Application of L-2-HG in embryo quality evaluation, embryo quality evaluation method and application thereof

Abstract

The invention provides application of L-2-HG in embryo quality evaluation, an embryo quality evaluation method and application thereof, and relates to the technical field of biology. According to the research of the inventor, the metabolite L-2-HG generated by the embryo in the culture medium can be used as an embryo quality evaluation index, and non-invasive, accurate and reliable embryo quality evaluation can be realized according to the change of the L-2-HG in the culture medium in the culture process, so that the efficiency and success rate of embryo screening are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of L-2-HG in embryo quality assessment, an embryo quality assessment method and an application thereof. Background Art

[0002] In assisted reproductive technology (ART), embryo quality assessment and selection are crucial for clinical outcomes. Successfully selecting embryos with developmental potential for transfer is the ultimate goal of embryo evaluation. Embryo selection must be standardized and non-invasive at the very least. Among the numerous scoring systems currently available, the most commonly used rely on developmental rate and morphological assessment under a light microscope. The 2011 European Society of Human Reproduction and Embryology (ESHRE) guidelines on embryo evaluation primarily focused on morphological assessment in 2011 and time-lapse imaging in 2017. During morphological assessment, researchers have further combined oocyte morphology and early cleavage timing to predict embryo quality. This combined morphological assessment approach has significantly improved the accuracy of embryo assessment. Automated time-lapse imaging has also made this method more convenient. These guidelines provide standardized methods and clinical recommendations for embryo evaluation, helping to improve the success rate of ART. While morphological assessment is less invasive, embryo selection involves a degree of subjectivity and experience. In 2023, Cell Research reported on the application of preimplantation DNA methylation screening (PIMS) in clinical assisted reproductive technology. DNA methylation refers to a chemical modification on DNA that can alter genetic expression without altering the DNA sequence. DNA methylation is a key mechanism for controlling gene expression and one of the most extensively studied epigenetic regulatory mechanisms. Previous studies have shown that DNA methylation modifications on the parental genome can affect the health of the offspring. Errors in DNA methylation during embryonic development can lead to abnormal or arrested embryonic development. Furthermore, erroneous DNA methylation can also cause epigenetic disorders through imprinting genes. Therefore, researchers examined DNA methylation levels in trophoblast cells during assisted reproductive technology. The study hopes to identify embryos with a higher birth rate within a specific methylation threshold, based on the pregnancy-specific methylation range. This approach will facilitate the use of epigenetic information to screen embryos in assisted reproductive technology, improve IVF birth rates, and reduce miscarriage rates. This method, to a certain extent, improves the subjective method of embryo selection based on empirical morphological scoring. However, since this method requires obtaining blastocyst trophoblast cells, it is a minimally invasive detection technology for embryos, which increases the economic and time costs of patients.

[0003] In addition to the aforementioned embryo assessment methods, non-invasive techniques are being used to predict embryo quality based on changes in metabolic and protein compounds in the culture medium. Early on, embryo viability is estimated by relying on the consumption of metabolites in the culture medium, such as glucose and pyruvate. Later, embryo oxygen consumption is used to minimize damage to the embryo while improving the accuracy of embryo selection and pregnancy rates. For example, metabolic characteristics in the culture medium, such as glucose and pyruvate uptake, can be used to assess developmental potential. Alternatively, oxygen consumption can be used to indicate metabolically active embryos, which are associated with higher clinical pregnancy and live birth rates after transfer. Furthermore, relevant hormones, metabolites, and proteins in follicular fluid can be used to predict the quality of later embryos. However, these non-invasive testing methods have low sensitivity. For example, it is difficult to detect differences in pyruvate consumption before and after embryo culture, and oxygen consumption testing is also susceptible to environmental influences.

[0004] Therefore, given the problems existing in the above-mentioned detection methods, it is necessary to find a non-invasive method that can accurately predict embryo quality in order to improve the efficiency of embryo screening and protect embryo development.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first object of the present invention is to provide an application of L-2-HG in a culture medium in the quality assessment of in vitro cultured embryos, so as to solve the above technical problems.

[0007] A second object of the present invention is to provide a method for evaluating embryo quality.

[0008] The third object of the present invention is to provide application of the above-mentioned evaluation method in in vitro embryo culture.

[0009] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a use of 2-hydroxyglutarate (L-2-HG) in a culture medium for evaluating the quality of in vitro cultured embryos.

[0010] As a further technical solution, the embryo is a blastocyst stage embryo.

[0011] As a further technical solution, the embryo quality is evaluated based on the changes in the L-2-HG content in the culture medium during the in vitro culture of the embryo.

[0012] In a second aspect, the present invention provides a method for evaluating embryo quality, comprising the following steps: The fertilized eggs or embryos are cultured in a culture medium. When the blastocyst is formed, the L-2-HG in the culture medium is detected, and the embryo quality is evaluated based on the L-2-HG content in the culture medium.

[0013] As a further technical solution, compared with healthy embryos, if the content of L-2-HG in the culture medium is reduced, the quality of the embryos in the culture medium is poor.

[0014] As a further technical solution, liquid chromatography-mass spectrometry was used to detect L-2-HG in the culture medium.

[0015] As a further technical solution, the culture medium is an animal embryo culture medium.

[0016] As a further technical solution, the culture medium is an animal embryo culture medium that does not contain L-2-HG.

[0017] In a third aspect, the present invention provides application of the above-mentioned evaluation method in in vitro embryo culture.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. High detection accuracy. By detecting L-2-HG secreted by embryos, the sensitivity of metabolite detection in culture medium is significantly improved. For example, only 30 μl is required to detect L-2-HG.

[0019] 2. Low cost. Targeted testing of α-hydroxy acids such as L-2-HG in embryo culture media is low-cost, reducing patient financial costs while enabling personalized testing for each embryo.

[0020] 3. Safety. Compared to testing trophoblast cells, the present invention only requires discarded embryo culture medium, which helps ensure embryo vitality.

[0021] 4. High detection efficiency. Instead of relying on the detection of embryonic trophoblasts, mass spectrometry data can be obtained directly from discarded embryo culture medium within 2 hours, which can quickly provide multi-dimensional evaluation indicators for clinical screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 A: Schematic diagram of collecting mouse eggs at different stages for LC-MS analysis; B: 2-HG content in mouse eggs at different stages; C: L-2-HG content in mouse eggs and granulosa cells; Figure 2 :A: Schematic diagram of oocyte collection and culture in vitro; B: Detection of 2-HG content in oocytes at different stages of in vitro culture; C: Detection of 2-HG content in GV oocytes at different time points of in vitro culture; Figure 3 :A: Contents of four lactate dehydrogenase (LDH) mRNAs in oocytes during the GV, MI, and MII periods; B: Schematic diagram of L-2-HG production and regulation in oocytes; C: Changes in NAD+ content in GV, MI, and MII; D: Changes in GSH content in GV, MI, and MII; E: Effect of the LDH inhibitor GNE140 on the contents of L-2-HG and D-2-HG in oocytes; F: Effect of the LDH inhibitor GNE140 on the NAD+ content in oocytes; Figure 4 : A: Working principle of the L-2-HG biosensor LHGFR0N3C; B: L-2-HG biosensor showing changes in L-2-HG content after GNE-140 treatment; C: Representative image of fluorescence changes in eggs detected by the L-2-HG biosensor after GNE140 treatment; Figure 5A: Schematic diagram of siLdhb injection into GV eggs; B: qPCR verification of siLdhb interference efficiency in eggs; C: LC-MS detection of changes in 2-HG content in eggs after siLdhb treatment; D: L-2-HG biosensor showing changes in L-2-HG content after siLdhb treatment; E: Representative graph of fluorescence changes in eggs after L-2-HG biosensor detection of siLdhb; Figure 6 A: LC-MS analysis of changes in NAD+ content in eggs after siLdhb addition; B: NAD+ content detection under hypoxic conditions (20%, 5%); C: L-2-HG content detection under hypoxic conditions (20%, 5%); D: Changes in 2-HG content in eggs after α-KG replaced pyruvate in the culture medium; E: Changes in 2-HG content in the culture medium after α-KG replaced pyruvate in the culture medium; F: Changes in egg maturation rate after α-KG replaced pyruvate; G: Representative images of changes in egg morphology after α-KG replaced pyruvate; Figure 7 : A: Schematic diagram of the detection of L-2-HG changes in culture medium and eggs after treatment with the inhibitor GNE140; B: Changes in 2-HG content in eggs after GNE140 intervention; C: Changes in 2-HG content in culture medium M16 after GNE140 intervention; Figure 8 :A: Changes in NAD+ content in different numbers of 2C embryos; B: Changes in L-2-HG and D-2-HG in different numbers of 2C embryos; C: Changes in L-2-HG and D-2-HG in the culture medium of different numbers of 2C embryos; Figure 9 :A: Flowchart of the culture medium surrounding BC embryos, BC embryo collection, and L-2-HG detection; B: LC-MS detection of changes in L-2-HG content in 4.5dpc embryos with different outcomes (0BC, 1BC, 2BC); C: LC-MS detection of changes in L-2-HG content in 4.5m of the culture medium surrounding 4.5dpc embryos with different outcomes (0BC, 1BC, 2BC); Figure 10 :A: Flowchart of collection of culture medium surrounding BC and BC embryos and L-2-HG detection; B: LC-MS detection of changes in L-2-HG content in 4.5dpc embryos with different outcomes (0BC, 1BC, 2BC); C: LC-MS detection of changes in L-2-HG content in 4.5m culture medium surrounding embryos with different outcomes (0BC, 1BC, 2BC). DETAILED DESCRIPTION

[0024] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0025] In a first aspect, the present invention provides a use of 2-hydroxyglutarate (L-2-HG) in a culture medium for evaluating the quality of in vitro cultured embryos.

[0026] The inventors have discovered that L-2-HG, a metabolite produced by the embryos themselves in the culture medium, can be used as an indicator for embryo quality assessment. Based on the changes in L-2-HG in the culture medium during the culture process, non-invasive, accurate and reliable embryo quality assessment can be achieved, thereby improving the efficiency and success rate of embryo screening.

[0027] In some optional embodiments, the embryo is a blastocyst stage embryo.

[0028] In a second aspect, the present invention provides a method for evaluating embryo quality, comprising the following steps: The fertilized eggs or 2C embryos are cultured in a culture medium. When the blastocyst is formed, the L-2-HG in the culture medium is detected. The embryo quality is evaluated based on the L-2-HG content in the culture medium.

[0029] The evaluation method provided by the present invention is simple and convenient, low in cost, safe, and has high detection accuracy and efficiency.

[0030] In some optional embodiments, liquid chromatography-mass spectrometry is used to detect L-2-HG in the culture medium.

[0031] In some optional embodiments, the culture medium is animal embryo culture medium.

[0032] In some optional embodiments, the culture medium is an animal embryo culture medium that does not contain L-2-HG. Using an animal embryo culture medium that does not contain L-2-HG avoids the influence of L-2-HG contained in the culture medium itself on embryo quality assessment.

[0033] In a third aspect, the present invention provides application of the above-mentioned evaluation method in in vitro embryo culture.

[0034] The evaluation method provided by the present invention can accurately predict the quality of embryos and is used for in vitro embryo culture, thereby helping to improve the efficiency of embryo culture.

[0035] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0036] Example 1 Dynamic changes of L-2-HG during oocyte maturation and its production mechanism Previous work has shown that eggs and early embryos contain α-hydroxy acids such as L-2-HG. Given the role of 2-HG in tumors and immune cells, which has not been reported in eggs or culture, this study investigated the production and effects of this compound in eggs and embryos. The egg and embryo acquisition methods are as follows: Oocyte retrieval: Oocytes of varying maturity were collected from 3-week-old female C57 mice according to their growth and maturation stages. Fully grown oocytes (GV) were harvested from ovaries 44-48 hours after injection of 5 IU PMSG (pregnant mareserum gonadotropin). Mi stage oocytes were harvested from ovaries 3-6 hours after injection of 5 IU hCG 48 hours after 5 IU PMSG. Mature Mi stage oocytes were harvested from the oviduct 12-14 hours after injection of 5 IU hCG 48 hours after 5 IU PMSG. To determine the metabolite levels at different stages, mass spectrometry was performed on GV, Mi stage, and Mi stage oocytes collected in vivo. Furthermore, to verify the source of L-2-HG in oocytes, GV oocytes were cultured in M16 medium until the Mi stage and Mi stage in vitro.

[0037] Mass spectrometry detection process for 2-HG in a small amount of egg samples, such as Figure 1 A in: 1) Oocyte sample collection: Wash three times in 0.9% NaCl and then add 80% methanol.

[0038] 2) Add internal standard: add isotope-labeled 2-HG internal standard [ 13 C5] D / L-2-HG, the mixture was centrifuged at 14000 g for 15 min at 4°C, and the supernatant was collected and dried.

[0039] 3) Derivatization: Add 50 μL of TSPC (containing 12.5 mM CAN) and derivatize for 20 min. Then add 5% pyridine to terminate the derivatization to distinguish L-2-HG and D-2-HG with different chiral structures.

[0040] 4) Mass Spectrometry Detection: TSPC-labeled L-2-HG and D-2-HG in oocytes were determined using liquid chromatography-tandem mass spectrometry (LC-MS) in the negative ion mode on a Waters ACQUITY UPLC I-Class PLUS system. High-performance liquid chromatography (HPLC) separation was performed at 30°C on a CORTECS-C18 column (2.1 × 100 mm, 1.6 μm). Mobile phase A consisted of 15 mM ammonium acetate and 0.1% ammonia (v / v) in water, and mobile phase B was acetonitrile. The optimized gradient was: 10% to 31% B from 0 to 4 min, 31% to 32% B from 4 to 15.5 min, 32% to 95% B from 15.5 to 16 min, 95% B from 16 to 18 min, and a final 4-min equilibration at 10% B.

[0041] 5) Mass spectrometry was performed in the negative ion mode using multiple reaction monitoring (MRM). L-2-HG and D-2-HG were at 448.1>155.1, and isotope-labeled ([ 13 C5] D / L-2-HG was subjected to MRM transition in the 453.1>155.1 mode, and the contents of L-2-HG and D-2-HG were calculated.

[0042] The test found that the L-2-HG content increased significantly from 53.88 nmol / 10 6 The egg rose to 129.69 nmol / 10 6 From MI to MII stage, the content of L-2-HG also increased significantly. In GV to MII stage eggs, the total amount of molecular D-2-HG was only 16.01 nmol / 10 6 eggs, and there is no obvious dynamic change during egg development ( Figure 1 B) in.

[0043] It is well known that oocytes exchange substances with surrounding granulosa cells. Therefore, oocytes were collected from mice according to the above method. After removing the cumulus granulosa cell clumps using a 1ml syringe tip, the cumulus granulosa cells were collected and the 2-HG content was measured. The results showed that the L-2-HG content in oocytes was 14.64 pmol / μg protein per unit mass, while the L-2-HG content in cumulus granulosa cells was only 1.88 pmol / μg protein ( Figure 1 C), indicating that L-2-HG is mainly produced by the egg itself. In addition, direct detection of in vitro culture GV to MII stage eggs ( Figure 2The results showed that L-2-HG increased significantly during in vitro oocyte maturation ( Figure 2 B in the figure) also proves that L-2-HG originates from the egg itself. During the in vitro culture of the egg, 2.5 μM Mildew was added to keep the egg in the GV stage ( Figure 2 C in the figure) detected the L-2-HG content in GV eggs cultured for different times (0h, 6h, 18h). The results showed that L-2-HG was not related to egg maturity, and its content gradually increased with the extension of culture time.

[0044] To explore the mechanism of L-2-HG production in eggs, we further analyzed the enzymes and conditions for L-2-HG production. We analyzed the transcriptional data of eggs at different maturation stages (GV, MI, and MII), which showed that during the egg maturation process, from the fully grown egg GV to the mature egg MII stage, Ldhb The content is much higher than Ld Other family members Ldha、Ldhc and Ldhd ( Figure 3 A in the figure). Studies have shown that LDHB can not only reduce pyruvate to lactate, but also reduce α-ketoglutaric acid (α-KG) to L-2-HG, both accompanied by the conversion of NADH to NAD. + The transformation ( Figure 3 B). Detection of NAD in cells during in vitro culture + The content of GSH was related to the content of NAD. + and GSH contents increased significantly ( Figure 3 C and D in the figure).

[0045] To verify the relationship between L-2-HG production and NAD + In vitro culture of GV culture medium M16, LDHB inhibitor (2.5 μM GNE-140) was added to interfere with LDHB activity ( Figure 3 After inhibiting LDHB activity, NAD in the egg was detected using LC-MS in positive ion mode. + The content of L-2-HG in eggs was detected by LC-MS negative ion mode. The results showed that the inhibition of LDHB activity led to a significant decrease in L-2-HG and NAD+ content ( Figure 3 In addition, we used L-2-HG-sensing fluorescent reporter (LHGFR) 0N3C Studying the L-2-HG content in viable eggs. L-2-HG biosensor LHGFR0N3C The combination of L-2-HG oxidase LhgR, which specifically senses L-2-HG, and FRET technology is a highly sensitive tool for detecting L-2-HG. After expressing LHGFR in cells, laser activation is performed and fluorescence intensity is measured at emission wavelengths of 465-495nm (mTFP, unbound L-2-HG) and 505-700nm (Venus, bound L-2-HG). The Venus / mTFP ratio can be used to determine the relative L-2-HG content ( Figure 4 A in the figure). 0N3C The oocytes expressing mRNA were cultured in M16 medium containing the inhibitor GNE-140 and milrinone, and DMSO was used as a control to study the effect of GNE140 on L-2-HG production in the oocytes. The results showed that after GNE140 treatment, L-2-HG content was significantly decreased ( Figure 4 B and C in ). Alternatively, use Ldhb siRNA intervention in oocytes Ldhb The cells were transcribed and cultured in the medium M16 containing milrinone, and a control group siNC (negative control) was set up. qPCR showed that siRNA could significantly reduce the expression of GV oocytes. Ldhb content( Figure 5 Consistent with the LC-MS results, the results of the L-2-HG biosensor showed that the Ldhb After ovulation, the L-2-HG content in GV eggs decreased significantly ( Figure 5 C, D, and E in Figure 4). LC-MS analysis revealed that the silencing Ldhb Post-NAD + The content was significantly reduced ( Figure 6 A).

[0046] In addition, due to the effects of hypoxia on L-2-HG in oocytes, we collected GV oocytes and cultured them in M16 medium containing milrinone. One group was cultured in 20% O2 as a control, while the hypoxia experimental group was cultured in a 5% O2 incubator. After 20 hours of culture, oocytes from both culture conditions were collected and analyzed by LC-MS. The results showed that L-2-HG content increased under hypoxia, while NAD decreased. + unchanged, indicating that LDHB produces L-2-HG, which helps maintain NAD in a hypoxic environment. + Stability ( Figure 6To further verify whether the precursor of L-2-HG in eggs is α-KG, complete culture medium PGL (containing pyruvate, glucose, and lactate), conditioned culture medium GL (with pyruvate removed), and culture medium α-KG (in which 0.3 mM of the precursor of L-2-HG, α-KG, was added to the conditioned culture medium to replace pyruvate, denoted as GL+α-KG (0.3 mM)) were prepared. It was found that the amount of L-2-HG produced by eggs remained unchanged, and the amount of L-2-HG in the culture medium increased significantly ( Figure 6 D and E in the figure (in the figure, the L-2-HG content in the egg is represented by GL and α-KG, and the L-2-HG content in the culture medium is represented by mGL and mα-KG). Therefore, it is speculated that the main precursor of L-2-HG in the egg is α-KG. It is speculated that under conditions such as hypoxia or insufficient energy, α-KG can be reduced to L-2-HG in the egg. After the addition of α-KG, the MI formation rate gradually increased, but the MII formation rate did not return to the level of the pyruvate-containing group ( Figure 6 F and G in ).

[0047] In conclusion, LDHB reduces α-KG to generate L-2-HG, which promotes NAD + LDHB is produced, alleviating the effects of pyruvate deficiency on oocyte maturation. LDHB not only participates in the reduction of pyruvate and α-KG, but may also participate in the reduction of other α-keto acids, alleviating reductive stress. We will further explore the content of α-keto and α-hydroxy acids in oocytes and their role in oocyte maturation.

[0048] Example 2 Study on the correlation between secretory α-hydroxy acid and embryonic development potential Currently, in clinical assisted reproduction, embryo quality assessment has an important impact on clinical pregnancy outcomes. Currently, embryo morphology is mainly used to assess embryo quality and predict pregnancy outcomes. Recently, epigenetic information has also been applied to PIMS, a technology for clinical embryo screening in assisted reproduction. In addition, non-invasive protein and metabolic content are used as potential biomarkers for clinical embryo development quality assessment, but because many metabolites are easily interfered with by components in the culture medium, changes in their content are not sufficient to reflect the quality of the embryo. Based on previous embryo metabolomics research, the present invention further discovered that α-keto acids can be reduced to α-hydroxy acids in eggs, thereby increasing NAD in eggs. + The content of L-2-HG in the culture medium ensures oocyte maturation. The metabolite, α-hydroxy acid, can be secreted into the culture medium, indirectly reflecting the oocyte's metabolic status. Therefore, the present invention uses the detection of the metabolite L-2-HG, secreted by oocytes and embryos in the culture medium, to predict embryo quality.

[0049] To determine the relationship between the L-2-HG content in the culture medium and embryo quality, we conducted the following experiments: (1) Detection of metabolites in embryos and culture medium M16; (2) Detection of the L-2-HG content in different amounts of embryo culture medium KSOM to further determine the accuracy of L-2-HG detection; (3) Detection of the L-2-HG content secreted in KSOM from the fertilized egg to the blastocyst stage to determine its relationship with embryo development potential; (4) Detection of the L-2-HG content secreted in KSOM from the 2-cell embryo stage to the blastocyst stage to determine its relationship with embryo development potential.

[0050] (1) Detection of metabolites in embryos and culture medium M16 To confirm that the α-hydroxy acids in the M16 medium were derived from the oocytes, we used 2.5 mM miltiorrhiza to inhibit meiosis and added GNE-140 to inhibit LDHB activity in the oocytes. After 20 hours of treatment, the oocytes and the surrounding 25 μl M16 medium were collected and the L-2-HG content in the oocytes and the surrounding M16 medium was measured. See the flow chart. Figure 7 A in Figure 1. The method for detecting L-2-HG in oocytes is as described previously. The procedure for detecting L-2-HG in culture medium M16 is as follows: 40 embryos are cultured in a single microdroplet to enrich the L-2-HG content in M16. 25 μl of M16 is collected and centrifuged at 14,000 rpm at 4°C to remove impurities. 20 μl of the sample is then added to 80 μl of pure methanol pre-chilled on dry ice and stored at -80°C. After all samples are collected, LC-MS is used to analyze changes in 2-HG in oocytes and culture medium.

[0051] The results showed that after GNE-140 inhibited LDHB in the eggs, the L-2-HG content in the eggs decreased significantly from 4115.44 nmol / 10 6 The egg dropped to 1488.22 nmol / 10 6 After treatment with the inhibitor GNE140, L-2-HG in the surrounding culture medium M16 also decreased from 3.35 μM to 2.72 μM. This result shows that after GNE140 treatment of eggs, not only did the L-2-HG content in the eggs decrease, but the L-2-HG content in the surrounding culture medium M16 also decreased significantly ( Figure 7 B and C in ).

[0052] (2) Detection of L-2-HG content in different amounts of embryo culture medium KSOM Establishing a very small amount of embryonic L-2-HG and cellular NAD + Mass spectrometry detection method and 2-HG detection method in KSOM.

[0053] NAD + The specific detection method is as follows: 1) Centrifuge the sample at 14,000 rpm for 15 min at 4°C. 2) Vacuum dry the supernatant at room temperature; 3) Prepare the reconstitution solution with acetonitrile: water = 3:1; 4) After draining the sample, add reconstitution solution (approximately 80 μl / aliquot) and incubate in a mixer at 4°C, 1600 rpm, for 15 minutes to promote reconstitution. 5) Centrifugation: 15,000 rpm, 4°C, 15 min; 6) Take 60 μl and transfer it into the inner liner for injection testing; 7) Prepare a 50 μg / ml basal NAD+ solution and prepare solutions A, B, C, D, E, and F according to the recipes in the table below.

[0054] .

[0055] 8) HPLC-LCMS detection of NAD + content.

[0056] Detection of NAD + Afterwards, the remaining metabolites were collected and the 2-HG isotope internal standard was added to the culture medium KSOM. After being dried, TSPC derivatization was performed, and the L-2-HG content in the embryos and culture medium was detected by LC-MS in negative ion mode.

[0057] In 30 μl of KSOM culture medium, 1, 5, 10, and 20 fertilized eggs were cultured, and 24 hours later, 2-cell embryos (2C) were collected and surrounded by KSOM culture medium. After collecting the embryos with 80% methanol, the supernatant was vacuum-dried after centrifugation, and the NAD in the embryos was detected. + and 2-HG content as well as the content of 2-HG in the culture medium.

[0058] The experimental results showed that after 24 hours of culture, as the number of 2-cell embryos increased, NAD + and L-2-HG content gradually increased ( Figure 8 The L-2-HG content in the KSOM culture medium also accumulated with the increase in the number of embryos. The fitted regression curve showed that the L-2-HG content in KSOM was close to saturation when the number of embryos reached 20 ( Figure 8 C). The results of the gradient embryo test indicate that six embryos and the surrounding culture medium KSOM can be collected for L-2-HG content testing to determine the relationship between L-2-HG content in the embryo surrounding culture medium KSOM and embryo quality.

[0059] (3) Relationship between the content of L-2-HG secreted in KSOM from the fertilized egg to the blastocyst stage and the embryonic developmental potential Using the above-mentioned 2-HG detection method in embryos and KSOM, the content of L-2-HG secreted into the surrounding culture medium KSOM from the fertilized egg to the blastocyst was studied, thereby studying the relationship between the L-2-HG content in KSOM and the embryonic developmental potential, that is, establishing the relationship between the L-2-HG content in the culture medium and blastocyst formation.

[0060] Three-week-old female C57 mice were injected with PMSG 48 hours after hCG injection. The females were then co-housed with male C57 mice aged 8 weeks or older in a 1:1 ratio. After 20 hours of co-housing, the mice were separated. The cumulus granulosa cell complex was harvested from the oviduct, and the granulosa cells surrounding the fertilized eggs were removed using hyaluronidase digestion. Fertilized eggs at 0.5 days per week were cultured in vitro in 20 μl of KSOM microdroplets. At 4.5 days per week, the embryos were collected and stored at -80°C (with 10 μl of KSOM culture medium). Figure 9 A in the figure). At 4.5dpc, the cells were divided into three groups according to the blastocyst formation (whether a blastocyst was formed was determined by whether there was a cavity), namely, no blastocyst formation (0BC), one blastocyst (1BC), and two blastocysts (2BC) formation groups. The culture medium KSOM for the three groups was recorded as 0BC-4.5m, 1BC-4.5m, and 2BC-4.5m. According to the BC formation situation, each group was combined with 3 microdroplets for a total of 6 embryos and the corresponding 30ul culture medium KSOM. The results of testing the L-2-HG content in the embryos and culture medium KSOM showed that there was no significant difference among the three groups of 0BC, 1BC, and 2BC at 4.5dpc ( Figure 9 This indicates that the L-2-HG content in the blastocysts is similar, but the L-2-HG detection intensity in the three groups of KSOM is significantly different ( Figure 9 The L-2-HG content in the 2BC group was significantly higher than that in the 0BC group. These results indicate that the L-2-HG content excreted by embryonic metabolism is closely related to the developmental potential of the zygote and 2C embryos, providing an important indicator for noninvasive assessment of embryo quality.

[0061] (4) Detection of the relationship between the content of L-2-HG secreted in KSOM from the 2C to blastocyst stage and the embryonic developmental potential Since clinical fertilized eggs were placed in single microdrop culture at 1.5 dpc, we further studied the relationship between the L-2-HG content secreted by embryos into the culture medium KSOM and the embryonic developmental potential during the development period from 2C (1.5 dpc) to BC (4.5 dpc). 2C were transferred to a new culture medium KSOM at 1.5 dpc, and embryos and KSOM were collected at 4.5 dpc. Figure 10 The results showed that the L-2-HG content in the culture medium of m0BC without blastocyst formation and the culture medium of m2BC with two blastocysts increased significantly from 1.26μM to 1.79μM at 4.5dpc. Figure 10There was no significant difference in L-2-HG content among the three groups of embryos: 0BC, 1BC, and 2BC ( Figure 10 C in the blastocyst). This indicates that the L-2-HG content in the blastocyst is similar, but the L-2-HG secreted into KSOM by embryos of different developmental potentials varies. The results show that the L-2-HG content excreted by embryo metabolism is closely related to the developmental potential of the zygote and 2C embryos, providing an important indicator for non-invasive assessment of embryo quality. It is speculated that NAD + The high content helps the development of early embryos.

[0062] In summary, the purpose of the present invention is to provide a method for predicting early embryo quality based on the L-2-HG content in the culture medium. By detecting the concentration changes of L-2-HG in the culture medium and combining the morphological and metabolic characteristics of embryonic development, a correlation model between L-2-HG content and embryo quality is established, providing a scientific basis for embryo screening.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of L-2-HG in culture medium to evaluate the quality of in vitro cultured embryos.

2. The use according to claim 1, characterized in that The embryo is a blastocyst stage embryo.

3. The use according to claim 1, characterized in that Embryo quality was assessed based on changes in L-2-HG content in the culture medium during in vitro embryo culture.

4. A method for evaluating embryo quality, characterized in that: The following steps are involved: The fertilized eggs or embryos are cultured in a culture medium. When the blastocyst is formed, the L-2-HG in the culture medium is detected, and the embryo quality is evaluated based on the L-2-HG content in the culture medium.

5. The evaluation method according to claim 4, characterized in that Compared with healthy embryos, if the L-2-HG content in the culture medium is reduced, the embryos in the culture medium are of poor quality.

6. The evaluation method according to claim 4, characterized in that Liquid chromatography-mass spectrometry was used to detect L-2-HG in the culture medium.

7. The evaluation method according to claim 4, characterized in that The culture medium is an animal embryo culture medium.

8. The evaluation method according to claim 7, characterized in that The culture medium is an animal embryo culture medium that does not contain L-2-HG.

9. Use of the evaluation method according to any one of claims 4 to 8 in in vitro embryo culture.

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