a first culture medium, wherein the first culture medium contains NR at a concentration of 1 mM. According to an embodiment of the present invention, the NR concentration that can be used is 0.5 to 10 mM, for example, 0.5, 1, 1.5, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 mM, wherein 1 mM is preferred.

[0019] According to an embodiment of the present invention, before step (i), it further includes: (ia) contacting sperm cells and egg cells in vitro in a fertilization culture medium to obtain the fertilized egg cell, wherein the fertilization culture medium contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0020] According to an embodiment of the present invention, the sperm cells and the egg cells are thawed in a thawing solution in the form of a freezing solution, and the thawing solution contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0021] According to an embodiment of the present invention, the freezing solution contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0022] According to an embodiment of the present invention, before contacting with the sperm cell, the egg cell is incubated in an egg incubation solution for 4-48 hours, wherein the incubation solution contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0023] According to an embodiment of the present invention, the oocyte is obtained by culturing immature oocytes in a maturation culture medium, wherein the maturation culture medium contains the signal pathway agonist at a concentration of 1 mM.

[0024] According to an embodiment of the present invention, before contacting with the egg cell, the sperm cell is washed with a sperm washing solution, wherein the sperm washing solution contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0025] According to an embodiment of the present invention, in step (i), the cleavage stage embryo is cultured using embryo culture medium, wherein the embryo culture medium contains the NAD signaling pathway agonist at a concentration of 1 mM, so as to obtain a blastocyst; and the blastocyst is cultured using blastocyst culture medium, wherein the blastocyst culture medium contains the NAD signaling pathway agonist at a concentration of 1 mM.

[0026] In the second aspect of the present invention, the present invention proposes the use of an NAD signaling pathway agonist in preparing a culture medium, wherein the culture medium is used for at least one of the following: culturing fertilized egg cells; washing sperm cells; maturation culture of immature eggs; fertilization of eggs and sperm cells; freezing of eggs or sperm cells; thawing of eggs or sperm cells; culturing cleavage stage embryos; and culturing blastocysts.

[0027] In other words, the present invention also proposes a culture medium for at least one of the following: culturing fertilized egg cells; washing sperm cells; maturation culture of immature eggs; fertilization of egg and sperm cells; freezing of egg or sperm cells; thawing of egg or sperm cells; culturing cleavage stage embryos; and culturing blastocysts.

[0028] By adopting the above culture medium, the quality of in vitro cultured embryos, such as developmental potential, can be effectively improved, thereby avoiding or alleviating the phenomenon of human embryo arrest during the in vitro fertilization of fertilized eggs. Specifically, by adding nicotinamide riboside NMN-related compounds in various links, the developmental potential of sperm, eggs, and embryos can be improved. Overall, human "test tube babies" can be improved by 20-30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A microscope photograph of an embryo according to an embodiment of the present invention is shown. NR means that NR is added during the culture process, and NR- means that NR is not added during the culture process;

[0030] Figure 2 Microscopic photographs of embryos of the NR group according to one example of the present invention are shown. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, and the described embodiments are exemplary. It should be noted that, unless otherwise specified, the reagents used in this article are all commercially available. The operating means or conditions not clearly listed in the following embodiments are also well known in the art, or can be known by reference.

[0032] Prepare the culture medium according to the following formula:

[0033] 1. Human immature oocyte in vitro maturation culture medium, add 1mM NR

[0034] 2. Human sperm washing solution, add 1mM NR

[0035] 3. Human egg incubation medium and fertilization medium, add 1mM NR

[0036] 4. Add 1mM NR to human egg and sperm freezing and thawing solutions respectively

[0037] 5. Human cleavage embryo culture, add 1mM NR

[0038] 6. Human blastocyst culture medium, add 1mM NR

[0039] Specifically,

[0040] 1. Culture medium for egg fertilization

[0041] 1) Egg washing culture medium: Take G-MOPS PLUS (add 1mM NR) into a 14ml round-bottom test tube (Falcon352001), use 5ml of culture medium per person, and place it in a 37℃ incubator for equilibrium overnight.

[0042] 2) Fertilization culture medium and fertilization dish: Take G-IVF PLUS (add 1mM NR) in a 14ml round-bottom test tube (Falcon352001), use 3ml culture medium per person, and equilibrate overnight in a 37℃ 5% CO2 incubator. Add 1ml G-IVF PLUS to each fertilization culture dish (3037), and cover with 500ul mineral oil; add 1ml G-IVF PLUS (for washing) to the outer circle.

[0043] 2. Cleavage culture medium and cleavage culture dish

[0044] Cleavage culture medium and culture droplets: Take G-1PLUS (add 1mM NR) as needed in a 14ml round-bottom test tube (Falcon352001), and place it in a 37℃ CO2 incubator for equilibrium overnight. Prepare several culture dishes (Falcon3001) per person (depending on the number of eggs), use a pipette to make 10 20μl culture droplets in a culture dish (Falcon 3001), and cover with 3ml of mineral oil (ART-4008). Place it in a 37℃, 5% CO2 incubator for equilibrium. Note: When making droplets, avoid the tip of the gun from touching the tube wall and the bottom of the dish, and cover with oil in time.

[0045] 3. Transplantation culture medium and blastocyst culture medium: Take G-2PLUS (add 1 mM NR) into a 14 ml round-bottom test tube (Falcon352001) and place it in a 37°C 5% CO2 incubator for equilibrium overnight.

[0046] 1) Transplantation dish: Before use, add 1 ml of the central area and 1 ml of the peripheral area (for washing) to the culture dish (Falcon 3037), and place in a 37°C, 5% CO2 incubator for equilibrium.

[0047] 2) Blastocyst culture dish: Ten 20 μl droplets were made in a culture dish (Falcon 3001), covered with 3 ml of mineral oil (ART-4008), and placed in a 37° C., 5% CO 2 incubator for equilibrium.

[0048] It should be noted that the in vitro culture time used in this article does not exceed 14 days, for example, no more than 10 days, no more than 8 days, and no more than 6 days; among them, the in vitro culture time of the embryos in this application does not exceed 8 days, specifically, the in vitro culture time of the blastocysts in this application is 5-6 days.

[0049] Example 1 Oocyte maturation culture

[0050] Ovulation is induced in female subjects and the size of their follicles is monitored. When the diameter of the follicles reaches 6 to 12 mm, immature eggs are obtained by puncture through B-ultrasound.

[0051] On the day of egg retrieval, the 3037 insemination dish is routinely prepared according to the "IVF-ET Laboratory Procedures and Laboratory Preparation", and the patient's name and medical record number are marked on the lid and bottom of the dish, and placed in a three-gas incubator for balance. At the beginning of egg collection, the laboratory egg collection operator and the nurse jointly check the patient's three certificates. During egg retrieval, the male enters the semen collection room to collect semen. Before egg retrieval, a certain amount of round-bottomed Falcon 2001 test tubes are placed in a 37°C constant temperature test tube rack for preheating. Prepare 5-10 egg collection dishes (Falcon 3003) and place them in the clean bench and preheat at 37°C. Add 2-3ml of preheated G-MOPS PLUS (containing 1mM NR) to the 3001 dish and place it on the clean bench at 37°C for standby use. Clinically, the test tubes containing flushing fluid and follicular fluid are placed in a constant temperature test tube rack. The laboratory pours the liquid in the test tube into the egg picking dish as soon as possible, and identifies the oocyte / cumulus complex (OCCC) under a microscope. The OCCC is moved into the G-MOPS PLUS dish marked with the patient's information and temporarily placed on the constant temperature platform of the IVF workstation. After the egg picking is completed, the OCCC is quickly moved into the outer circle of the double-well dish of in vitro fertilization solution (G-IVF PLUS, containing 1mM NR) and blown several times to remove blood and residual G-MOPS PLUS as much as possible. After fertilization, the OCCC is transferred to the central circle of the dish, and the dish containing the OCCC is immediately placed in the incubator. In general, a maximum of 20 OCCCs are placed in each dish. After culturing for 24-48 hours at 37°C and 5% CO2, the maturity of the eggs is evaluated, and it is found that the maturity rate of immature eggs is significantly improved compared with the culture medium without NR added.

[0052] Example 2 Washing human sperm

[0053] After traditional testicular sperm is obtained, it is placed in Modified-HTF (Irvine Scientific company) containing 1mM NR and then cultured in a 5% CO2 chamber for 10 to 24 hours. It was found that the motility of sperm samples obtained from the testicles and weak sperm in peripheral semen was significantly improved.

[0054] Example 3 Sperm freezing and thawing

[0055] Each 100mL of human semen cryoprotectant contains the following substances:

[0056] Glucose 1.5g, trisodium citrate dihydrate 1.3g, glycerol 20ml, NR 1mM, egg yolk 20ml, dimethyl sulfoxide 0.1mL, and the balance is double distilled water.

[0057] Human semen cryoprotectant and semen were mixed in a volume ratio of 1:3, added to the test tube, and frozen using a programmable cooling device. The freezing program was set as follows: (1) equilibration at 20°C for 5 minutes; (2) from 20°C to -2°C, freezing rate -1°C / min; (3) from -2°C to -30°C, freezing rate -7.2°C / min; (4) from -30°C to -90°C, freezing rate -30°C / min.

[0058] After freezing, the frozen human sperm was revived, and the sperm motility after freezing and thawing and the sperm freezing recovery rate were evaluated, using the percentage of sperm with forward motility as an indicator. The inventors found that adding NR to the freezing and thawing recovery fluid can effectively improve the recovery rate of sperm recovery.

[0059] Example 4 Fertilization culture

[0060] After the eggs are obtained, they are inseminated in a fertilization dish with a volume of 1 ml and a ratio of 10,000 sperm per egg. In short, sperm are separated and washed by density gradient centrifugation, and the volume required for fertilization is calculated according to density and motility. Insemination is performed using an eppendorf pipette. The dish containing the oocytes is taken out of the incubator, and two people check the patient information on the dish and the sperm suspension tube, carefully aspirate the upstream sperm suspension, and then put the fertilization dish back into the incubator. Record the time of insemination, the operator, and the certificate.

[0061] Studies have found that adding NR to fertilization culture medium can effectively improve the normal fertilization rate.

[0062] Example 5 Embryo Culture

[0063] In this embodiment, dysplastic embryos (before S phase) were added to embryo culture medium (HTF or G-1 containing 1 mM NR, and the embryo culture medium was balanced overnight in an incubator at 37°C, 6% CO2, 5% O2 before use), and it was found that the dysplastic embryos had good embryonic continued development performance. Specifically, in the 11 patients who had completed the study, it was found that after adding NR, the embryos with stagnant development showed the ability to continue development: 6 / 13=46%, and the ability to continue development to varying degrees, that is, from the embryos that were slowly developing and almost stagnant, they developed into morula or early blastocysts. Without the addition of NR, the probability of embryos continuing to develop was <10%. Figure 1 The microscope photo of the embryo according to one embodiment of the present invention is shown. NR means that NR was added during the culture process, and NR- means that NR was not added during the culture process. Among them, the patient who failed multiple "IVF" treatments had good blastocyst formation and became pregnant after incubation with NR-added culture medium.

[0064] Example 6 Human Blastocyst Culture

[0065] The embryos on D3 were transferred to G-2PLUS blastocyst culture droplets (containing 1mM NR) for further culture, and the blastocyst culture dish was placed in a tri-gas incubator for further culture. The embryonic development was observed on D5 and D6. Blastocyst rating: Currently, the blastocysts are scored using Gardner's blastocyst scoring system, in which the scoring criteria mainly include three indicators: the size of the blastocyst cavity, the size of the inner cell mass, and the trophectoderm cells. Based on the above scoring criteria, it was found that the blastocysts scored significantly higher with the addition of NR than without the addition of NR.

[0066] Example 7: Human embryo culture results

[0067] In this embodiment, patients of advanced age (42 years and above) who have failed multiple IVF implantations were selected. After egg retrieval, they were divided into two groups, the NR group and the Control group. After fertilization and fertilized egg culture, they were cultured in embryo culture medium. The only difference between the NR group and the Control group was that the embryo culture medium of the NR group contained 1mM NR, while the embryo culture medium of the Control group did not contain NR. The embryo culture results are shown in Table 1. Microscopic photos of the embryos in the NR group are shown in Table 1. Figure 2 As shown, the results confirmed that NR has the effect of significantly improving the blastocyst formation rate, especially the blastocyst formation rate of elderly patients with multiple IVF implantation failures.

[0068] Table 1 Blastocyst formation rate of NR group and Control group

[0069] Blastocyst formation rate NR Group 8 / 22(36.36％) Control Group 0 / 30(0％)

[0070] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for culturing embryonic cells in vitro for purposes other than disease diagnosis and treatment, characterized in that: include: (ia) contacting a sperm cell and an egg cell in vitro in a fertilization medium to obtain the fertilized egg cell, wherein the fertilization medium contains the NAD signaling pathway agonist at a concentration of 1 mM, wherein the sperm cell and the egg cell are thawed in a thawing solution in the form of a freezing solution, wherein the thawing solution contains the NAD signaling pathway agonist at a concentration of 1 mM, and the freezing solution contains the NAD signaling pathway agonist at a concentration of 1 mM; Before contacting with the sperm cell, the egg cell is incubated in an egg incubation solution for 4 to 48 hours, wherein the incubation solution contains the NAD signaling pathway agonist at a concentration of 1 mM; The oocytes are obtained by culturing immature oocytes in a maturation culture medium, wherein the maturation culture medium contains the NAD signaling pathway agonist at a concentration of 1 mM; Before contacting with the egg cell, the sperm cell is washed with a sperm washing solution, wherein the sperm washing solution contains the NAD signaling pathway agonist at a concentration of 1 mM; (i) culturing a fertilized egg cell or a derivative thereof in vitro in the presence of an NAD signaling pathway agonist, wherein the in vitro culturing is performed by placing the fertilized egg cell or a derivative thereof in a first culture medium containing the NAD signaling pathway agonist at a concentration of 1 mM; Cultivating cleavage-stage embryos using an embryo culture medium containing the NAD signaling pathway agonist at a concentration of 1 mM to obtain blastocysts; as well as, Cultivating the blastocyst using a blastocyst culture medium, wherein the blastocyst culture medium contains the NAD signaling pathway agonist at a concentration of 1 mM; Wherein, the NAD signaling pathway agonist is NR; The fertilized egg cell derivative is a dysplastic embryo; The dysplastic embryos are at the blastocyst stage.

2. Use of an NAD signaling pathway agonist in preparing a culture medium, wherein the culture medium is used for washing sperm cells, and / or freezing sperm cells; and / or thawing sperm cells; in, The NAD signaling pathway agonist is NR.

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