Application of rutin hydrate in prevention and treatment of recurrent embryo planting failure

Abstract

The invention discloses application of rutin hydrate in preventing and treating repeated embryo planting failure, and belongs to the technical field of assisted reproduction. It is found for the first time that CD36 is a key membrane protein for lipid transport and uptake of EECs, rutin hydrate competitively inhibits combination of CD36 and natural ligands of CD36, abnormal lipid excessive uptake is remarkably blocked, lipid accumulation and lipid toxicity damage in cells are effectively relieved, and the effect of inhibiting EECs lipid transport and uptake is achieved. And finally, the endometrial receptivity defect related to RIF is reversed, and the embryo adhesion and implantation capability is remarkably improved. Therefore, rutin hydrate provides a brand new strategy for developing targeted therapy drugs for RIF, especially for lipid metabolism disorder type endometrial receptivity badness, and has remarkable clinical application and transformation prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of assisted reproductive technology, and particularly relates to application of hydrous rutin in prevention and treatment of recurrent embryo implantation failure. BACKGROUND

[0002] In recent years, reproductive technology has developed rapidly, and in vitro fertilization-embryo transfer technology has gradually developed into an important method for treating infertility. However, studies have found that the failure rate of infertile women to become pregnant through assisted reproductive technology (ART) is high, and 75% of all unsuccessful pregnancies are caused by embryo implantation failure. Recurrent implantation failure (RIF) is a complex disease characterized by the failure to achieve clinical pregnancy after multiple high-quality embryo transfers, and RIF affects about 10% of in vitro fertilization-embryo transfer patients worldwide. The latest consensus defines it as: a 40-year-old adult woman who has not achieved clinical pregnancy after transferring at least 3 high-quality embryos in 3 fresh or frozen cycles, and the conditions for high-quality embryos include: 3-day embryos (cell number ≥ 8, blastomere size uniformity, and fragment rate < 10%) and blastocysts (≥ 3BB). After multiple failed in vitro fertilization cycles, RIF inevitably brings psychological, economic and physical multiple blows to infertile couples, and is one of the major challenges in assisted reproductive technology.

[0003] The causes of RIF are complex, and poor endometrial receptivity (ER) is considered to be one of the key factors. Endometrial epithelial cells (EECs) as the "gatekeeper" directly involved in embryo adhesion are the main target cells of estrogen and progesterone, which accurately receive the instructions of ovarian steroid hormones by expressing corresponding nuclear receptors (ERa, PR). The most striking feature of ER is the dramatic change in the expression of EECs surface molecules, which is the basis for the embryo to recognize and attach to the endometrium. FoxO1 (Forkhead box O1) is a recognized "receptivity core marker" in EECs. During the "implantation window period" in humans and mice, FoxO1 must be transferred from the cytoplasm into the nucleus of the glandular cavity / luminal epithelial cells, and its absence or misplacement will cause the embryo to fail to break through the epithelial barrier and implantation failure.

[0004] Currently, the treatment strategies for RIF in clinic are still limited and lack of high specificity, and the mainstream schemes are mainly focused on uterine cavity perfusion (such as perfusion of human chorionic gonadotropin, peripheral blood mononuclear cells or granulocyte colony-stimulating factor, etc.), immune regulation (including the use of glucocorticoids, hydroxychloroquine and the like to inhibit possible abnormal immune response) and endometrial scraping (to stimulate endometrial repair and ER improvement through local mechanical damage theory) and other empirical interventions. Although these methods show certain effects in some patients, their mechanisms are generally broad, and the efficacy evidence is mainly from small-scale studies with inconsistent conclusions, so it is difficult to be stable and popularized. In essence, most of the existing strategies are not directly aimed at the core pathological link of "ER abnormality", resulting in significant blindness and uncertainty in clinical treatment, and new therapeutic drugs that can precisely regulate key target points in the establishment process of EECs are urgently needed to target the functional disorder of EECs, the molecular expression timing disorder and the embryo-mother dialogue disorder.

[0005] Hydrated rutin is a hydrate form of natural flavonoids rutin, which is a generally recognized safe (Generally Recognized As Safe, GRAS) dietary flavonoid, and is also known as "vitamin P" due to its role in maintaining the integrity and permeability of blood vessels. Rutin, as an active ingredient widely existing in plants such as buckwheat and citrus, has been verified for its safety in clinical application; it has good human tolerance under conventional doses, and the adverse reactions are mild and rare, which provides an important safety basis for its potential application in specific populations (such as women in the pre-pregnancy period). However, although rutin and its derivatives are known to have significant antioxidant, anti-inflammatory, and improved microcirculation activities, whether they can specifically target EECs to regulate cell function, such as reducing oxidative stress damage, inhibiting abnormal inflammatory response, or enhancing adhesion molecule expression to improve endometrial receptivity and thus intervene in the pathological process of RIF, has not been reported so far. SUMMARY

[0006] The present application researches and finds that CD36 is a key membrane protein for lipid transport and uptake of EECs, and hydrated rutin can significantly block excessive uptake of abnormal lipids by competitively inhibiting the binding of CD36 to its natural ligand, thereby effectively reducing intracellular lipid accumulation and lipotoxicity damage, and improving cell energy metabolism. Based on the mechanism of action of the drug, the present application verifies that hydrated rutin has the function of regulating metabolic dysfunction and molecular expression timing disorder of EECs (endometrial epithelial cells), and as a new drug, it has a significant effect on improving endometrial receptivity and promoting embryo adhesion to prevent and treat RIF. In view of this, the present application provides the pharmaceutical use of hydrated rutin in improving endometrial receptivity and preventing and treating embryo implantation failure, i.e. the application of hydrated rutin in preparing a drug for preventing and treating repeated embryo implantation failure.

[0007] The present application comprises the following technical solutions: In a first aspect, the present application provides use of CD36 or an agent for detecting CD36 in the preparation of a product for predicting recurrent failed embryo implantation.

[0008] The product includes but is not limited to reagents, kits, chips, test papers, membrane strips, models or detection platforms.

[0009] The agent for detecting CD36 includes any reagent required for detecting CD36 protein level or gene expression level by RT-PCR method, RT-qPCR method, biochip detection method, Southern blotting method, in situ hybridization method, Western Blot, immunohistochemical method (IHC), immunofluorescence staining method, enzyme-linked immunosorbent assay (ELISA), spatial transcriptome technology.

[0010] In some embodiments of the present application, the agent for detecting CD36 is an agent for detecting CD36 protein level by immunofluorescence staining method.

[0011] In a second aspect, the present application provides a model for predicting recurrent failed embryo implantation, wherein the model takes the detection result of immunofluorescence staining of CD36 protein in the endometrial tissue of a subject as a variable, is trained and verified by a machine learning model, and is constructed to obtain the model.

[0012] In the specific embodiments of the present application, the model comprises an information acquisition module and a diagnosis module.

[0013] The information acquisition module is used to perform the step of acquiring the CD36 protein expression level information of the endometrial tissue of the subject. The diagnosis module is used to perform the step of determining whether the subject is a recurrent failed embryo implantation patient according to the CD36 protein expression level information.

[0014] In a third aspect, the present application provides use of a CD36 binding inhibitor in at least one of the following: 1) in the preparation of a drug for preventing and / or treating recurrent failed embryo implantation; 2) in the preparation of a drug for improving endometrial receptivity.

[0015] The CD36 binding inhibitor refers to a substance that can specifically bind to CD36 and thereby inhibit the binding of CD36 to its natural receptor.

[0016] In one specific embodiment of the present application, the CD36 binding inhibitor is rutin hydrate, which is determined by SPR technology to have an affinity constant of 2.13 µM for binding to CD36, indicating that rutin hydrate as a CD36 binding inhibitor can effectively prevent CD36 from binding to its receptor.

[0017] In the present application, the subject to which the rutin hydrate is administered is a patient who has undergone in vitro fertilization-embryo transfer and has recurrent implantation failure (RIF).

[0018] Further, the subject is a patient with unexplained recurrent implantation failure (RIF), i.e., further, the subject is a strictly screened patient with unexplained recurrent implantation failure. It must meet the following conditions at the same time: multiple high-quality embryo transfer failures have been experienced, and factors such as male factors, hydrosalpinx, moderate to severe endometriosis, intrauterine adhesions, uterine fibroids, or specific autoimmune diseases that cause implantation failure have been ruled out. The drug has at least one of the following effects: 1) Inhibiting lipid uptake by endometrial cells and reducing lipid accumulation; 2) Promoting FoxO1 nuclear translocation and improving endometrial receptivity; 3) Improving embryo adhesion and implantation capacity; 4) Promoting endometrial thickening and uterine cavity closure.

[0019] Decreased endometrial receptivity is often accompanied by dysregulation of the microenvironment, including metabolic disorders, inflammatory responses, and abnormal lipid accumulation, which are important pathological bases for RIF and female infertility. One of the core mechanisms by which the rutin hydrate provided in the present application prevents and treats RIF is to significantly improve endometrial receptivity. Specifically, rutin hydrate can promote the translocation of transcription factor FoxO1 from the cytoplasm to the nucleus, thereby regulating the expression of downstream target genes, inducing cell cycle arrest in EECs, promoting their transition to a functional differentiation state, and causing remodeling of cell morphology and function, ultimately converting EECs from a non-receptive state to a receptive state that can effectively accept, adhere to, and promote embryo invasion, significantly improving the success rate of embryo adhesion and implantation.

[0020] In the present application, the application includes administering to the patient a therapeutically effective amount of rutin hydrate or a pharmaceutical composition having rutin hydrate as an active molecule. The pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0021] The term "pharmaceutically acceptable" as used herein means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated with it. Preferably, the term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The pharmaceutically acceptable excipients described herein can include any solvent, solid excipient, diluent, or other liquid excipient, etc. suitable for a particular target dosage form.

[0022] The pharmaceutical composition with rutin hydrate as the active molecule provided by the present application can be used for the treatment of diseases and in vitro cell culture experiments. When used for the treatment of diseases, the pharmaceutical composition usually refers to a unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical field. All the methods include the step of combining the active ingredient with the excipients constituting one or more accessory ingredients.

[0023] The dosage form of the pharmaceutical composition includes oral preparations, injections, uterine cavity perfusion or topical vaginal administration. The advantage of rutin hydrate as the raw material is that it significantly reduces hygroscopicity, improves powder flowability and compressibility in a stable crystal form, greatly optimizing the preparation process and stability.

[0024] The pharmaceutical composition with rutin hydrate as the active molecule provided by the present application is used for the preparation of endometrium before embryo transfer in assisted reproductive technology, and for the prevention and treatment of repeated embryo implantation failure, including improving embryo implantation.

[0025] Further, in some application modes of the present application, the application also includes the combined administration of rutin hydrate or the pharmaceutical composition with rutin hydrate as the active molecule and other conventional agents or conventional treatment methods in the field. The other conventional agents in the field include but are not limited to antibiotics, low molecular weight heparin, immunosuppressive drugs, and hormone drugs.

[0026] The technical solution provided by the present application has the following advantages: RIF is caused by a complex mechanism, and it is particularly necessary to elucidate the pathogenesis and screen effective potential targets for diagnosis and treatment for the prediction, diagnosis and intervention of embryo implantation failure. Rutin hydrate shows clear anti-RIF pharmacological activity and therapeutic potential in in vivo experiments. The animal experiments provided by the present application show that in the high-fat uterine animal model, after a certain dose of rutin hydrate intervention, the pregnancy outcome of the test mammal is significantly improved, specifically manifested as a significant increase in the number of implanted embryos and an improvement in the degree of uterine cavity closure. Histological and molecular biological analysis of endometrial tissue further shows that rutin hydrate can effectively reduce the abnormal accumulation of lipids in EECs and promote the translocation of transcription factor FoxO1 from the cytoplasm to the nucleus, ultimately confirming that rutin hydrate can significantly enhance endometrial receptivity and improve embryo implantation rate. Rutin hydrate effectively restores the homeostasis of the endometrial microenvironment through the above mechanism, providing a novel and potential drug strategy for the treatment of RIF and related fertility disorders, and is expected to improve the clinical pregnancy rate after embryo transfer.

[0027] The application of rutin hydrate provided by the present application not only has important targeted therapeutic value for preventing and treating RIF, but also shows potential in the broader maintenance of women's reproductive health. The application provides a new treatment strategy and drug tool with clear mechanism and clear target for related infertility for the first time from the perspective of regulating endometrial lipid metabolism disorder, which makes up for the defects of the existing treatment scheme in lack of pertinence, and provides a new choice and intervention approach for clinical treatment. Therefore, rutin hydrate provides a new strategy for developing targeted therapeutic drugs for RIF, especially for lipid metabolism disorder type endometrial receptivity, and has a significant clinical application and conversion prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Abnormal accumulation of lipid droplets in the endometrial tissue of RIF patients; wherein A is a typical microscopic image of BODIPY staining method for detecting the distribution of lipid droplets in the endometrial samples of RIF group and CTR group; B is the ratio of lipid droplet area to total cell area (lipid droplet / cell area ratio) in EECs of the two groups and the difference in the constituent ratio of lipid droplet positive (lipid droplet / cell area ratio > 10%) population in RIF and CTR groups; C is the receiver operating characteristic (ROC) curve for predicting the risk of RIF in subjects using lipid droplet positive area as an index.

[0029] Figure 2Expression of CD36 in endometrial tissue and its lipid uptake function; wherein, A is single cell analysis of CD36 expression in EECs of RIF patients; B is the result of CD36 staining in endometrial samples of RIF and CTR groups; C is the receiver operating characteristic (ROC) curve of CD36 mean fluorescence intensity as an index to predict the risk of RIF; D is the BODIPY staining result after overexpression of CD36 in EECs.

[0030] Figure 3 Pharmacodynamic experiment of rutin hydrate and CD36; wherein, A is the result of CD36 protein virtual screening and molecular docking; B-C is the comprehensive result of kinetic simulation of rutin hydrate and CD36; D is the result of surface plasmon resonance (SPR) of rutin hydrate and CD36 (KD=2.13 μM); E is the BODIPY staining result of the effect of different concentrations of rutin hydrate on lipid uptake of EECs.

[0031] Figure 4 Effect of rutin hydrate on EECs receptivity; wherein, A is the 1-hour adhesion rate of lipid accumulation and embryo spheres to EECs monolayer after treatment with rutin hydrate; B is the 24-hour flattening rate of embryo spheres to EECs monolayer after lipid accumulation and intervention with rutin hydrate; C is the BODIPY and FoxO1 staining result after lipid accumulation and treatment with rutin hydrate.

[0032] Figure 5 Rutin hydrate improves mouse embryo implantation and endometrial receptivity; wherein, A is BODIPY and FoxO1 staining of the control group, high-fat uterine animal model group, and different concentrations of rutin hydrate high-fat uterine animal model treatment group; B is the number of embryo implantation sites (blue dye labeling) in the uterine cavity of mice in each group; C is the uterine cavity closure of E4.5d mice in each group. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0034] Research conclusion 1: Lipid accumulation in endometrial epithelial cells of RIF patients Test purpose: Detection and analysis of abnormal accumulation of lipid droplets in endometrial tissue of RIF patients The present application evaluates lipid uptake by BODIPY staining. BODIPY™ 493 / 503 is a lipid fluorescent probe that can pass through the cell membrane into the interior of the cell, and specifically stains by locating on neutral lipids in the cell, so the dye can be used to label living cells and fixed cells, and to stain the lipids of the cells.

[0035] Test method: From November 2024 to October 2025, 30 patients with repeated embryo implantation failure (RIF) were included in the study (RIF group) in the China Welfare Foundation International Peace Maternal and Child Health Hospital.

[0036] Inclusion criteria: Patients who have experienced ≥3 high-quality embryo transfer (blastocyst score ≥3BB or cleavage stage embryo ≥7 cells II) and each time the endometrial thickness is ≥7mm, the morphology is normal (confirmed by ultrasound) and still have not obtained clinical pregnancy.

[0037] Exclusion criteria: Uncontrolled endocrine diseases (such as hyperthyroidism / hypothyroidism, uncontrolled diabetes); active genital tract infection or TORCH acute infection; antiphospholipid syndrome (APS) or other autoimmune diseases (such as systemic lupus erythematosus); known thrombotic disease; use of immunosuppressants or anticoagulant drugs within the past 3 months; other major diseases (heart, liver, and kidney failure, malignancy); women who do not understand or agree to participate in the study.

[0038] At the same time, 30 patients with male factor or tubal factor infertility but normal endometrial function were included as a control group (CTR group). This study was approved by the Human Research Ethics Committee of the China Welfare Foundation International Peace Maternal and Child Health Hospital (approval number: GKLW-A-2024-087-01; GKLW-A-2025-090-01), the research protocol followed the guidelines established by the Declaration of Helsinki, and written informed consent was obtained from all subjects.

[0039] Endometrial biopsy samples from the RIF and CTR groups were fixed in 4% paraformaldehyde, frozen sectioned after OCT embedding, and the thickness was 8 μm. After washing with PBS, BODIPY™ 493 / 503 was used for room temperature and light-free staining for 30 minutes, DAPI was used for nuclear staining, and after mounting, the samples were observed under a confocal microscope and images were collected. Figure 1 A). ImageJ software was used to analyze the ratio of EEC lipid droplet area to total cell area, and the difference between the RIF and CTR groups was statistically analyzed. Figure 1 B). R Studio software was used to draw the ROC curve to evaluate the predictive performance of lipid droplet accumulation for RIF. Figure 1C). The results show that EECs (endometrial epithelial cells) of RIF patients have excessive lipid accumulation, and lipid droplets have certain predictive efficiency for RIF.

[0040] Research conclusion 2: CD36 is highly expressed in endometrial epithelial cells of RIF patients, and CD36 can promote lipid uptake Test purpose: verification of the expression of CD36 in the endometrium of RIF patients and its relationship with lipid uptake Based on the analysis of the public single-cell RNA database (GSE183837), it was found that CD36 was significantly up-regulated in EECs of RIF patients (p < 0.05) Figure 2 A). CD36 immunofluorescence staining was performed on endometrial tissue sections (tissue sections were obtained as described above) Figure 2 B), the mean fluorescence intensity was analyzed and the ROC curve was drawn Figure 2 C). The results show that EECs (endometrial epithelial cells) of RIF patients have excessive lipid accumulation, and lipid droplets have certain predictive efficiency for RIF.

[0041] CD36 overexpression plasmid was constructed, and after EECs (endometrial epithelial cells) were infected for 72 hours, BODIPY staining was used to evaluate lipid uptake Figure 2 D). The results show that overexpression of CD36 in EECs can significantly promote lipid uptake.

[0042] Research conclusion 3: Rutin has high affinity with CD36 Test purpose: pharmacodynamic experiment verification of the binding of rutin and CD36 The terms involved in this study are explained as follows: XP GScore: It is an empirical scoring function in Schrodinger software used to evaluate the binding affinity of ligands and receptors. The lower the numerical value (in kcal / mol), the stronger the predicted binding ability, which takes into account factors such as hydrophobic interaction, hydrogen bonding, and steric hindrance.

[0043] MM-GBSA dG Bind: It is the binding free energy calculated by molecular mechanics / generaized Born surface area method (unit kcal / mol), the stronger the negative value, the more stable the binding, which reflects the energy balance of ligand-receptor complex in solvent environment.

[0044] RMSD: RMSD (Root Mean Square Deviation) is an index to measure the difference in atomic positions between a molecular structure (such as a protein or ligand) and a reference structure, usually in Å, the smaller the value, the higher the structure coincidence, which is often used to evaluate the stability of simulation or docking results.

[0045] SPR: is a kind of biosensor technology based on optical physics principle, used for real-time, label-free detection of interaction between biomolecules (such as protein and small molecule, antibody and antigen binding). When the molecular binding leads to the change of chip surface quality, the refractive index changes, and the SPR instrument can accurately determine the dissociation constant (KD) and other kinetic parameters of molecular binding by monitoring this change.

[0046] 1, CD36 protein virtual screening Rutin hydrate goes deep into the binding pocket of CD36 protein, and CD36 protein residues PHE379, PHE383, PHE266, etc. form hydrophobic force with Rutin hydrate. The ligand forms one hydrogen bond with each of the residues ASN53, LYS385, ASP209, and ASP250, two hydrogen bonds with the residue THR380, and one π-π bond with the residue PHE266. Figure 3 A).

[0047] 2, Kinetic simulation results of Rutin hydrate and CD36 protein Rutin hydrate forms hydrogen bonds directly with CD36 protein A chain residues ASN53 (83%), GLY58 (65%), THR380 (88%), THR59 (55%), ARG96 (109%), ASP250 (99%), and ASP209 (61%), forms a water bridge with residue GLN116 (53%) through a water molecule, and forms a π-cation bond directly with residue ARG96 (83%) Figure 3 B).

[0048] Rutin hydrate and CD36 protein were subjected to 100 ns MD simulation, and the molecular dynamics trajectory was analyzed Figure 3 C). As shown in the figure, the conformational stability of RMSD to simulation time, wherein smaller fluctuations indicate that all the complexes have obtained stable conformation, the results show that Rutin hydrate and CD36 protein are relatively stable after 25 ns, the system is in equilibrium state, and the RMSD values of protein and ligand are very small (<3 angstrom), indicating that the complex conformation changes little.

[0049] 3, Affinity determination of Rutin hydrate and CD36 The binding affinity constant of CD36 protein immobilized on CM5 chip and Rutin hydrate was measured by SPR technology analysis to be 2.13 µM Figure 3 D), which indicates that there is a strong specific interaction between them.

[0050] 4, Intervention of different concentrations of Rutin hydrate on EEC lipid uptake function Figure 3 E) Established lipid accumulation EECs model (100 mM oleic acid combined with 50 mM palmitic acid treatment for 24 hours), after intervention with different concentrations of rutin hydrate, it was found that 25 mM rutin hydrate could significantly inhibit lipid uptake by BODIPY staining.

[0051] Research conclusion 4: Rutin hydrate reduces EECs lipid uptake and improves receptivity Test method: Establish a lipid accumulation EECs model, and intervene with rutin hydrate (25 mM) in the treatment group (Lipid+Rutin hydrate), the Lipid group is the lipid accumulation EECs model group, and the CTR group is the conventional EECs. Use Bewo cells (human choriocarcinoma cells) to form embryonic spheres and co-culture with EECs monolayer, and detect 1 hour adhesion rate (A) and 24 hour flattening rate (B) respectively. The results suggest that compared with the Lipid group, rutin hydrate treatment significantly restores the adhesion rate and flattening rate of the embryonic spheres formed by Bewo cells in the high-fat EECs model. Figure 4 A) and 24 hours flattening rate (B). The results suggest that compared with the Lipid group, rutin hydrate treatment significantly restores the adhesion rate and flattening rate of the embryonic spheres formed by Bewo cells in the high-fat EECs model. Figure 4

[0052] Observe the lipid droplet accumulation and FoxO1 subcellular localization changes (C) by BODIPY staining and FoxO1 immunofluorescence co-staining. The results suggest that rutin hydrate effectively blocks lipid uptake and promotes FoxO1 expression and nuclear entry, allowing EECs to enter the receptive state. Figure 4

[0053] Research conclusion 5: Rutin hydrate improves embryo implantation in high-fat uterine animal models Test method: C57BL / 6 female mice were selected, and 300 mM tribromoethanol was injected intraperitoneally to anesthetize the mice. After making an incision on the abdomen of the mouse to remove the uterine horn, a microsyringe was used to inject 10 mM of a mixture of oleic acid and palmitic acid into the unilateral uterine cavity to construct an endometrial high-fat model, i.e. the Lipid group, and the other side was injected with the same volume of PBS as a control. After the mouse recovered from anesthesia, 20 mM of PBS was continuously perfused into the uterine cavity for three days as a control group, and rutin hydrate at low (10 mg / kg / d) and high (50 mg / kg / d) doses was used as a treatment group (n=8). The high and low dose treatment groups are referred to as Lipid+Rutin (10 mg / kg) and Lipid+Rutin (50 mg / kg) groups, respectively. Drug preparation: Rutin hydrate preparation uses physiological saline as a solvent, is sterilized by a 0.22 mM filter membrane, and is prepared and used immediately.

[0054] The above treated female mice were randomly mated with similar week C57BL / 6 male mice in the next estrus cycle, and the day when the plug was seen was recorded as 0.5 days of pregnancy. The uterus was taken on the 4.5th day of pregnancy (E4.5), OCT embedded to prepare frozen sections, and BODIPY / FoxO1 staining was used. Figure 5 ​​A). The results showed that high-dose rutin can significantly inhibit uterine lipid uptake and promote FoxO1 nuclear translocation of EECs. On the 5th day of pregnancy, the bed site was labeled by tail vein injection of Chicago blue dye, and the number of embryos was counted by dissection Figure 5 B), the results showed that high and low doses of rutin did not affect the pregnancy of the control side uterus, the number of embryos in the high-fat side uterus was significantly decreased, and high-dose rutin can improve the number of embryos in the high-fat side uterus. Hematoxylin-eosin (H&E) staining was used to observe the uterine cavity closure (H&E) Figure 5 C), the results suggest that the high-fat endometrium is thin and poorly closed, and high-dose rutin can significantly promote endometrial thickening and uterine cavity closure.

[0055] In summary, the present application verifies that rutin can inhibit abnormal lipid accumulation in uterine endothelial cells, promote FoxO1 nuclear translocation, enhance endometrial receptivity, and ultimately improve embryo implantation outcomes by targeting CD36, from clinical tissue verification, molecular mechanism research, cell function experiments to animal in vivo studies. The present application provides an innovative drug candidate with a clear mechanism for the prevention and treatment of RIF.

[0056] The above embodiments are only used to illustrate the technical solutions of the present application, and not to limit it. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements to some or all of the technical features, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of CD36 or an agent for detecting CD36 in the preparation of a product for predicting recurrent implantation failure, wherein the agent for detecting CD36 includes any agent required for detecting CD36 protein level or gene expression level by RT-PCR method, RT-qPCR method, biochip detection method, Southern blotting method, in situ hybridization method, Western blotting method, immunohistochemical method, immunofluorescence staining method, enzyme-linked immunosorbent assay, spatial transcriptome technology.

2. Use according to claim 1, characterized in that, The product includes reagents, kits, chips, test papers, membrane strips, models or detection platforms.

3. A model for predicting recurrent implantation failure, wherein the model is trained and verified by a machine learning model with the detection result of immunofluorescence staining of CD36 protein in endometrial tissue of a subject as a variable, and the model is constructed.

4. The model of claim 3, wherein, The model includes an information acquisition module and a diagnosis module; The information acquisition module is configured to perform the step of acquiring CD36 protein expression level information of endometrial tissue of a subject; The diagnosis module is configured to perform the step of determining whether the subject is a recurrent implantation failure patient according to the CD36 protein expression level information.

5. Use of a CD36 binding inhibitor in at least one of the following: 1) in the preparation of a drug for preventing and / or treating recurrent implantation failure; 2) in the preparation of a drug for improving endometrial receptivity; The CD36 binding inhibitor refers to a substance that can specifically bind to CD36 and inhibit the binding of CD36 to its receptor.

6. Use according to claim 5, characterized in that, The CD36 binding inhibitor is hydrous rutin, and the subject for administration of the hydrous rutin is a patient receiving in vitro fertilization-embryo transfer and suffering from recurrent implantation failure.

7. Use according to claim 6, characterized in that, The subject is a patient with unexplained recurrent implantation failure, i.e., excluding recurrent implantation failure caused by male factors or tubal factors or uterine ectopic pregnancy factors, and the subject has normal endometrial function.

8. Use according to claim 5, characterized in that, The drug has at least one of the following effects: 1) inhibiting lipid uptake of endometrial cells and reducing lipid accumulation; 2) promoting FoxO1 nuclear translocation and improving endometrial receptivity; 3) improving embryo adhesion and implantation capacity; 4) promoting endometrial thickening and uterine cavity closure.

9. Use according to any one of claims 5 to 8, characterized in that, The application includes administering a therapeutically effective amount of hydrous rutin or a pharmaceutical composition with hydrous rutin as an active molecule to the patient; the dosage form of the pharmaceutical composition includes oral preparations, injections, uterine cavity perfusion or local vaginal administration dosage forms.

10. Use according to claim 9, characterized in that, The application also includes administering hydrous rutin or a pharmaceutical composition with hydrous rutin as an active molecule in combination with other conventional agents or conventional treatment methods in the art.

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