A combined drug for improving embryo implantation efficiency of rif patients and a screening method

Abstract

The application provides a combined drug for improving embryo implantation efficiency of RIF patients and a screening method, and belongs to the technical field of embryo implantation. In order to solve the problem of how to screen a general drug combination on the basis of a large heterogeneous RIF population, the application locks 1119 kinds of drugs that may have a therapeutic effect in a large range through a six-step screening method combined with advanced RNA sequencing technology, and then further screens the drugs by using a highly simulated endometrial epithelial organoid and stromal cell model, tests the influence of the 1119 kinds of drugs on the activity of the endometrial epithelial organoid and stromal cell, and finally screens 7 kinds of effective drugs which can form a broad-spectrum drug with significant therapeutic effect on RIF in a combined form. The 7 kinds of effective drugs screened by the application are icotinib, fumaric acid propofol tenofovir tablets, clopidogrel, tizacat, osimertinib, venetoclax and rebastinib.

Description

Technical Field

[0001] This invention belongs to the field of embryo implantation technology, specifically relating to a combination drug and screening method for improving embryo implantation efficiency in RIF patients using a spectral approach. Background Technology

[0002] Human life begins with the successful implantation of the embryo in the mother's uterus—a biological milestone. This process showcases the precision and fragility of life's evolution: the fertilized egg does not move passively, but undergoes a series of strictly programmed cell divisions and differentiations within the fallopian tube, progressing through the morula stage, and finally forming a structurally prepared blastocyst on the fifth day after fertilization, which then enters the uterine cavity. At this time, the blastocyst and the mother's endometrium enter a brief window of mutual recognition and "dialogue." Around the seventh day after fertilization, the blastocyst breaks free of the zona pellucida and initiates its most crucial developmental process—first attaching to the pre-acceptable endometrial epithelium through trophoblast cells, and then invading the endometrial matrix through protease action, establishing a direct maternal-fetal blood circulation connection. This is not only the prelude to the birth of new life, but also a successful maternal-fetal dialogue, the absolute cornerstone for the establishment and maintenance of pregnancy.

[0003] However, this delicate initial step in life presents insurmountable physiological and emotional challenges for tens of millions of families worldwide. According to a recent large-scale epidemiological survey report led by Peking University Third Hospital, covering multiple provinces, the incidence of infertility among couples of childbearing age in my country has climbed to approximately 18%. This enormous number has evolved from a private health issue into a significant public health concern affecting family stability, social well-being, and population structure. Against this backdrop, in vitro fertilization-embryo transfer (IVF-ET), a milestone in modern reproductive medicine, has illuminated the path for countless families. It simulates the in vivo environment to complete gamete fertilization and early embryonic development, aiming to bypass some of the obstructive and functional barriers in natural conception. However, IVF-ET is far from the ultimate solution, and its technological bottlenecks are clearly visible: despite continuous optimization of embryo laboratory techniques, the global average embryo implantation rate still mainly hovers between 30% and 40%. This stark data reveals a harsh reality: of the embryos transferred back to the mother, more than half, even those with "high-quality" morphology, fail to continue their life cycle due to implantation failure. This highlights the high complexity of the implantation process and the limitations of current technology.

[0004] What troubles clinicians and patients even more deeply is a particularly challenging group embedded in this bottleneck—repeated implantation failure (RIF). They account for approximately 10% to 15% of the total population undergoing assisted reproductive technology (ART) and are a typical representative of "refractory infertility" in the field of reproductive medicine. RIF has a strict and operational definition in clinical practice: it usually refers to patients who have undergone 2 to 6 cycles of comprehensive ART, with a cumulative transfer of no fewer than 10 morphologically assessed "high-quality" embryos, or have experienced at least 3 different transfer cycles (each cycle with 1-2 high-quality embryos transferred), yet have consistently failed to achieve any clinical pregnancy (a gestational sac is visible on ultrasound), or have only experienced a brief biochemical pregnancy (a transient increase in serum hCG levels, indicating that the embryo attempted to "knock on the door" but failed to "enter the room" and thus failed).

[0005] For couples desiring children, recurrent reproductive tract infections (RIF) represent a recurring physical and psychological ordeal. It brings not only immense financial burdens but also profound emotional exhaustion and psychological trauma, plunging families into a cycle of hope and despair. Therefore, a deep dive into the multifactorial and heterogeneous causes of RIF—its roots potentially lying in genetic abnormalities of the embryo itself, spatiotemporal misalignments of endometrial receptivity, maternal immune intolerance disorders, or microscopic thrombosis—and a systematic improvement in embryo implantation efficiency for RIF patients, goes far beyond simply fulfilling individual fertility desires. It directly addresses the core blind spots and breakthroughs in current assisted reproductive technologies, representing an essential path from "empirical implantation" to "precision reproductive medicine." Furthermore, it is a strategically crucial link in reducing adverse pregnancy outcomes, ensuring long-term maternal and infant health, and ultimately improving the quality of the nation's newborn population, carrying immense clinical value and profound social significance.

[0006] Currently, recurrent implantation failure (RIF) has become a core challenge and a major obstacle in the field of assisted reproductive technology (ART). The root of this predicament lies in the fact that RIF is not a single disease, but a complex clinical syndrome resulting from the interplay of multiple factors, including those related to the embryo, endometrium, maternal immunity, and endocrine function. This high degree of heterogeneity, coupled with the lack of a unified and clear international "gold standard" for etiological classification and diagnosis, leaves clinicians navigating in a fog, lacking clear targets for the assessment and intervention of RIF, thus posing a significant challenge.

[0007] While current mainstream treatment strategies can be broadly categorized into four main areas—(i) uterine intervention (e.g., treating intrauterine lesions, improving endometrial blood flow); (ii) embryo laboratory optimization (e.g., blastocyst culture, PGT-A screening, modified culture medium); (iii) immunomodulatory therapy (e.g., using glucocorticoids, lymphocyte immunotherapy, immunosuppressants); and (iv) endometrial receptivity assessment and intervention (e.g., ERA testing to guide individualized implantation timing, using granulocyte colony-stimulating factor, etc.)—these strategies are inherently fragmented and exploratory in their application. Because it is impossible to precisely pinpoint the core failure point for each patient, treatment is significantly "blind" and "trial-and-error." Doctors often have to adopt an "experience-based" approach, trying or combining multiple potentially effective treatments for each patient. This not only increases the time and economic cost of treatment but also leads to extreme individualization of efficacy: the same treatment may be a "miracle" for some patients but completely ineffective for others. The overall lack of stable and predictable efficacy leaves both patients and doctors suffering in a cycle of hope and despair.

[0008] Therefore, despite the numerous existing methods, there is still a lack of a universally applicable and effective standard treatment pathway based on a clear etiological classification worldwide. Summary of the Invention

[0009] In view of the above-mentioned problems, and in order to overcome the shortcomings and deficiencies of the existing technology, this application proposes to perform transcriptome differential expression analysis on endometrial epithelial cells and stromal cells of RIF patients, and initially screen 1119 compounds from the MCE-HY-L022 drug library; these 1119 drugs were co-cultured with endometrial epithelial organoids (EEOs) and stromal cells obtained from in vitro culture of RIF patients, and the sum of cell activity of each drug in all groups and the effectiveness coverage of each drug were calculated. Taking both of these indicators into account, the drug effectiveness was ranked, and the following 7 effective drugs were selected: Icotinib, tenofovir alafenamide fumarate tablets, etc. The drugs included alafenamide, clopidogrel, tezacaftor, osimertinib, venetoclax, and ribociclib; an in vitro embryo implantation model of RIF patients was constructed to verify the implantation capability of the above seven drugs.

[0010] The technical solution adopted in this invention is as follows:

[0011] The first aspect of this invention protects a combination drug for improving embryo implantation efficiency in patients with recurrent embryonic fibrosis (RIF), the combination drug comprising:

[0012] Icotinib, tenofovir alafenamide fumarate tablets, clopidogrel, tezacotto, osimertinib, venetok, riboteninib.

[0013] Furthermore, the molar concentrations of icotinib, tenofovir alafenamide fumarate tablets, clopidogrel, tezacotto, osimertinib, venetoclax, and ribotetinib were all 10 µM.

[0014] A second aspect of this invention protects a method for screening combination drugs to improve embryo implantation efficiency in patients with recurrent embryonic fibrosis (RIF), the method comprising the following steps:

[0015] Step 1: Bulk RNAseq was performed on endometrial epithelial cells and stromal cells from 22 RIF patients. Through transcriptome differential expression analysis, 1119 drugs were initially screened in the MCE-HY-L022 drug library.

[0016] Step 2: Endometrial epithelial organoids and stromal cells were isolated and cultured from ex vivo endometrial tissue of RIF patients. The isolated and cultured endometrial epithelial organoids and stromal cells were used as experimental groups, and the 1119 drugs screened in Step 1 were used to treat the endometrial epithelial organoids and stromal cells respectively.

[0017] Step 3: Calculate the sum of cell activity of each drug across all experimental groups and the effectiveness coverage of each drug across all experimental groups. Rank the effectiveness of the drugs based on these two indicators and select 7 effective drugs.

[0018] Step 4: Preliminary verification: Endometrial epithelial organoids and stromal cells isolated and cultured from RIF patients were co-cultured with combinations of 7 effective drugs for 48 hours to observe the growth efficiency of endometrial epithelial organoids and stromal cells.

[0019] Step 5: Validation of in vitro embryo implantation model. An in vitro embryo implantation model of RIF patients was established. The in vitro embryo implantation model was treated with the 7 combination drugs screened in Step 3, and the blastocyst implantation efficiency was detected.

[0020] Step 6: Verify embryo implantation using human preimplantation blastocysts in an in vitro embryo implantation model of RIF patients.

[0021] Furthermore, the specific processing procedure for step 2 is as follows:

[0022] Endometrial epithelial organoids containing matrix gel or stromal cells were seeded into 96-well plates at a density of 2 µl per well or 3000 stromal cells per well. 1119 drugs were added to the 96-well plates, and each drug was repeated three times.

[0023] Each 96-well plate included a non-drug treatment control group and a blank control group; the non-drug treatment control group was supplemented with an equal amount of DMSO as the drug, while the blank control group contained only culture medium.

[0024] Furthermore, the specific processing procedure for step 3 is as follows:

[0025] Five days after treatment, the cell viability of endometrial epithelial organoids and stromal cells was detected using a CCK-8 assay kit.

[0026] The sum of cell activity of each drug in all experimental groups and the effectiveness coverage of each drug were calculated, and the top 7 effective drugs were screened.

[0027] Furthermore, 1119 drugs were added to the 96-well plate at a concentration of 10 µM.

[0028] Furthermore, the sum of cell activity for each drug across all experimental groups and the effectiveness coverage of each drug across all experimental groups were calculated. Based on these two indicators, the effectiveness of the drugs was ranked, and the seven most effective drugs were selected. The specific procedures are as follows:

[0029] Load the Excel file and parse each worksheet;

[0030] When processing data, groups with a fold change < 1 are recorded as having a fold change of 0.

[0031] Calculate the sum and ranking of the fold change values ​​of each drug in all experimental groups of endometrial epithelial organoids or stromal cells;

[0032] Calculate the number of coverage groups for each drug with a fold change greater than 1;

[0033] The sum of the fold change values ​​of each drug in the endometrial epithelial organoids or stromal cells of all experimental groups and the number of covered groups were combined and ranked with each group having a 50% weight. The top 7 drugs were selected as effective drugs.

[0034] Furthermore, the seven drugs screened included Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib.

[0035] Furthermore, the endometrial organoids and stromal cells of RIF patients were treated with a combination of seven effective drugs, and their growth changes were observed to confirm the promoting effect of this combination on the survival of endometrial organoids and stromal cells in RIF patients.

[0036] Furthermore, the process of establishing an in vitro embryo implantation model for RIF patients is as follows:

[0037] Fabrication of in vitro embryo implantation chips;

[0038] Artificial extracellular matrix was prepared using hydrogel and matrix gel. The artificial extracellular matrix was pre-prepared onto the in vitro embryo implantation chip through the gel / blastocyst loading window on the chip as a base layer and incubated at 37°C for 30 minutes.

[0039] 5 μl of hydrogel and matrix gel mixture was mixed with stromal cells and endometrial epithelial organoids and incubated at 37°C for 30 minutes to solidify the scaffold as an intermediate layer;

[0040] Coat the surface of the intermediate layer with a 1 μl 50% matrix gel and incubate at 37°C for 30 minutes.

[0041] After the matrix gel layer solidifies, fragmented endometrial epithelial organelles are laid onto the matrix gel layer through the gel / blastocyst loading window;

[0042] Endometrial epithelial organoid culture was performed by adding endometrial epithelial organoid culture medium through the culture medium reservoir.

[0043] Furthermore, the stromal cells and endometrial epithelial organoids used in the establishment of the in vitro embryo implantation model were all obtained by isolating and culturing endometrial tissue isolated from RIF patients.

[0044] Furthermore, after pretreating the in vitro embryo implantation model with a combination of 7 effective drugs for 3 days, blastocysts / blastocysts were added to the middle region of the in vitro embryo implantation model through the gel / blastocyst loading window, and the 7 effective drugs were continuously added during the co-culture period.

[0045] Beneficial effects:

[0046] This invention employs a highly efficient drug screening strategy based on an in vitro implantation model of recurrent embryo implantation failure with endometritis to ensure the reliability and functional relevance of the screening results. Transcriptome differential expression analysis of endometrial epithelial and stromal cells from RIF patients initially screened 1119 drugs from the MCE-HY-L022 drug library. These 1119 drugs were then co-cultured with endometrial epithelial organoids and stromal cells obtained from in vitro culture of RIF patients. The sum of cell viability and efficacy coverage of each drug across all groups were calculated. Taking both indicators into account, the drugs were ranked for efficacy, and the following 7 effective drugs were selected: Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib. Two validation trials were then conducted: initial validation was performed by co-culturing endometrial epithelial organoids and stromal cells from in vitro culture of RIF patients with the 7 effective drugs; subsequently, an in vitro embryo implantation model of RIF patients was constructed to verify the implantation ability of the 7 effective drugs on blastocysts / blastocysts. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0048] Figure 1 A heatmap of gene expression from the bulk RNAseq transcriptome of 22 RIF patients.

[0049] Figure 2 This is a drug library type.

[0050] Figure 3 The selected drug type from the drug library.

[0051] Figure 4 This is a diagram from the second round of drug screening experiments.

[0052] Figure 5 Representative bright-field images of the seven selected combination drugs applied to endometrial epithelial organoids and stromal cells in five RIF patients for 48 hours.

[0053] Figure 6 This is a schematic diagram of a 3D implanted chip.

[0054] Figure 7 Image of a 3D implanted chip.

[0055] Figure 8 A schematic diagram of the generation of a blastocyst-endometrial epithelial organoid implantation model.

[0056] Figure 9 This is a representative immunofluorescence (IF) image of endometrial epithelial organoids, showing the epithelial cell markers CK7 and KI67.

[0057] Figure 10 Representative IF images of mesenchymal cells show the mesenchymal cell markers N-Cadherin and Vimentin.

[0058] Figure 11 This is a representative IF image of the downstream gene IHH of endometrial epithelial organoids during the proliferative phase after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0059] Figure 12 This is a representative IF image of HSD17β2, a downstream gene of endometrial epithelial organoids during the secretory phase, after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0060] Figure 13 This is a representative IF image of SPP1, a downstream gene of endometrial epithelial organoids during the secretory phase, after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0061] Figure 14 This is a QPCR for the expression of downstream genes of endometrial epithelial organoids during the proliferative or secretory phases after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0062] Figure 15 This is a representative IF image of the translocation of the progesterone receptor (PGR) from the cell membrane to the cell nucleus during the secretory phase of endometrial epithelial organoids after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0063] Figure 16 This is a representative IF image showing the morphological changes of secretory cells after decidualization following co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0064] Figure 17 qPCR was used to express the secretory genes PRL and IGFBP1 in stromal cells after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0065] Figure 18CCK-8 OD values ​​represent the proliferation of endometrial epithelial organoids in the proliferative or secretory phase after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0066] Figure 19 The CCK-8 OD value represents the proliferation of mesenchymal cells in the proliferative or secretory phase after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0067] Figure 20 This is a cluster diagram of endometrial receptivity gene expression in endometrial organoids and stromal cells during the proliferative or secretory phases after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0068] Figure 21 Hardness tests were conducted on three different base adhesives.

[0069] Figure 22 Representative IF images of the unfolding of three different matrix glial intermediate cells.

[0070] Figure 23 A statistical graph showing the unfolded area of ​​three different matrix glial intermediate cells.

[0071] Figure 24 Representative IF images showing the unfolding of mesenchymal cells under different mixing ratios of matrix gel and hydrogel.

[0072] Figure 25 A statistical graph showing the proportion of mesenchymal cells unfolded under different mixing ratios of matrix gel and hydrogel.

[0073] Figure 26 Hardness tests were conducted for different mixing ratios of the matrix adhesive and hydrogel.

[0074] Figure 27 Representative IF images showing the development of endometrial epithelial organoid fragments in different proportions of Matrices.

[0075] Figure 28 Representative IF images of a 3D radiograph of an endometrial model; E-Cad: glandular epithelium (GE) / luminal epithelium (LE), VIM: stromal cells.

[0076] Figure 29 Representative IF images of a real uterus and endometrial model; E-Cad: glandular epithelium (GE) / luminal epithelium (LE), VIM: stromal cells.

[0077] Figure 30 Representative IF images of luminal epithelium (LE:LGR5) and glandular epithelium (GE:PAEP).

[0078] Figure 31 A statistical graph showing the differences in blastocyst (Q-tracker655 labeled) adhesion rate (24 hours) and extended implantation rate (48 hours) between normal and RIF patients on the implantation model.

[0079] Figure 32 Representative bright-field plots showing the implantation effects of the seven selected combination drugs in five RIF patient implantation models.

[0080] Figure 33 The results show the implantation efficiency of the seven selected combination drugs before and after treatment in five RIF patient implantation models. Ctrl: Implantation efficiency in normal individuals.

[0081] Figure 34 Immunofluorescence staining images of the development of the seven selected combination drugs after implantation in a RIF patient implantation model. Intraembryonic structures: pseudo-amniotic cavity (OCT4+), pseudo-yolk sac cavity (GATA6+), mesenchymal cells (VIM+); Extraembryonic structures: chorionic trophoblastic ectoderm (HLA-G+), syncytiotrophoblastic ectoderm (hCG+).

[0082] Figure 35 Bright field image showing the implantation of human blastocysts in an endometrial model composed of the endometrium of five RIF patients treated with a combination of seven drugs. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0084] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0085] This embodiment specifically discloses a method for screening combination drugs to improve embryo implantation efficiency in patients with recurrent embryonic fibrosis (RIF). Although various RIF treatments are currently used clinically, their effectiveness varies greatly due to the heterogeneity of RIF patients, resulting in limited overall results. In this study, high-throughput testing of thousands of drugs was conducted on multiple patients using suitable in vitro models. A set of drug combinations applicable across multiple patients was discovered.

[0086] The following is the specific drug screening process:

[0087] Step 1: Bulk RNAseq was performed on endometrial epithelial cells and stromal cells from 22 RIF patients. Through transcriptome differential expression analysis, 1119 drugs were initially screened in the MCE-HY-L022 drug library.

[0088] Step 2: Endometrial epithelial organoids and stromal cells were isolated and cultured from ex vivo endometrial tissue of RIF patients, and the endometrial epithelial organoids and stromal cells were treated with 1119 drugs screened in step 2.

[0089] Step 3: Calculate the sum of cell activity for each drug and the coverage of each drug's effectiveness. Rank the drugs based on these two indicators and select 7 effective drugs.

[0090] Step 4: Preliminary verification: Endometrial epithelial organoids and stromal cells isolated and cultured from other RIF patients were co-cultured with a combination of 7 effective drugs for 48 hours to observe the growth efficiency of endometrial epithelial organoids and stromal cells.

[0091] Step 5: Validation of in vitro embryo implantation model. An in vitro embryo implantation model of RIF patients was established. The in vitro embryo implantation model was treated with the 7 combination drugs screened in Step 3, and the blastocyst implantation efficiency was detected.

[0092] Step 6: Use human preimplantation blastocysts in an in vitro embryo implantation model of RIF patients to verify the embryo implantation status after drug treatment of the implantation model of RIF patients.

[0093] Round I: Screening the drug library and drugs, the specific steps are as follows:

[0094] First, bulk RNAseq transcriptome sequencing was performed on endometrial epithelial cells and stromal cells from 22 patients with recurrent uterine fibroids (RIF). Differentially expressed genes were identified through transcriptome analysis. Figure 1 Analysis revealed that endometrial epithelial cells and stromal cells in RIF patients exhibited phenotypic expression patterns, with distinct expression levels between the two groups. This further demonstrates the high consistency of RIF patients at the transcriptional level. The upregulated genes in the endometrial epithelial cells of these RIF patients are associated with the following pathways: toxicity tolerance, humoral immune response, and defense against bacterial infection; the upregulated genes in the stromal cells are associated with the following pathways: cell polarity, tight junctions, and ubiquitin ligase activity.

[0095] Based on the results of transcriptome differential gene enrichment analysis, we compared the results with a library of FDA-approved drugs ( Figure 2This included various drug types, mechanisms of action, and pathways of action, ultimately selecting the MCE-HY-L022 drug library as the initial target library for drug screening. These drug libraries offer the following advantages: these drugs have completed extensive preclinical and clinical studies, possessing good biological activity, safety, and pharmacokinetic properties, shortening the development cycle and reducing development costs. Through comparison, we initially screened 1119 drugs from the MCE-HY-L022 drug library, as shown in Tables 1-14 below.

[0096] Table 1. Drug names on the first shelf of the drug database (out of 14 shelves):

[0097] ;

[0098] Table 2. Drug names on the second panel of the drug database:

[0099] ;

[0100] Table 3. Drug names on the third board of the drug database:

[0101] ;

[0102] Table 4. Drug names on the fourth panel of the drug database:

[0103] ;

[0104] Table 5. Drug names on the fifth panel of the drug database:

[0105] ;

[0106] Table 6. Drug names on the sixth panel of the drug database:

[0107] ;

[0108] Table 7. Drug Names on the Seventh Board of the Drug Database:

[0109] ;

[0110] Table 8. Drug Names on the Eighth Board of the Drug Library:

[0111] ;

[0112] Table 9. Drug Names on the Ninth Board of the Drug Library:

[0113] ;

[0114] Table 10. Drug names on the tenth panel of the drug library:

[0115] ;

[0116] Table 11. Drug names on the eleventh panel of the drug database:

[0117] ;

[0118] Table 12. Drug names on the twelfth shelf of the drug database:

[0119] ;

[0120] Table 13. Drug names on the thirteenth shelf of the drug database:

[0121] ;

[0122] Table 14. Drug names on the fourteenth panel of the drug database:

[0123] ;

[0124] These drugs are associated with the following pathways, such as Figure 3 As shown, the distribution of targets and mechanisms of action in the MCE-HY-L022 drug library mainly includes:

[0125] 1. Pathogen-related targets: anti-infective drugs such as Bacterial, Antibiotic, Fungal, Parasite, SARS-CoV, HIV, HBV, and Influenza Virus are used for antibacterial, antiviral, antifungal, and antiparasitic research.

[0126] 2. Signaling Pathways and Cellular Processes: NF-κB, JAK, MAPK, ERK, PI3K, Akt, AMPK, MEK, mTOR, STAT, HIF / HIF Prolyl Hydroxylase, involved in core signaling pathways such as cell growth, metabolism, immune response, hypoxia, and stress. Commonly used in research on cancer, metabolic disorders, inflammation, autophagy, and apoptosis.

[0127] 3. Programmed death and metabolism: Apoptosis, Autophagy, Ferroptosis, Mitochondrial metabolism, Reactive Oxygen Species (ROS).

[0128] 4. Nervous system targets: 5-HT Receptor, Dopamine Receptor, GABA Receptor, Adrenergic Receptor, mAChR, Histamine Receptor, Opioid Receptor, Adenosine Receptor, covering key receptors in the central nervous system, are important targets for research on psychotropic drugs and neuroprotective agents.

[0129] 5. Ion channels and physiological regulation: Calcium Channel, Potassium Channel, Sodium Channel, TRP Channel, corresponding to cardiovascular, electrophysiological, and pain transmission studies; COX, PDE, HMG-CoA Reductase, HDAC, Carbonic Anhydrase, involving classic targets such as inflammation, blood lipids, and enzyme inhibitors.

[0130] 6. Cancer and Signal Transduction Targets: Bcr-Abl, FGFR, c-Kit, c-Met / HGFR, PDGFR, EGFR, VEGFR, CDK, MEK, ERK, etc., are involved in tumor signaling pathways and proliferation regulation, and include various clinical anticancer drugs and investigational inhibitors. HDAC, Topoisomerase, DNA Alkylator, Antifolate, are involved in DNA damage repair and epigenetic regulation.

[0131] 7. Nuclear receptors and metabolism-related: PPAR, Progesterone Receptor, Androgen Receptor, Estrogen Receptor, Glucocorticoid Receptor, corresponding to hormone regulation and metabolic syndrome research; P-glycoprotein, Cytochrome P450, NO Synthase, involved in drug metabolism and drug resistance mechanisms.

[0132] Round II: Screening for effective drugs in endometrial epithelial organoids and stromal cells, with the following specific steps:

[0133] 1. Culture of human endometrial epithelial organoids and stromal cells:

[0134] Endometrial tissue biopsies were collected from 22 patients with recurrent intrauterine febrile fibrosis (RIF) during the proliferative phase of the menstrual cycle (days 3–7 after menstruation, i.e., days 7–13 of the menstrual cycle). Endometrial epithelial organoids and stromal cells were isolated and cultured, resulting in a total of 44 experimental groups.

[0135] Endometrial tissue was washed with DMEM / F12 culture medium and then digested at 37°C with a mixture of Dispass and collagenase for 40 minutes. Digestion was terminated with DMEM / F12 containing 10% FBS. The digestion solution was separated through a 40 μm filter to obtain endometrial epithelial cells (greater than 40 μm) and stromal cells (less than 40 μm).

[0136] The obtained stromal cells were resuspended in culture medium and seeded into culture dishes, and cultured at 37°C and 5% CO2. After standing for 15 minutes, the supernatant was discarded, and fresh culture medium was added for further culture to obtain adherent endometrial stromal cells. Endometrial epithelial cells were resuspended in matrix gel (Corning 356231), and seeded in culture dishes at 20 μl per dish. After standing at 37°C for 15 minutes to allow the gel to solidify, endometrial epithelial organoid culture medium was added for further culture to form endometrial epithelial organoids with a three-dimensional lumen-like structure.

[0137] The required culture medium is as follows:

[0138] Mesenchymal cell culture medium: DMEM / F-12 + 10% FBS + 1% anti-anti;

[0139] Endometrial epithelial organoid culture medium: Advanced DMEM / F-12 + 1x N2 supplement + 1x B27 supplement - Vitamin A + 1x ITS-X + 100 μg / ml Primocin + 1.25 mM N-acetyl-L-cysteine ​​+ 2 mM L-glutamine + 50 ng / ml recombinant human EGF + 100 ng / ml recombinant human Noggin + 500 ng / ml recombinant human Rspondin-1 + 100 ng / ml recombinant human FGF-10 + 50 ng / ml recombinant human HGF + 50 nM ALK-4, -5, -7 inhibitor, A83-01 + 10 nM nicotinamide.

[0140] 2. 1119 drugs for treating endometrial epithelial organoids and stromal cells:

[0141] Endometrial epithelial organoids containing Matrigel were seeded into 96-well plates at a density of 2 µl per well / 3000 stromal cells per well. Then, 1119 drugs (final drug concentration 10 µM) were added to the 96-well plates. Each drug was tested in triplicate. Each plate included a 0-drug control (non-drug treatment control group) with DMSO as the drug solvent and a blank control containing only culture medium (no cells and no drugs). Figure 4 As shown.

[0142] 3. CCK-8 kit (cell counting kit-8) for detecting endometrial cell viability:

[0143] Patients with recurrent fibrosis (RIF) have low endometrial cell viability and proliferation capacity. Therefore, our criterion for evaluating drug efficacy was whether endometrial cell viability was increased compared to the control group without medication. Five days after drug treatment, endometrial cell viability was assessed using a CCK-8 assay kit. The core substrate of CCK-8 is the water-soluble tetrazolium salt WST-8. Viable cells can reduce WST-8 to an orange, water-soluble substance—formazan—and its production is approximately proportional to the intensity of cellular metabolic activity. The absorbance (OD value) of each well was then read at the visible light absorption peak (450 nm) using a microplate reader. The OD value represents cell viability, thus assessing the drug's effectiveness.

[0144] The OD values ​​of each experimental group, the control group with no drug, and the blank control group were exported. The method for calculating cell viability is shown in the figure below. A ratio greater than 1 indicates that cell viability has improved after drug administration.

[0145] Cell viability (%) = [A (drug-treated) - A (blank)] / [A (0-drug-treated) - A (blank)] × 100

[0146] A (Drug Addition): Absorbance of the pores containing cells, CCK solution, and drug solution.

[0147] A (Blank): Absorbance of wells containing culture medium and CCK solution but without cells.

[0148] A (0 drug added): Absorbance of the pore containing cells and CCK solution but no drug solution.

[0149] Cell activity: Cell proliferation activity or cytotoxic activity:

[0150] The above formula can be simplified to: fold change = (OD_treated - OD_blank) / (OD_control_mean - OD_blank); where OD_treated represents A (medication added); OD_blank represents A (blank); and OD_control_mean represents A (0 medication added).

[0151] Round III: Effective drugs are selected using algorithms. The specific steps are as follows:

[0152] An algorithm was developed to automatically read and calculate the sum of cell viability for each drug across all groups, as well as the effectiveness coverage of each drug. Based on the results of step two, the drugs were ranked for effectiveness, taking into account both indicators, and the following seven effective drugs were selected: Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib. These seven effective drugs had the highest sum of cell viability across all groups, indicating that they best improved endometrial cell viability. They also covered the most combinations with viability values ​​greater than 1, indicating that they exerted a broad effect on the epithelial and stromal cells of various RIF patients.

[0153] The basic process of the algorithm is as follows:

[0154] 1. Load the Excel file and parse each worksheet;

[0155] 2. Process the data; groups with a fold change < 1 are recorded as having a fold change of 0.

[0156] 3. Calculate the sum and ranking of the fold change values ​​of each drug for endometrial epithelial organoids or stromal cells across all experimental groups;

[0157] 4. Calculate the number of coverage groups where each drug has a fold change > 1;

[0158] 5. Based on the sum of the foldchange values ​​of each drug in the endometrial epithelial organoids or stromal cells of different experimental groups and the number of covered groups, the drugs were ranked with a weight of 50% each, and the top 7 drugs were selected.

[0159] The corresponding code for the above algorithm is as follows:

[0160] import pandas as pd

[0161] import numpy as np

[0162] import matplotlib.pyplot as plt

[0163] # Step 1: Load the Excel file and parse each worksheet

[0164] file_path = 'C: / Users / recordarse / Desktop / pythonlearning / data / lq.xlsx' # Path to the Excel file

[0165] data = pd.ExcelFile(file_path) # Load the Excel file

[0166] sheets_data = {sheet: data.parse(sheet) for sheet in data.sheet_names} # Parse each worksheet

[0167] # Step 2: Process the data and filter drugs with fold change > 1

[0168] combined_matrix = pd.DataFrame() # Used to store the processed matrix

[0169] original_combined_matrix = pd.DataFrame() # Used to store the original fold change value

[0170] for sheet_name, df in sheets_data.items():

[0171] If 'drug name' is in df.columns and 'fold change' is in df.columns:

[0172] df = df.set_index('drug name') # Set the drug name as the index

[0173] filtered_df = df['fold change'].apply(lambda x: x if x>1 else 0) # Set the value of fold change<= 1 to 0

[0174] combined_matrix[sheet_name] = filtered_df # Save the processed data

[0175] original_combined_matrix[sheet_name] = df['fold change'] # Save the original data

[0176] # Fill in missing values ​​of 0 (indicating drugs not present in some patient groups)

[0177] combined_matrix = combined_matrix.fillna(0)

[0178] original_combined_matrix = original_combined_matrix.fillna(0)

[0179] # Step 3: Calculate the total fold change value and ranking

[0180] combined_matrix['Total Fold Change'] = combined_matrix.sum(axis=1) # Calculate the total fold change value for each drug

[0181] combined_matrix['Rank'] = combined_matrix['Total Fold Change'].rank(ascending=False, method='min') # Rank by total value

[0182] # Step 4: Calculate the ranking of each group. Groups with a fold change of 0 are ranked as 0.

[0183] group_ranks = pd.DataFrame(index=combined_matrix.index)

[0184] for sheet_name in combined_matrix.columns[:-2]: # Ignore Total FoldChange and Rank columns

[0185] group_ranks[sheet_name] = combined_matrix[sheet_name].rank(ascending=False, method='min')

[0186] group_ranks[sheet_name] = group_ranks[sheet_name].where(combined_matrix[sheet_name]>0, 0) # Set the rank of sheets with a fold change of 0 to 0.

[0187] # Step 5: Generate Output

[0188] output_df = combined_matrix[['Total Fold Change', 'Rank']].copy()

[0189] output_df['Covered Groups'] = combined_matrix.iloc[:,:-2].astype(bool).sum(axis=1) # Calculate the number of covered groups

[0190] output_df['Group Fold Changes'] = combined_matrix.iloc[:, :-2].apply(lambda row: row[row>0].to_dict(), axis=1) # Record the fold change values ​​for each group

[0191] output_df['Group Ranks'] = group_ranks.apply(lambda row: row[row>0].to_dict(), axis=1) # Record the rankings of each group

[0192] # Save to CSV file

[0193] output_file = "Drug_plan1_veison3.csv"

[0194] output_df.to_csv(output_file, index=True)

[0195] print(f"Total drug fold change value and ranking have been saved to: {output_file}")

[0196] The targets, mechanisms, uses, and related drug / metabolic characteristics of the seven effective drugs selected by the above algorithm are shown in Table 15.

[0197] Table 15. Targets, mechanisms of action, uses, and related drug / metabolic characteristics of 7 drugs:

[0198] ;

[0199] Round IV: Validating the effect of a combination of 7 effective drugs on endometrial cells in patients with recurrent uterine fibrosis (RIF). The specific procedures are as follows:

[0200] Based on the patient transcriptome heatmap obtained from previous bulk-RNAseq analysis, one patient from each expression category was selected, totaling five patients, for efficacy validation of the seven effective drugs. Each drug was mixed at 10 μM to form a mixture of the seven effective drugs, which was then used to treat endometrial organoids and stromal cells of RIF patients (the isolation and culture methods for endometrial organoids and stromal cells were as described in step one). Proliferation and apoptosis of endometrial organoids and stromal cells of RIF patients were monitored after 48 hours. Figure 5 As shown, the addition of seven effective drugs greatly improves the growth efficiency of endometrial epithelial organoids and stromal cells.

[0201] Round V: Verify the effects of seven effective drugs on the implantation of blastocysts in an endometrial model. The specific procedures are as follows:

[0202] 1. The method for isolating and culturing endometrial epithelial organoids and their stromal cells is as described in step one.

[0203] 2. Culture of blastocysts:

[0204] 2.1 Culture of human pluripotent stem cells (hPSCs) in a naïve state:

[0205] Initiating cells: Primed WIBR3 and RUES2 hESCs;

[0206] Culture medium: Initially cultured in mTeSR Plus, then replaced with modified 5i / L / A or PXGL medium to reset to naïve state;

[0207] Culture conditions: adhered to inactivated mouse fibroblasts (MEF), 37°C, 5% O2, 5% CO2;

[0208] Passage: Using Accutase;

[0209] 5i / L / A formulation:

[0210] DMEM / F12: Neurobasal 1:1, N2, B27, GlutaMAX, non-essential amino acids, β-mercaptoethanol, 0.5% KSR, antibiotics;

[0211] Small molecules / cytokines: PD0325901, IM-12, SB590885, WH-4-023, LIF, Activin A, Y-27632;

[0212] PXGL formulation:

[0213] DMEM / F12:Neurobasal 1:1, half-strength N2 / B27, GlutaMAX, β-mercaptoethanol, antibiotics;

[0214] Small molecules / cytokines: PD0325901, LIF, XAV939, Gö6983, Y-27632;

[0215] 2.2 Thylakoid formation:

[0216] Naïve hESCs (gifted free of charge by Ali H. Brivanlou's laboratory) were single-celled (Accutase, 37°C, 3 minutes);

[0217] MEF removal: 0.1% gelatin-coated petri dishes at 37°C for 30 minutes;

[0218] hPSCs were collected and filtered to 40 μm.

[0219] Cells were resuspended in BFM-I (5i / L / A + CEPT cocktail, without Y-27632 and BSA);

[0220] AggreWell-400 pretreatment: Add anti-adhesion solution, centrifuge at 1900 g for 5 minutes, and incubate at room temperature for ≥30 minutes;

[0221] Cultivation: 100,000 cells / well, centrifuged at 100 g for 3 minutes, 37°C, 5% O2, 5% CO2;

[0222] Training process:

[0223] Day 0: Vaccination;

[0224] Day 1: Replace BFM-I with BFM-II (half volume, two replacements each time);

[0225] Days 2-6: Replace BFM-II with half the dose daily;

[0226] BFM-I Formula: DMEM / F12: Neurobasal, 0.25× N2, 0.25× B27, 0.5× GlutaMAX, 0.5× Non-essential amino acids, 0.1 mM β-mercaptoethanol, 0.5% KSR, 0.5% P / S, 1 μM PD0325901, 0.5 mM MIM-12, 0.5 μM SB590885, 1 μM WH-4-023, 20 ng / ml recombinant human LIF, 10 ng / ml Activin A, Chroman 1, 5 μM Emricasan, 1x polyamine supplement, 0.7 μM TransISRIB.

[0227] BFM-II formulation: DMEM / F12:Neurobasal 3:1, 0.25x N2 / B27, 1 mM GlutaMAX, 0.5x non-essential amino acids, β-mercaptoethanol, 0.5% KSR, 1 μM PD0325901, 2 μM A83-01, 0.5 μM SB590885, 1 μM WH-4-023, 10 ng / ml LIF, 0.5 μM LPA, CEPT cocktail.

[0228] 3. Design and fabrication of in vitro embryo implantation microarrays:

[0229] like Figure 6 As shown, the in vitro embryo implantation chip (microfluidic chip) has a parallel three-channel structure: the middle channel is a gel / blastocyst loading channel, and the left and right sides are culture medium channels (medium channels); the size of the middle channel is 6 mm long × 1.5 mm wide × 5 mm deep.

[0230] The central channel is connected to the other two channels, and a 0.1 mm long barrier branch at the top of the chip separates the central channel from the bilateral channels, allowing the gel to be confined within the central space by surface tension. This design separates the endometrial epithelial organoid culture medium within the side channels from the implantation culture medium within the top window.

[0231] The left and right channels are used to separate endometrial epithelial organoid culture medium and implantation culture medium.

[0232] The mold for the microfluidic chip was designed in AutoCAD and imported into Fusion 360 to generate a CNC machining program. A plastic mold was then generated using micro-CNC. PDMS curing agent and base polymer were mixed at a 1:10 ratio and poured onto the mold to produce a polydimethylsiloxane (PDMS, Dow Corning, 01673921)-based microfluidic chip. The PDMS mold was vacuum-treated for 4 hours and baked at 65°C for 12 hours. PDMS chips were removed, and holes were punched at predetermined locations. The bottom of the chip and a clean cover glass were treated with plasma at 150W for 3 minutes, then the bottom and the activated cover glass surface were tightly adhered for 1 minute, and baked at 65°C for 12 hours. The chip was applied within 1-7 days after UV irradiation. A schematic diagram of the fabricated microfluidic chip structure is shown below. Figure 4 As shown in the image, the microfluidic chip is as follows: Figure 7 As shown.

[0233] 4. Preparation of in vitro embryo implantation model:

[0234] 4.1 Construction of the endometrial model (e.g.) Figure 8 (as shown)

[0235] Embryo implantation occurs in the endometrium, involving a single layer of luminal epithelial cells (LE), glandular epithelium (GE) embedded among stromal cells, stromal cells, blood vessels, and immune cells. An endometrial model was established combining epithelial and stromal cells obtained from endometrial biopsies of fertile women. Epithelial cells were cultured in streptavidin to form endometrial epithelial organoids capable of differentiating into LE and GE; while stromal cells were maintained in a monolayer culture. Endometrial epithelial organoids expressed CK7 and E-cadherin (E-CAD) markers. Figure 9 ), while mesenchymal cells showed positive staining for Vimentin (VIM) and N-cadherin (N-CAD). Figure 10 Both cell types were in a proliferative state, which was confirmed by KI67 and PCNA staining. Figure 9 and Figure 10 ).

[0236] Normal implantation occurs during the "implantation window," corresponding to the mid-secretory phase of the menstrual cycle. To simulate this phase in vitro, endometrial epithelial organoids and stromal cells were treated with estrogen (E2) alone to simulate the proliferative phase, and E2, progesterone (P4), and cyclic adenosine monophosphate (cAMP) were added during the secretory phase. Following hormone treatment, endometrial epithelial organoids significantly upregulated E2-responsive genes, including estrogen receptor (ESR), Indian hedgehog signaling molecule (IHH), and olfactomedin 4 (OLFM4), while simultaneously upregulating P4-responsive genes such as progesterone receptor (PGR), 17β-hydroxysteroid dehydrogenase (HSD17β2), secretory phosphoprotein 1 (SPP1), and paired-box gene 8 (PAX8). These results confirm that endometrial epithelial organoids can simulate the hormonal responses of the menstrual cycle in vitro. Figures 11-14 Notably, PGRs shift from membrane localization to the nucleus in endometrial epithelial organoids, marking the beginning of the P4-dominated secretory phase. Figure 15 ).

[0237] Interstitial cells respond to E2, P4, and cAMP, undergoing decidualization and transforming from elongated to round shapes. Figure 16 It also upregulated markers such as prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1), indicating that it had entered the implantation receptive state. Figure 17 Hormone treatment also enhanced the proliferative capacity of endometrial epithelial organoids and stromal cells. Figure 18 and Figure 19 Furthermore, endometrial receptivity assay (ERA) identified 248 genes related to endometrial receptivity using next-generation sequencing, verifying that endometrial cells treated with E2, P4, and cAMP had entered a receptive state. Figure 20 ).

[0238] To optimize the extracellular matrix (ECM) for both endometrial cell spread and blastocyst invasion, we tested three commercially available Matrigel variants with different stiffnesses. The results showed that Matrigel 354277 was best suited for the spread and growth of stromal cells in a three-dimensional environment (hereinafter referred to as Matrigel). Figures 21-23 However, when used alone, the matrix gel did not facilitate blastocyst invasion, suggesting the need for further optimization of the ECM.

[0239] During implantation, trophoblast cells infiltrate the endometrium by secreting matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM). To better reproduce this process in our model, we combined an MMP-degradable hydrogel with matrix gel. We tested different dilutions and mixing ratios to determine the optimal combination that supports the three-dimensional growth of stromal cells and glandular epithelial (GE) cells within the gel while allowing luminal epithelial (LE) cells to form a monolayer on the surface. The results showed that the optimal mixing ratio was 80% degradable hydrogel (1:3 dilution) with 20% matrix gel, a combination that supported the spread of stromal cells in a three-dimensional environment without aggregation. Figure 24 and Figure 25 The Young's modulus of this artificial extracellular matrix is ​​approximately 184 Pa, which closely matches the stiffness of uterine tissue in vivo (171–250 Pa). Figure 26 For the amplification of endometrial epithelial organoids on the surface to form LE, a 50% Matrigel concentration is most effective. Figure 27 ).

[0240] To construct an endometrial model in a microfluidic chip, an artificial extracellular matrix was first prepared by mixing 1:3 (v / v) hydrogel (THE WELLBIOSCIENCE, TWG010) + 20% matrix gel (Corning, 354277) (v / v). 2 μl of this matrix was pre-coated onto the chip as a base layer through the gel / blastocyst loading wells and incubated at 37°C for 30 minutes. Another 5 μl mixture of 1:3 hydrogel (25%) + 20% matrix gel was then mixed with 6.5 x 10 μL of matrix gel. 3 Interstitial cells (normal individuals) and 2x10 3 Endometrial epithelial organoids (from normal individuals) were mixed and incubated at 37°C for 30 minutes to solidify the scaffold, serving as the central portion. A 1 μl layer of 50% matrix gel was coated onto the central surface of the microfluidic chip through the gel / blastocyst loading window, and the microfluidic chip was placed in a 37°C incubator for 30 minutes. After the matrix gel layer solidified, fragmented endometrial epithelial organoids (approximately 3 × 10⁻⁶) were loaded through the gel / blastocyst loading window. 3 (Normal individuals) were seeded onto 50% Matrigel. EXM + 10% FBS (Endometrial Epithelial Organoid Culture Medium) was added through the culture reservoir for endometrial-like cell culture. Stromal cells and glands expanded within one day, and the luminal epithelium formed a monolayer within two days. The resulting endometrial model was structurally similar to the in vivo endometrium, containing luminal epithelial cells (E-CAD). + LGR5 + ), glandular cells (E-CAD) + PAEP + ) and interstitial cells (VIM) + ), Figures 28-30 .

[0241] 4.2 RIF-type in vitro embryo implantation model:

[0242] The endometrial model established in 4.1 is structurally similar to the in vivo endometrium, and in turn, an in vitro embryo implantation model conforming to the RIF type is established.

[0243] First, an artificial extracellular matrix was prepared by mixing 1:3 (v / v) hydrogel (THE WELL BIOSCIENCE, TWG010) + 20% matrix gel (Corning, 354277) (v / v). 2 μl of this matrix was pre-coated onto the chip as a base layer through the gel / blastocyst loading wells and incubated at 37°C for 30 minutes. Another 5 μl mixture of 1:3 hydrogel (25%) + 20% matrix gel was then mixed with 6.5 x 10... 3 Interstitial cells (RIF) and 2x10 3 Endometrial epithelial organoids (RIFs) were mixed and incubated at 37°C for 30 minutes to allow the scaffold to solidify, serving as the central portion. A 1 μl layer of 50% matrix gel was coated onto the central surface of the microfluidic chip through the gel / blastocyst loading window, and the microfluidic chip was placed in a 37°C incubator for 30 minutes. After the matrix gel layer solidified, fragmented endometrial epithelial organoids (approximately 3 × 10⁶) were loaded through the gel / blastocyst loading window. 3 (RIF) was spread onto 50% Matrigel. Endometrial-like cells were cultured by adding EXM + 10% FBS (Endometrial Epithelial Organoid Culture Medium) through the culture reservoir. Stromal cells and glands expanded within 1 day, and luminal epithelium formed a monolayer within 2 days.

[0244] 4.3 First, the implantation kinetics of blastocysts in in vitro embryo implantation models derived from RIF (section 4.2) and control (section 4.1) sources were compared, and significant differences were observed after 24 and 48 hours of co-culture. In the control endometrium, blastocysts effectively attached, proliferated, and invaded. Conversely, blastocysts in RIF-derived endometrial-like bodies exhibited poor adhesion, limited invasion and growth, and significant cell death was observed in both endometrial-like bodies and blastocysts near the attachment site. Figure 31 In summary, these findings suggest that abnormalities in the endometrial cells of RIF patients lead to implantation failure.

[0245] 4.4 Next, the combination of the above 7 effective drugs was added to the in vitro embryo implantation model of the same batch of RIF patients in Part 1, and the blastocyst implantation efficiency was tested before and after drug administration.

[0246] (1) Drug pretreatment and co-culture:

[0247] The combination of the above seven effective drugs was used to pretreat the in vitro implantation model established in Part 4.2 for 3 days. After that, blastocysts were added to the central region of the microfluidic chip to form an embryo implantation chip. 30 blastocysts were placed in each embryo implantation chip, and the 30 blastocysts were added through the loading window. Drug treatment was continued during co-culture (i.e., drugs were continuously added during the co-culture period to maintain the same treatment conditions as the pretreatment).

[0248] (2) Observation indicators and judgment time points:

[0249] Attachment determination (attachment rate): Determined after 24 hours. Determination criteria: The blastocyst / blastocyst adheres firmly to the surface of the endometrial model and does not shift when the slide / chip is slightly shaken (based on microscopic observation records).

[0250] Outgrowth assessment (outgrowth rate): Determined after 48 hours. Assessment criteria: The blastocyst / blastocyst spheroid clearly ruptures and trophoblast cells spread outwards in a single layer, evolving from a spherical shape.

[0251] Record the number of attachments and expansions of 30 blastocysts in each embryo implanted into the chip, and calculate rate = number_positive / number_seeded × 100%.

[0252] The study results showed that when blastocysts were added to the implantation model, in the endometrial model where blastocysts successfully implanted after combination treatment with seven effective drugs ( Figure 32 After co-culturing for 48 hours, the expansion and invasion rate of blastocysts was significantly higher than that of the untreated group (). Figure 33 ).

[0253] In this batch of RIFs, after 7 days of in vitro co-culture using a blastocyst-uterus model and treatment with a combination of 7 effective drugs, the blastocysts formed intraembryonic structures—the protoamniotic cavity and yolk sac cavity—and extraembryonic structures—syncytiotrophoblastic ectoderm and chorionic exotrophoblastic cells. Figure 34 ).

[0254] Round VI: Validating the effect of a combination of 7 effective drugs on the implantation of real human blastocysts in an endometrial model. The specific procedures are as follows:

[0255] Finally, we further explored the potential impact of the combination of these seven effective drugs on the implantation ability of blastocysts in RIF patients.

[0256] like Figure 35As shown, when the RIF patient implantation model was pretreated with this combination of 7 effective drugs 3 days in advance, and these 7 effective drugs were continuously added after blastocyst implantation, blastocysts that would otherwise not implant could undergo significant implantation. This indicates that the combination of these 7 drugs can significantly and broadly improve the embryo implantation ability of different RIF patients.

[0257] Several points to note during the fifth and sixth rounds of drug screening:

[0258] First embryo substitute:

[0259] To circumvent ethical restrictions on the use of human embryos, lower the application threshold, and increase experimental throughput, the embryo samples used in in vitro embryo implantation models can be replaced with trophoblast spheroids or other embryo-mimetic structures to replace human embryonic organoids (blastocysts) or actual human blastocysts, in order to achieve similar functional validation effects.

[0260] Second drug dosage and treatment time:

[0261] During drug screening, the dosage of the drug, following the validation method of a uniform starting concentration of 10 μM in the second round, can be adjusted according to the specific drug properties. If necessary, further optimal concentration validation experiments can be conducted without affecting the overall screening process.

[0262] Consumables for the third drug screening process:

[0263] The 96-well plate screening system used can be replaced with a 384-well plate, or operated in conjunction with an automated liquid handling workstation, to increase screening throughput and reduce human error. Simultaneously, the embryo implantation chip can employ a microfluidic design to improve data volume and reproducibility.

[0264] This study found that a combination of seven effective drugs—itotinib, tenofovir alafenamide, clopidogrel, tezacaftor, osimertinib, venetoclax, and ribociclib—treated endometrial epithelial organoids (models), and continued administration of these seven drugs after blastocyst implantation, resulted in successful blastocyst implantation. However, the RIF (recurrent intraepithelial neoplasia) population exhibits heterogeneity in clinical practice, and this combination remains ineffective for some patients, failing to achieve stable and predictable efficacy. Therefore, based on this study, the next stage of our research will focus on how to accurately biotype the large and heterogeneous RIF population and, based on this, match corresponding targeted treatment regimens. This will be a key question for our clients, and subsequent research will focus on developing precision medicines for the heterogeneous RIF population.

[0265] The above-described embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A combination drug for improving embryo implantation efficiency in patients with recurrent embryonic fibrosis (RIF), characterized in that, The combination of drugs includes: icotinib, tenofovir alafenamide fumarate tablets, clopidogrel, tezacotto, osimertinib, venetoclax, and ribotetinib.

2. The combination drug for improving embryo implantation efficiency in RIF patients according to claim 1, characterized in that, The molar concentrations of icotinib, tenofovir alafenamide fumarate tablets, clopidogrel, tezacotto, osimertinib, venetoclax, and ribotetinib were all 10 µM.

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