A culture medium additive for improving embryonic developmental potential and its application

Abstract

This invention discloses a culture medium additive for improving embryonic developmental potential and its application, belonging to the field of in vitro embryo culture technology. This invention is the first to discover that CPI-455, a KDM5 histone demethylase inhibitor, can significantly improve the blastocyst rate, developmental speed, and embryo quality of preimplantation embryos by increasing H3K4me3 levels within a certain concentration range. Simultaneously, the addition of CPI-455 and NMN can substantially increase the rate of high-quality / high-quality blastocysts and embryo quality in in vitro embryo culture. The embryonic developmental potential additive provided by this invention is of great significance for improving in vitro embryo culture efficiency and embryo quality, accelerating the process of breeding superior embryos, and assisting in research on embryo-related mechanisms.

Description

Technical Field

[0001] This invention relates to the field of in vitro embryo culture technology, and in particular to a culture medium additive for improving embryo developmental potential and its application. Background Technology

[0002] In vitro embryo production in animals is a modern reproductive technology widely used in livestock production and biological research. It plays a crucial role in promoting animal breed improvement and efficient breeding. Furthermore, it allows for the study of early human embryonic development (especially the critical stage of 2-3 weeks after conception) using in vitro models, filling gaps in our understanding of the "embryo black box" and providing new tools for research on birth defects and genetic diseases. In vitro embryo production (… in vitro In vitro fertilization (IVP) refers to the process of retrieving immature oocytes from the ovaries of donors of different ages and physiological states, maturing them in vitro, fertilizing them, and further culturing and developing them to ultimately obtain embryos suitable for transplantation or cryopreservation. Oocyte in vitro maturation (IVP) in vitro maturation (IVM), in vitro fertilization (IVF) in vitro fertilization (IVF) and in vitro culture ( in vitro Culture (IVC) is one of the three key technological steps in in vitro embryo production. IVC involves culturing fertilized eggs to the desired embryonic stage. As the final step in IVP, it directly affects the quality of subsequent embryonic development.

[0003] The culture medium serves as the external environment for the growth and development of in vitro embryos, directly providing them with nutrients and energy. In recent years, researchers have added various substances to the basal in vitro culture medium to more realistically simulate the in vivo embryonic development environment, thereby obtaining higher-quality blastocysts. Although in vitro embryo production technology has made significant progress with the continuous development of molecular biology, biochemistry, and cell biology techniques, bottlenecks such as low blastocyst rates, low embryo production efficiency, unsatisfactory embryo quality, and poor developmental potential still exist, greatly limiting the large-scale application of in vitro embryo technology.

[0004] NMN (Nicotinamide Mononucleotide) is an important biomolecule, specifically nicotinamide adenine dinucleotide (NAD). + One of the precursors of NAD. + As a key coenzyme in cellular energy metabolism and various biological processes, NMN, as a synthetic intermediate, has received widespread attention in recent years due to its potential anti-aging and health benefits. Research on the application of NMN in the reproductive field is still in its early stages, but some preliminary findings suggest that it may have a positive effect on reproductive health.

[0005] CPI-455 is a specific inhibitor of the KDM5 histone demethylase. It regulates gene expression by inhibiting KDM5 enzyme activity and increasing H3K4me3 levels. It has significant research value and application potential in fields such as cancer research, stem cell differentiation, and neuroscience. However, there are currently no reports on CPI-455 in the field of embryonic development. Summary of the Invention

[0006] The purpose of this invention is to provide a culture medium additive that improves the developmental potential of embryos and its application, so as to provide new additives for in vitro embryo culture, and contribute to more realistically simulating the in vivo embryo development environment, obtaining high-quality blastocysts, and improving the efficiency and quality of in vitro embryo culture.

[0007] To achieve the above objectives, the present invention provides a culture medium additive for improving embryonic developmental potential, wherein the additive is a reagent that increases H3K4me3 levels and / or inhibits oxidative stress levels.

[0008] Preferably, the in vitro embryonic development is early embryonic development; Day 1 to Day 6; the additive for improving embryonic developmental potential is applied on the day the fertilized egg is obtained.

[0009] Preferably, the additive is a Kdm5 inhibitor and / or a nicotinamide derivative.

[0010] Preferably, the Kdm5 inhibitor is one or more of KDM5-C70, KDM5-C49, KDM5-IN-1, CPI-455, KDOAM-25, CPI-455 hydrochloride, KDOAM-25 trihydrochloride, JQKD82, JQKD82 and JQKD82.

[0011] Preferably, the Kdm5 inhibitor is CPI-455, and the working concentration of CPI-455 is 1 μM to 10 μM.

[0012] Preferably, the nicotinamide derivative is one or more of nicotinic acid, nicotinic acid mononucleotide, nicotinamide mononucleotide, nicotinamide ribose, nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate.

[0013] Preferably, the nicotinamide derivative is nicotinamide mononucleotide, and the working concentration of nicotinamide mononucleotide is 100 μM.

[0014] A culture medium for improving embryonic developmental potential, wherein the culture medium is a basal medium to which the above-mentioned culture medium additive for improving embryonic developmental potential is added; wherein the basal medium is one of M16, KSOM, CZB, G1-PLUS and G2-PLUS.

[0015] The application of a culture medium additive as described above for improving embryonic developmental potential in in vitro embryo culture for non-medical purposes.

[0016] Application of a culture medium, as described above, for improving embryonic developmental potential in in vitro embryo culture for non-medical purposes.

[0017] Therefore, the present invention provides a culture medium additive for improving embryonic developmental potential and its application, the specific technical effects of which are as follows:

[0018] (1) This invention first discovered that the KDM5 histone demethylase inhibitor CPI-455 can significantly improve the blastocyst rate, development speed and embryo quality of preimplantation embryos by increasing the level of H3K4me3 within a certain concentration range.

[0019] (2) The addition of CPI and NMN can significantly improve the rate of high-quality / high-quality blastocysts and embryo quality in in vitro embryo culture. The additives for improving embryo development potential provided by this invention are of great significance for improving the efficiency and quality of in vitro embryo culture, accelerating the breeding process of superior varieties, and assisting in the study of embryo-related mechanisms.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the result of the effect of adding different concentrations of CPI-455 on the blastocyst rate of mice in Example 1 of the present invention; wherein A is a schematic diagram of adding different concentrations of CPI-455 during in vitro culture; B is a photograph of fertilized eggs on day 4 after adding different concentrations of CPI-455 during in vitro culture; C is the statistical result of the blastocyst rate on day 4 after adding different concentrations of CPI-455 during in vitro culture.

[0023] Figure 2 This is the effect of adding different concentrations of CPI-455 on H3K4me3 in IVF blastocysts in Example 1 of this invention; where A is an immunofluorescence image; B is the result of immunofluorescence quantitative analysis.

[0024] Figure 3This invention relates to the effect of adding CPI-455 on gene expression in preimplantation blastocysts in Example 1; where A represents the DEGs analysis results of transcriptome sequencing of IVF blastocysts (represented by IVF in the figure), IVO blastocysts (represented by IVO in the figure), and blastocysts after adding CPI-455 (represented by CPI in the figure); B represents the enrichment analysis results of upregulated genes after adding CPI-455; and C represents the enrichment analysis results of downregulated genes after adding CPI-455.

[0025] Figure 4 This is Example 2 of the present invention, showing the effect of adding different concentrations of NMN on the blastocyst rate in mice; where A is a schematic diagram of adding different concentrations of NMN during in vitro culture; B is a photograph of fertilized eggs on day 4 after adding different concentrations of NMN; and C is the statistical result of the blastocyst rate on day 4 after adding different concentrations of NMN during in vitro culture.

[0026] Figure 5 This is the result of the effect of adding NMN on oxidative stress in IVF blastocysts in Example 2 of the present invention; where A is the detection result of ROS content in IVF blastocysts of the NMN-added group and the control group, green represents ROS, scale bar 60μm; B is the detection result of GSH content in IVF blastocysts of the NMN-added group and the control group, blue represents GSH, scale bar 60μm; C is the detection result of ATP content in IVF blastocysts of the NMN-added group and the control group; D is the detection result of NAD in IVF blastocysts of the NMN-added group and the control group. + Content detection results; E represents the NADH content detection results in IVF blastocysts of the NMN-added group and the control group; ns indicates no significance, with no statistically significant difference; * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001;

[0027] Figure 6 This is the result of the effect of adding NMN on gene expression in IVF blastocysts in Example 2 of the present invention; where A is the transcriptome sequencing DEGs analysis results of IVF blastocysts (represented by IVF in the figure), IVO blastocysts (represented by IVO in the figure), and blastocysts after adding NMN (represented by NMN in the figure); B is the enrichment analysis results of upregulated genes after adding NMN; C is the enrichment analysis results of downregulated genes after adding NMN.

[0028] Figure 7This is the result of the effect of adding CPI455 or NMN on the rate of high-quality embryos on Day 3 in Example 3 of the present invention; where A is the effect of adding CPI-455 or NMN on the proportion of high-quality embryos on Day 3; B is the effect of adding CPI-455 or NMN on embryos at different developmental stages on Day 3; Ctl is the control group, CPI is the group with added CPI-455, and NMN is the group with added NMN.

[0029] Figure 8 This is the result of the effect of adding CPI-455 on the human blastocyst rate and high-quality blastocyst rate in Example 3 of the present invention; where A is the effect of adding CPI-455 or NMN on the human blastocyst rate; B is the effect of adding CPI-455 or NMN on the human high-quality blastocyst rate; C is the effect of adding CPI-455 or NMN on embryos at different developmental stages; D is the light micrograph of human embryos cultured with added CPI-455 or NMN before implantation; Ctl is the control group, CPI is the group with added CPI-455, and NMN is the group with added NMN; the statistical criteria for Day 5 / 6 blastocysts are all blastocysts from stage 1 to stage 6, where stage 1 and stage 2 blastocysts are early blastocysts, stage 3 blastocysts are fully expanded blastocysts, stage 4 blastocysts are expanded blastocysts, and embryos that have not formed blastocysts are non-blastocysts;

[0030] Figure 9 This is the result of the effect of simultaneous addition of CPI-455 and NMN on mouse embryonic development in Example 4 of the present invention; where A is the effect of simultaneous addition of CPI-455 and NMN on mouse blastocyst rate; B is the effect of simultaneous addition of CPI-455 and NMN on the proportion of high-quality embryos on Day 3; C is the effect of simultaneous addition of CPI-455 and NMN on embryos at different developmental stages on Day 3; D is the effect of simultaneous addition of CPI-455 and NMN on human blastocyst rate; E is the effect of simultaneous addition of CPI-455 and NMN on high-quality blastocyst rate; F is the effect of simultaneous addition of CPI-455 and NMN on the proportion of embryos at different developmental stages; the statistical standard for Day 5 / 6 blastocysts is all blastocysts from stage 1 to stage 6, where stage 1 and stage 2 blastocysts are early blastocysts, stage 3 blastocysts are fully expanded blastocysts, stage 4 blastocysts are extended blastocysts, and embryos that have not formed blastocysts are non-blastocysts. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels; G-IVF plus fertilization solution, G-1 PLUS culture medium and M2 culture medium were all commercially available products.

[0034] The human gametes and embryos involved in the examples were all from the affiliated reproductive hospital, and all samples were donated by infertile patients with full informed consent. The experiments in the examples were approved by the Reproductive Medicine Ethics Committee of the affiliated reproductive hospital (March 1, 2023) and strictly complied with the "Administrative Measures for Human Assisted Reproductive Technology" (2001 edition), "Specifications for Human Assisted Reproductive Technology" (2003 revised edition), "Measures for Ethical Review of Biomedical Research Involving Humans" (National Health Commission, 2016 edition), "Guidelines for Stem Cell Research and Clinical Translation" (International Society for Stem Cell Research, 2016 edition), "Ethical Guidelines for Human Embryonic Stem Cell Research" (2003 edition), and the Declaration of Helsinki, as well as other laws, regulations, and ethical norms.

[0035] The methods and / or steps not described in detail in the embodiments are all conventional techniques in the art.

[0036] Example 1

[0037] The effects of CPI-455 inhibitors on in vitro embryo development and blastocyst rate were investigated as follows:

[0038] S1. Preparation of CPI-455 stock solution: Carefully open the cap of the purchased CPI-455 bottle in a clean bench to avoid powder spillage. Add 359.3 μL of DMSO to fully dissolve the powder, prepare a 10 mM stock solution, and dispense into 1.5 mL centrifuge tubes for storage at -20°C.

[0039] S2, mouse in vitro fertilization and early embryo culture.

[0040] (1) Superovulation: 6-8 week old SPF grade ICR female mice were selected and injected with PMSG 10 IU intraperitoneally at 15:00 and hCG 10 IU 46-48h.

[0041] (2) Liquid preparation: Add 600 μL of G-IVF plus fertilization medium to wells 1 and 2 of a four-well dish, and add 600 μL of G-1 PLUS culture medium to wells 3 and 4. Cover each well with 8 drops of mineral oil and mark with a non-toxic marker pen. Place the dish in a 37℃, 6% CO2 incubator for at least 6 hours to equilibrate. Dispense M2 culture medium and preheat it in a 37℃, CO2-free incubator. Add 200 μL of G-IVF plus fertilization medium to a 1.5 mL centrifuge tube as the sperm capacitation medium and place it in a 37℃, 6% CO2 incubator for 6 hours to equilibrate.

[0042] (3) Oocyte collection: 16 hours after hCG, the mice were euthanized by cervical dislocation, the oviducts were removed and placed in M2 culture medium, and then the ampulla of the oviduct was torn open with forceps under a stereomicroscope to collect the cumulus-oocyte complex (COC). After being thoroughly rinsed with G-IVF PLUS, the COCs were transferred to a pre-balanced G-IVF PLUS four-well dish and placed in a 37℃, 6% CO2 incubator for fertilization.

[0043] (4) Sperm capacitation: Adult male ICR rats aged 12 weeks or older were selected, euthanized by cervical dislocation, and mature sperm from the epididymal tail were collected and placed at the bottom of a centrifuge tube containing G-IVF PLUS culture medium. The sperm were capacitation was performed in a 37°C, 6% CO2 incubator for 30 min to 1 h.

[0044] (5) In vitro fertilization: Capacitated sperm are added to a four-well fertilization dish containing COC, and the sperm density is adjusted to 1~2×10⁻⁶. 6 The cells were placed in a desktop incubator at 37℃, 6% CO2, and 5% O2 for 6 hours to allow for fertilization.

[0045] (6) Early embryo culture: After fertilization, 2PN fertilized eggs with clearly visible pronuclei were selected and rinsed with G-1PLUS culture medium to remove granulosa cells and sperm. The fertilized eggs were then transferred to pre-balanced G-1PLUS culture dishes (the G-1 PLUS culture medium in the dishes was supplemented with CPI-455 stock solution prepared by S1, resulting in final CPI-455 concentrations of 0 μM, 1 μM, 5 μM, 10 μM, and 20 μM, respectively). The dishes were cultured for 4 days in a desktop incubator at 37℃, 6% CO2, and 5% O2, serving as the in vitro embryo culture experimental group. Each CPI-455 concentration was replicated in triplicate. Blastocyst formation in different groups was observed, photographed using an inverted fluorescence microscope, and the blastocyst rate was statistically analyzed.

[0046] The results are as follows Figure 1 As shown, 5 μM CPI-455 significantly increased the blastocyst rate of preimplantation genetic testing (PGT) in mice. Compared with the control group (Ctl) without CPI-455, the in vitro blastocyst rate of embryos treated with 10 μM CPI-455 was not different, while the in vitro blastocyst rate of embryos treated with 20 μM CPI-455 was significantly reduced.

[0047] Blastocysts with different concentrations of CPI-455 were collected and subjected to immunofluorescence staining.

[0048] The results are as follows Figure 2As shown, adding 1 μM CPI-455 did not effectively increase the level of H3K4me3 in mouse IVF blastocysts, while 5 μM, 10 μM, and 20 μM CPI-455 significantly increased the level of H3K4me3. Combined with the above embryonic development data, 5 μM CPI-455 can significantly increase the level of H3K4me3, thereby increasing the rate of preimplantation genetically modified (IVF) blastocysts in mice. Low levels of H3K4me3 in IVF blastocysts impair embryonic development, and the significant increases in H3K4me3 levels after adding 10 μM and 20 μM CPI-455 also impaired embryonic development. This indicates that H3K4me3 homeostasis is crucial for preimplantation embryonic development; both excessively high and low levels can damage embryonic development.

[0049] S3. Collection of naturally conceived mouse embryos.

[0050] (1) Superovulation and mating: Select 6-8 week old SPF grade ICR female mice, inject 10 IU of Pregnant Mare Serum Gonadotropin (PMSG) into the peritoneum at 15:00, and inject 10 IU of Human Chorionic Gonadotropin (hCG) into the peritoneum 46-48 hours later. Then, mat them with adult ICR male mice over 12 weeks old at a ratio of 2:1.

[0051] (2) Checking for vaginal plugs: At 8:00 a.m. the day after the mice are put together, use tweezers to observe whether there is a milky white solid gel-like substance in the vagina of the female mouse, i.e., a vaginal plug. If there is one, it means that the female mouse has successfully mated. Record the date of finding the plug as E0.5. Keep the female mouse with the plug separately.

[0052] (3) Embryo collection: Female mice with plugs were euthanized by cervical dislocation at hCG 27h, hCG 39h, hCG 48h, hCG 56h, hCG 65h, and hCG 77h, respectively. Both fallopian tubes were removed, and PN5 fertilized eggs, Early 2 Cells, Late 2 Cells, 4 Cells, 8 Cells, and Morulas were flushed out of the fallopian tubes using an insulin injection. At hCG 93h (E3.5), the female mice with plugs were euthanized by cervical dislocation, and both uteri were removed. M2 culture medium was drawn into a syringe, and then the blastocysts in the uterus were flushed out using a syringe. At this time, the embryos were in vivo blastocysts at E3.5.

[0053] (4) Embryo culture: The obtained E3.5 in vivo blastocysts were rinsed 3-5 times with G-1 PLUS culture medium and then transferred to a pre-balanced G-1 PLUS culture dish and cultured for 4 days in a desktop incubator at 37℃, 6% CO2, and 5% O2.

[0054] To investigate the effect of CPI-455 addition on gene expression in mouse preimplantation embryos and whether blastocysts with CPI-455 addition are closer to in vivo blastocysts (IVO blastocysts), in vitro cultured blastocysts (IVF blastocysts), IVO blastocysts, and IVF blastocysts with 5 μM CPI-455 addition were collected from S2 and subjected to micro-cell transcriptome sequencing.

[0055] The results are as follows Figure 3 As shown, the gene expression of IVF blastocysts in the CPI-455-added group was more similar to that of IVO blastocysts. Enrichment analysis of the upregulated genes after adding 5 μM CPI-455 successfully enriched multiple pathways related to oxidative stress and ATP synthesis. This indicates that adding CPI-455 to increase H3K4me3 levels may improve the expression of oxidative stress-related genes. In conclusion, there may be some interaction between H3K4me3 levels and oxidative stress, and adding CPI-455 to improve preimplantation embryo development in mice is a relatively effective strategy for improving IVF embryo quality.

[0056] Example 2

[0057] The effects of nicotinamide mononucleotide (NMN) on in vitro embryo development and blastocyst rate were investigated, as follows:

[0058] S1. NMN Stock Solution Preparation: Carefully open the received NMN bottle cap in a clean bench to avoid powder loss. Add 299.2 μL of DNase / RNase-free distilled water to fully dissolve the powder, prepare a 1M stock solution, and dispense into 1.5 mL centrifuge tubes for storage at -80°C.

[0059] S2, mouse in vitro fertilization and early embryo culture.

[0060] The method is exactly the same as (1) to (6) in S2 of Example 1, except that the NMN stock solution prepared in S1 is added to the G-1 PLUS culture dish in (6) so that the final concentrations of NMN are 0 μM, 10 μM, 100 μM, 200 μM and 400 μM respectively.

[0061] Inverted fluorescence microscope images of different groups of blastocysts and statistical results of blastocyst rate are as follows: Figure 4 As shown, 100 μM NMN can significantly increase the blastocyst rate of mouse preimplantation embryos.

[0062] To further verify whether oxidative stress levels were improved in IVF blastocysts from the NMN-added group, blastocysts from the control group (Ctl) and the group with 100 μM NMN (NMN group) were collected, and ROS and GSH levels were measured, as follows:

[0063] ROS content detection:

[0064] The ROS level in each blastocyst was quantified by measuring the endogenous H2O2 level in the cells using DCHFDA. The DCHFDA stock solution was 1 mM (dissolved in DMSO) and stored at -20°C.

[0065] (1) Dilute 1 mM DCHFDA concentrated stock solution 100 times with M2 operating solution to obtain 0.01 mM staining solution.

[0066] (2) Place the blastocyst in 0.01mM DCHFDA staining solution and incubate at 37°C for 30 min.

[0067] (3) Use M2 processing solution to wash the blastocyst 10 times to thoroughly wash away the fluorescent dye around the blastocyst.

[0068] (4) Mount the blastocysts and observe them using a laser confocal microscope (Dragonfly, Andor Technology, UK) with an excitation wavelength of 488 nm. Ensure that parameters such as laser intensity and exposure time are consistent each time you take a picture.

[0069] (5) The fluorescence intensity of each blastocyst was analyzed using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD).

[0070] GSH content detection:

[0071] Thiol Tracker Violet was used to detect the level of GSH in blastocysts. The Thiol Tracker Violet stock solution was 20 mM (dissolved in DMSO) and stored at -20°C.

[0072] (1) Dilute 20 mM Thiol Tracker Violet concentrated stock solution 2000 times with M2 operating solution to obtain 10 μM staining solution.

[0073] (2) Place the blastocyst in 10 μM Thiol Tracker Violet staining solution and incubate at 37°C for 30 min.

[0074] (3) Use M2 processing solution to wash the blastocyst 10 times to thoroughly wash away the fluorescent dye around the blastocyst.

[0075] (4) Mount the blastocysts and observe them using a laser confocal microscope (Dragonfly, Andor Technology, UK) with an excitation wavelength of 405 nm. Ensure that the laser intensity, exposure time, and other parameters are consistent each time you take a picture.

[0076] (5) The fluorescence intensity of each blastocyst was analyzed using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD).

[0077] The results are as follows Figure 5 As shown, adding 100μM NMN can effectively reduce ROS and increase GSH.

[0078] Using the EnSpire multimodal tablet reader (PerkinElmer, USA) and NAD + / NADH-Glo™ Assay Kit for NAD + And the measurement of NADH levels. Results are as follows: Figure 5 As shown, adding 100 μM NMN can increase ATP and NAD content in blastocysts. + This indicates that adding 100 μM NMN can effectively reduce oxidative stress in IVF blastocysts and improve preimplantation embryonic development in mice.

[0079] To reveal the effects of NMN supplementation on gene expression in mouse preimplantation embryos and to clarify whether NMN-supplemented IVF blastocysts are more similar to IVO blastocysts, IVF blastocysts, IVO blastocysts, and IVF blastocysts supplemented with 100 μM NMN (NMN for short) were collected for micro-cell transcriptome sequencing. Results are as follows: Figure 6 As shown, gene expression in the NMN-added group was more similar to that of IVO blastocysts. Enrichment analysis of the upregulated genes after NMN addition revealed the successful enrichment of multiple pathways related to oxidative stress and ATP synthesis. This indicates that adding NMN to improve preimplantation embryonic development in mice is a relatively effective strategy for improving IVF embryo quality.

[0080] Example 3

[0081] The effects of CPI-455 and NMN on the day-3 good embryo rate, day-5 / 6 blastocyst rate, and high-quality blastocyst rate of early human embryos were investigated, as follows:

[0082] S1. Collection and in vitro maturation of immature human oocytes.

[0083] All collected immature oocytes came from the affiliated reproductive hospital. During assisted reproductive treatment, patients donated immature oocytes that had no clinical value after signing informed consent forms. The collected immature oocytes were cultured in an in vitro maturation medium for 24 hours at 37°C with 6% CO2 and 5% O2. The in vitro maturation medium consisted of M199 (GIBCO, 11-150-059), 20% Systemic Serum Substitute (Irvine Scientific, 99193), and 75 mIU / mL recombinant follicle stimulating hormone (Merck Serono). The in vitro matured oocytes can be used for subsequent ICSI.

[0084] S2, Human intracytoplasmic sperm injection and early embryo culture.

[0085] The main procedures for fertilizing in vitro matured oocytes with donated research sperm using ICSI are as follows:

[0086] (1) Preparation of ICSI culture dishes.

[0087] 1) Preparation of ICSI operating dish: Prepare a thin drop of PVP solution, a slightly thicker round drop of PVP solution, a long drop of Hepes solution for egg washing, and several Hepes fertilization drops in the ICSI dish. Also prepare 2-3 thin drops of Hepes solution, and then cover them with mineral oil. PVP can slow down the sperm's movement and help to immobilize the sperm.

[0088] 2) Add 1~2μL of treated sperm to the PVP droplet.

[0089] (2) Install the injection system.

[0090] 1) Under low magnification, first install the fixation pin, ensuring its tip is horizontal. Then install the injection needle, with its tip slightly downward at a 30-degree angle. Switch to high magnification and align the injection needle and fixation pin to the same horizontal plane. Check that the tip of the fixation pin is smooth and that the tip of the injection needle is sharp.

[0091] 2) Place the ICSI dish on the stage and maintain a constant temperature of 37°C. Focus the PVP droplet at the edge and repeatedly aspirate PVP with the injection needle for cleaning. Clean the fixation needle with PVP as well.

[0092] (3) ICSI steps.

[0093] 1) Oocyte preparation: Mature MII oocytes are transferred to ICSI dishes for subsequent injection.

[0094] 2) Sperm immobilization: Select morphologically normal, motile sperm from the edge of the PVP strip. Aspirate the sperm tail-first, then head-first, into the injection needle and transfer it into the PVP droplet to release the sperm. Use a microinjection needle to press the sperm tail against the bottom of the dish and quickly pull it horizontally past the sperm tail; the sperm will be immediately immobilized. Gently pick up the immobilized sperm with the needle tip and aspirate it tail-first, then head-first, into the injection needle, transferring it to the microdroplet containing the egg cell.

[0095] 3) Fixing the oocyte: Fix the oocyte in the Hepes fertilization drop in the center of the field of view with a microfixation needle, so that the first polar body is located at the 12 o'clock or 6 o'clock position, and the oocyte is just touching the bottom of the culture dish.

[0096] 4) Sperm injection: Lower the injection needle in the Hepes fertilization droplet until the needle tip is at the same level as the center of the oocyte. Push the sperm to the tip of the needle. Insert the needle vertically into the zona pellucida and oocyte cytoplasm at the 3 o'clock position, entering the cytoplasm. Aspirate some of the cytoplasm (make sure the cytoplasmic membrane has ruptured). Then inject the aspirated cytoplasm along with the sperm into the cytoplasm. Withdraw the injection needle and release the oocyte. The oocyte cytoplasmic membrane will return to its normal morphology within 1-5 minutes after injection. Check if the sperm is inside the cytoplasm. If the sperm is injected into the perioval space, repeat the above procedure once more until all oocytes in the micromanipulation dish have been injected.

[0097] 5) After the injected oocytes are thoroughly washed with cleavage fluid, they are transferred into G-1 PLUS and cultured in a time difference incubator at 37°C, 6% CO2, and 5% O2.

[0098] (4) Observe the fertilization situation and select 2PN fertilized eggs for subsequent experiments.

[0099] S3. Add CPI-455 or NMN to culture early human embryos.

[0100] Two-next-phase (2PN) fertilized eggs were transferred to G-1 PLUS medium containing 0 μM, 5 μM CPI-455, or 100 μM NMN, and cultured for three days in a time-variety incubator at 37°C, 6% CO2, and 5% O2. The development of each embryo was observed and recorded on Day 3. Subsequently, the embryos were transferred to G-2 PLUS medium containing 0 μM, 5 μM CPI-455, or 100 μM NMN for 2–3 days, and the development of each embryo was observed and recorded on Day 5 and 6. The rate of high-quality embryos on Day 3, and the blastocyst rate and high-quality blastocyst rate on Day 5 and 6 were statistically analyzed for the control group, the CPI-455-added group, and the NMN-added group.

[0101] Day 3 high-quality embryos are scored between 6C3 and 10C3, for example, 6C3 / 4, 7C3 / 4, 8C3 / 4, 9C3 / 4, and 10C3 / 4. Day 5 / 6 blastocysts are defined as all blastocysts from stage 1 to stage 6, with stage 1 and 2 being early blastocysts, stage 3 being fully expanded blastocysts, and stage 4 being extended blastocysts. High-quality Day 5 / 6 blastocysts are defined as those with a score of 4BC or higher, such as 4BB, 4AB, 4BA, and 4AA.

[0102] The results are as follows Figure 7 As shown, adding CPI or NMN can improve the rate of high-quality embryos on Day 3; further analysis revealed that CPI or NMN promotes the development of some 4-6 cell embryos to the 7-10 cell stage.

[0103] Statistical results of blastocysts after CPI treatment are as follows: Figure 8 As shown, adding CPI or NMN can improve the blastocyst rate. Considering that the transferable blastocysts obtained by patients in clinical assisted reproductive treatment are all counted as high-quality blastocysts, this embodiment defines blastocysts with 4 or higher blastocyst counts as high-quality blastocysts, based on the Gardner scoring criteria and the high-quality blastocyst scoring rules in assisted reproductive clinical practice. The results show that adding CPI or NMN can significantly improve the high-quality blastocyst rate.

[0104] Example 4

[0105] The effects of simultaneous addition of CPI-455 and NMN on in vitro embryo development and blastocyst rate were investigated, as follows:

[0106] S1, CPI-455 and NMN reservoir preparation: The method is exactly the same as the S1 part in Example 1 and Example 2.

[0107] S2, mouse in vitro fertilization and early embryo culture.

[0108] The method is exactly the same as (1) to (6) in S2 section of Example 1 and (1) to (3) in Example 3, except that the CPI-455 and NMN stock solutions prepared in S1 are added to the G-1PLUS and G-2PLUS culture dishes at the same time, so that the final concentrations of CPI-455 and NMN are 5μM and 100μM, respectively.

[0109] Inverted fluorescence microscope images of different groups of blastocysts and statistical results of blastocyst rate are as follows: Figure 9 As shown, 5 μM CPI-455 and 100 μM NMN significantly improved the blastocyst rate, the rate of high-quality Day 3 embryos, the blastocyst rate, and the rate of high-quality Day 5 embryos in mice before implantation. Further analysis revealed that CPI and NMN promoted the development of some 4-6 cell embryos to the 7-10 cell stage.

[0110] Therefore, this invention is the first to discover that CPI-455, a KDM5 histone demethylase inhibitor, can significantly improve the blastocyst rate, developmental speed, and embryo quality of preimplantation embryos by increasing the level of H3K4me3 within a certain concentration range. Simultaneously, the addition of CPI-455 and NMN can greatly improve the rate of high-quality blastocysts and embryo quality in in vitro embryo culture. The additives for improving embryonic developmental potential provided by this invention are of great significance for improving the efficiency and quality of in vitro embryo culture, accelerating the breeding process of superior embryos, and assisting in the study of embryo-related mechanisms.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A culture medium additive for improving embryonic developmental potential, characterized in that: The additives are 1 μM to 10 μM CPI-455 and 100 μM nicotinamide mononucleotide; The embryonic development refers to early in vitro embryonic development, specifically Day 1 to Day 6. The additive that improves embryonic developmental potential is applied on the day the fertilized egg is obtained.

2. A culture medium for improving embryonic developmental potential, characterized in that: The culture medium is a basal culture medium supplemented with the culture medium additive for improving embryonic developmental potential as described in claim 1; the basal culture medium is one of M16, CZB, G1-PLUS and G2-PLUS.

3. The use of a culture medium additive for improving embryonic developmental potential as described in claim 1 in in vitro embryo culture for non-medical purposes.

4. The use of a culture medium for improving embryonic developmental potential as described in claim 2 in in vitro embryo culture for non-medical purposes.

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