Combined medicine for improving embryo planting efficiency of RIF patient and screening method

Abstract

The invention provides a combined medicine for improving the 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 general drug combinations on the basis of a huge heterogeneity RIF group, 1119 drugs which possibly have treatment effects are locked in a large range through a six-step screening method in combination with an advanced RNA (Ribonucleic Acid) sequencing technology; the method comprises the following steps: selecting 1119 types of medicines, then further utilizing a highly simulated endometrial epithelium organ and interstitial cell model to screen medicines, testing the influence of the 1119 types of medicines on the activity of the endometrial epithelium organ and interstitial cell, and finally screening 7 types of effective medicines to form a broad-spectrum medicine with a remarkable curative effect on RIF through a combination form. The seven effective drugs screened by the invention are respectively icotinib, a propofol fumarate tenofovir tablet, clopidogrel, tizacarbox, osimertinib, Vinetogram and ribotinib.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of embryo implantation, and particularly relates to a combined drug for improving embryo implantation efficiency of RIF patients and a screening method. BACKGROUND

[0002] The gestation of human life begins with the successful "settling down" of the embryo in the mother's uterus, a biological milestone event, i.e. embryo implantation. This process demonstrates the precision and fragility of life evolution: the zygote does not move passively, but undergoes a series of strict programmed cell division and differentiation in the fallopian tube, and finally forms a structurally preliminary blastocyst on the fifth day after fertilization and enters the uterine cavity. At this time, the blastocyst and the maternal endometrium enter a short window period of mutual recognition "dialogue". About seven days after fertilization, the blastocyst breaks through the zona pellucida and initiates its most critical development program - first attaching to the pre-accepting endometrial epithelium through trophoblast cells, and then invading the endometrial stroma through protease action to establish a direct maternal-fetal blood circulation connection. This is not only the prologue of new life, but also a successful maternal-fetal dialogue, and is the absolute cornerstone for the establishment and maintenance of pregnancy.

[0003] However, this precise initial step of life poses a physiological and emotional challenge that is difficult to overcome for millions of families around the world. According to the latest large-scale epidemiological survey report led by Peking University Third Hospital, covering multiple provinces, the incidence of infertility among childbearing age couples in China has risen to about 18%, and the absolute number is huge, which has evolved from a private health problem to a significant public health issue affecting family stability, social well-being and population structure. Under this background, in vitro fertilization-embryo transfer (IVF-ET) technology, as a milestone in modern reproductive medicine, has illuminated the way forward for countless families. It simulates the in vivo environment in vitro to complete gamete fertilization and early embryonic development, aiming to bypass some obstructive and functional obstacles in the natural conception process. However, IVF-ET technology is far from being a final solution, and its technical bottlenecks are clearly visible: despite continuous optimization of embryonic laboratory techniques, the average embryo implantation rate worldwide still mainly hovers in the range of 30% to 40%. This cold data reveals a grim reality: among the embryos implanted back into the mother, more than half, even if they are "high quality" morphologically, cannot continue the life story due to the failure of the implantation process, which highlights the high complexity of the implantation process and the limitations of current technology.

[0004] More deeply troubled clinicians and patients is embedded in this bottleneck of a particularly thorny group - repeated implantation failure patients (RIF). They account for about 10% to 15% of the total population of assisted reproductive treatment, is the typical representative of the field of reproductive medicine “refractory infertility”. RIF in clinical practice has its strict, operational definition: usually refers to patients experienced 2 to 6 cycles of comprehensive assisted reproductive treatment, cumulative implantation of not less than 10 in morphological evaluation as “high quality” of embryos, or at least experienced 3 different implantation cycle (1-2 high quality embryo implantation per cycle), but has not been able to obtain any clinical pregnancy (B ultrasound visible gestational sac), or only experienced a short biochemical pregnancy (serum hCG levels one-time increase, prompt embryo has tried to “knock”, but failed to successfully “enter the door”).

[0005] RIF occurs, for couples eager to have children, is a repeated physical and mental suffering. It brings not only a huge economic burden, but also a deep emotional consumption and psychological trauma, so that the family in the cycle of hope and despair are suffering. Therefore, to reveal the RIF of multiple factors, heterogeneous etiology - its root may be hidden in the genetic abnormalities of the embryo itself, the space-time dislocation of endometrial receptivity, maternal immune tolerance disorder, or microthrombosis, and so on - and on this basis, systematically improve the embryo implantation efficiency of RIF patients, its significance has far exceeded the category of meeting individual fertility wishes. It points directly to the core blind area and breakthrough point of the current assisted reproductive technology, is the only way to promote the field from “empirical transplantation” to “precision reproductive medicine”, is a strategic key link to reduce adverse pregnancy outcomes from the source, protect the long-term health of mother and child, and ultimately improve the quality of the national birth population, bearing extremely important clinical value and far-reaching social significance.

[0006] At present, repeated implantation failure has become the core problem and “ceiling” in the field of assisted reproduction, which is urgent to be overcome. Its difficulties are rooted in the fact that RIF is not an independent disease, but a complex clinical syndrome caused by the interaction of multiple factors such as embryo, endometrium, maternal immunity and endocrine. This high heterogeneity, combined with the lack of international unified and clear etiological classification and diagnostic “gold standard”, makes clinicians like to walk in the fog, and lacks clear targets for RIF evaluation and intervention, thus facing great challenges.

[0007] Current mainstream treatment strategies can be roughly summarized into four directions: (i) uterine intervention (such as treatment of intrauterine lesions and improvement of endometrial blood flow); (ii) embryo laboratory optimization (such as blastocyst culture, PGT-A screening, and improved culture medium); (iii) immunomodulatory therapy (such as the use of glucocorticoids, lymphocyte immunotherapy, and immunosuppressants); and (iv) endometrial receptivity assessment and intervention (such as ERA detection to guide individualized implantation timing and the use of granulocyte colony-stimulating factor). However, these strategies are essentially scattered and exploratory in application. Since the core link of failure cannot be accurately located for each patient, the treatment is significantly "blind" and "trial and error". Doctors often have to take an "empirical superposition" strategy and try or combine various possible effective solutions for each patient, which not only increases the time and economic cost of treatment, but also leads to extremely individualized efficacy: the same solution can be a "miracle" for some patients, but completely ineffective for others. The overall lack of stable and predictable efficacy makes patients and doctors suffer in the cycle of hope and disappointment.

[0008] Therefore, despite the apparent number of existing methods, there is still a lack of a standard treatment path that is based on clear etiological typing, universal, and effective worldwide. SUMMARY

[0009] In view of the above problems, in order to overcome the shortcomings and deficiencies of the prior art, the present application proposes to perform transcriptome differential expression analysis on endometrial epithelial cells and stromal cells of RIF patients, and preliminarily screen out 1119 compounds from the MCE-HY-L022 drug library. The 1119 drugs are used for co-culture with endometrial epithelial organoids (EEOs) and stromal cells obtained by in vitro culture of RIF patients, the sum of cell activity in all groups for each drug and the effective coverage range of each drug are calculated, both indicators are considered to sort the drug effectiveness, and the following 7 effective drugs are selected: Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib. An in vitro embryo implantation model of RIF patients is constructed to verify the implantation ability of the above 7 drugs.

[0010] The technical solution adopted by the present application is: The present application protects a combination drug for improving the embryo implantation efficiency of RIF patients in the first aspect of the application, and the combination drug comprises: Erlotinib, propofol tenofovir fumarate tablets, clopidogrel, tizacato, osimertinib, venetoclax, and ribociclib.

[0011] Further, the molar concentration of erlotinib, propofol tenofovir fumarate tablets, clopidogrel, tizacato, osimertinib, venetoclax, and ribociclib is 10 μM.

[0012] The present application protects a screening method for a combination drug for improving the embryo implantation efficiency of RIF patients in the second aspect of the application, and the method comprises the following steps: Step 1: Bulk RNAseq is performed on the endometrial epithelial cells and stromal cells of 22 RIF patients, and through transcriptome differential expression analysis, 1119 drugs are preliminarily screened from the MCE-HY-L022 drug library; Step 2: The endometrial epithelial organoids and stromal cells are obtained by isolating and culturing the endometrial tissues of RIF patients, and the endometrial epithelial organoids and stromal cells obtained by isolating and culturing are used as the experimental group, and the 1119 drugs screened in step 1 are used to treat the endometrial epithelial organoids and stromal cells, respectively; Step 3: The sum of cell activity of each drug in all experimental groups and the effective coverage range of each drug in all experimental groups are calculated, and the effectiveness of the drugs is sorted according to the two indexes, and 7 effective drugs are selected; Step 4: Preliminary verification, the endometrial epithelial organoids and stromal cells isolated and cultured from RIF patients are co-cultured with the combination of the 7 effective drugs for 48 hours, and the growth efficiency of the endometrial epithelial organoids and stromal cells is observed; Step 5: In vitro embryo implantation model verification, an in vitro embryo implantation model of RIF patients is established, and the 7 combination drugs screened in step 3 are used to treat the in vitro embryo implantation model, and the blastocyst implantation efficiency is detected.

[0013] Step 6: The embryo implantation situation is verified by using human pre-implantation blastocysts in the in vitro embryo implantation model of RIF patients.

[0014] Further, the specific treatment process of step 2 is as follows: According to the density of 2 μl of Matrigel containing endometrial epithelial organoids per well or 3000 stromal cells per well, inoculate into a 96-well plate, add 1119 drugs in the 96-well plate, and each drug is repeated three times; Each 96-well plate is provided with a non-drug treatment control group and a blank control group; wherein the non-drug treatment control group is added with an equal amount of DMSO as the drug, and the blank control group only contains the culture medium.

[0015] Further, the specific processing procedure of step 3 is as follows: After 5 days of treatment, the cell activity of endometrial epithelial organoids and the cell activity of stromal cells are detected by using a CCK-8 kit. The sum of cell activities of each drug in all experimental groups and the effective coverage range of each drug are calculated, and the top 7 effective drugs are screened.

[0016] Further, 1119 drugs are added to the 96-well plate at a concentration of 10 µM.

[0017] Further, the sum of cell activities of each drug in all experimental groups and the effective coverage range of each drug in all experimental groups are calculated, and the effectiveness of the drugs is ranked according to these two indicators. The specific operation of selecting 7 effective drugs is as follows: Load the Excel file and parse each worksheet; Process the data, and the fold change of the group with fold change <1 is recorded as 0; Calculate the sum of fold change values of each drug in all experimental groups of endometrial epithelial organoids or stromal cells and the ranking; Calculate the number of covered groups for each drug with fold change >1; Integrate the sum of fold change values of each drug in all experimental groups of endometrial epithelial organoids or stromal cells and the number of covered groups, rank them according to the weight of 50% each, and select the top 7 as effective drugs.

[0018] Further, the 7 screened drugs include Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib.

[0019] Further, the 7 screened drugs include Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, and Ribociclib.

[0020] Further, the process of establishing an in vitro embryo implantation model for RIF patients is as follows: Prepare an in vitro embryo implantation chip; Prepare an artificial extracellular matrix using hydrogel and Matrigel. Load the artificial extracellular matrix onto the in vitro embryo implantation chip through the gel / blastocyst loading window on the in vitro embryo implantation chip as the basement layer, and incubate at 37°C for 30 minutes. Mix 5 ul of hydrogel and matrigel mixture with mesenchymal cells and endometrial epithelial organoids, incubate at 37℃ for 30 minutes to solidify the scaffold as the middle layer; Coat 1 ul of 50% matrigel thin layer on the surface of the middle layer, and place it in a 37℃ incubator for 30 minutes; After the matrigel thin layer is solidified, the fragmented endometrial epithelial organoids are placed on the matrigel thin layer through the gel / blastocyst loading window; Add endometrial epithelial organoid culture medium through the medium reservoir to culture the endometrial epithelial organoids.

[0021] Further, the mesenchymal cells and endometrial epithelial organoids used in the process of establishing the in vitro embryo implantation model are obtained by isolating and culturing the endometrial tissue of RIF patients.

[0022] Further, after the in vitro embryo implantation model is pretreated with 7 effective drug combinations for 3 days, the blastocyst / blastocyst is added to the middle region of the in vitro embryo implantation model through the gel / blastocyst loading window, and the 7 effective drugs are continuously added during the co-culture period.

[0023] Beneficial effects: The present application adopts a set of high-efficiency drug screening strategy based on the in vitro implantation model of endometritis type repeated embryo implantation failure, to ensure the reliability and functional correlation of the screening results. Through transcriptome differential expression analysis of endometrial epithelial cells and mesenchymal cells of RIF patients, 1119 drugs are preliminarily screened from the MCE-HY-L022 drug library; the 1119 drugs are co-cultured with the endometrial epithelial organoids and mesenchymal cells obtained by culturing the endometrial tissue of RIF patients, the sum of cell activity of each drug in all groups and the effectiveness coverage range of each drug are calculated, both indicators are considered to sort the drug effectiveness, and the following 7 effective drugs are selected: Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, Ribociclib; then, preliminary verification is carried out by co-culturing the endometrial epithelial organoids and mesenchymal cells of RIF patients with the 7 effective drugs, and then the in vitro embryo implantation model of RIF patients is constructed to verify the implantation ability of the 7 effective drugs on the blastocyst / blastocyst. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained based on these drawings without creative labor.

[0025] Figure 1 Bulk RNAseq transcriptome gene expression heat map for 22 RIF patients.

[0026] Figure 2 Drug library type.

[0027] Figure 3 Selected drug library drug type.

[0028] Figure 4 Second round of drug screening experiment chart.

[0029] Figure 5 Representative bright field images of the 7 selected combination drugs after 48 hours of action on endometrial epithelial organoids and stromal cells of 5 RIF patients.

[0030] Figure 6 3D implant chip schematic diagram.

[0031] Figure 7 3D implant chip image.

[0032] Figure 8 Schematic diagram of blastocyst-endometrial epithelial organoid implantation model.

[0033] Figure 9 Representative immunofluorescence (IF) images of endometrial epithelial organoids showing epithelial cell markers CK7 and KI67.

[0034] Figure 10 Representative IF images of stromal cells showing stromal cell markers N-Cadherin and Vimentin.

[0035] Figure 11 Representative IF images of endometrial epithelial organoids after co-treatment of estrogen (E2), progesterone (P4) and cAMP downstream genes IHH in the proliferation phase.

[0036] Figure 12 Representative IF images of endometrial epithelial organoids after co-treatment of estrogen (E2), progesterone (P4) and cAMP downstream genes HSD17β2 in the secretory phase.

[0037] Figure 13Representative IF images of SPP1, a downstream gene of secretory phase, in endometrial epithelial organoids treated with estrogen (E2), progesterone (P4) and cAMP.

[0038] Figure 14 QPCR of downstream genes of proliferative phase or secretory phase in endometrial epithelial organoids treated with estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP.

[0039] Figure 15 Representative IF images of progesterone receptor (PGR) translocation from cell membrane to cell nucleus in secretory phase in endometrial epithelial organoids treated with estrogen (E2), progesterone (P4) and cAMP.

[0040] Figure 16 Representative IF images of cell morphological changes after decidualization in secretory phase in stromal cells treated with estrogen (E2), progesterone (P4) and cAMP.

[0041] Figure 17 QPCR of secretory phase genes PRL and IGFBP1 expressed in secretory phase in stromal cells treated with estrogen (E2), progesterone (P4) and cAMP.

[0042] Figure 18 CCK-8 OD values of cell proliferation in proliferative phase or secretory phase in endometrial epithelial organoids treated with estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP.

[0043] Figure 19 CCK-8 OD values of cell proliferation in proliferative phase or secretory phase in stromal cells treated with estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP.

[0044] Figure 20 Cluster plot of endometrial receptivity genes expression in proliferative phase or secretory phase in endometrial epithelial organoids and stromal cells treated with estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP.

[0045] Figure 21 Hardness test of three different Matrigel.

[0046] Figure 22 Representative IF images of stromal cell spreading in three different Matrigel.

[0047] Figure 23 Stromal cell spreading area statistics in three different Matrigel.

[0048] Figure 24Representative IF images of mesenchymal cells spreading in different ratios of Matrigel and hydrogel.

[0049] Figure 25 Statistical chart of mesenchymal cell spreading ratio in different ratios of Matrigel and hydrogel.

[0050] Figure 26 Stiffness test in different ratios of Matrigel and hydrogel.

[0051] Figure 27 Representative IF images of endometrial epithelial organoid fragments spreading in different ratios of Matrigel.

[0052] Figure 28 Representative IF images of endometrial model three-dimensional light sheet; E-Cad: Glandular epithelium (GE) / luminal epithelium (LE), VIM: mesenchymal cells.

[0053] Figure 29 Representative IF images of real uterus and endometrial model; E-Cad: Glandular epithelium (GE) / luminal epithelium (LE), VIM: mesenchymal cells.

[0054] Figure 30 Representative IF images of luminal epithelium (LE: LGR5) and glandular epithelium (GE: PAEP).

[0055] Figure 31 Statistical chart of the difference in blastocyst (Q-tracker655 labeled) adhesion rate (24 hours) and expanded implantation rate (48 hours) on the implantation model between normal and RIF patients.

[0056] Figure 32 Representative bright field images of the implantation effect of the 7 screened combination drugs on the implantation model of 5 RIF patients.

[0057] Figure 33 Statistical results of implantation efficiency of the 7 screened combination drugs on the implantation model of 5 RIF patients before and after treatment, Ctrl: normal human implantation efficiency.

[0058] Figure 34 Immunofluorescence staining images of the development of the 7 screened combination drugs on the implantation model of RIF patients after implantation, intraembryonic structures: pseudo-amniotic cavity (OCT4+), pseudo-yolk sac cavity (GATA6+), mesenchymal cells (VIM+); extraembryonic structures: extravillous trophoblast (HLA-G+), syncytial trophoblast (hCG+).

[0059] Figure 35 Bright field images of human blastocyst implantation on the endometrial model of 5 RIF patients' endometrium treated with 7 drug combinations. DETAILED DESCRIPTION

[0060] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application.

[0061] The test materials used in the following embodiments are all purchased from conventional biochemical reagent stores, unless otherwise specified.

[0062] The present embodiment specifically discloses a screening method of a combined drug for improving the embryo implantation efficiency of RIF patients. Although a plurality of RIF treatment methods are clinically applied at present, the effectiveness of these treatments is greatly different among individuals due to the heterogeneity of RIF patients, and the effect is very small. In the present study, a plurality of patients are subjected to high-throughput testing of thousands of drugs through a suitable in vitro model. A group of drug combinations commonly used among a plurality of patients is found.

[0063] The following is the specific drug screening process: Step 1: Bulk RNAseq is performed on the endometrial epithelial cells and stromal cells of 22 RIF patients, and through transcriptome differential expression analysis, 1119 drugs are preliminarily screened out from the MCE-HY-L022 drug library; Step 2: The endometrial epithelial organoids and stromal cells are obtained by isolating and culturing the endometrial tissues of RIF patients, and the 1119 drugs screened in step are used to treat the endometrial epithelial organoids and stromal cells; Step 3: The sum of cell activity of each drug and the effective coverage range of each drug are calculated, and the effectiveness of the drugs is sorted according to the two indexes, and 7 effective drugs are selected; Step 4: Preliminary verification, the endometrial epithelial organoids and stromal cells isolated and cultured from other RIF patients are co-cultured with the combination of the 7 effective drugs for 48 hours, and the growth efficiency of the endometrial epithelial organoids and stromal cells is observed; Step 5: In vitro embryo implantation model verification, an in vitro embryo implantation model of RIF patients is established, and the 7 combined drugs screened in step 3 are used to treat the in vitro embryo implantation model, and the blastocyst implantation efficiency is detected.

[0064] Step 6: The embryo implantation situation of the RIF patient's embryo implantation model after drug treatment is verified by using human pre-implantation blastocysts in the RIF patient's in vitro embryo implantation model.

[0065] Round I: Screening of drug library and drug, the specific operation steps are as follows: First, Bulk RNAseq transcriptome sequencing is performed on the endometrial epithelial cells and stromal cells of 22 RIF patients. Through transcriptome differential genes (Figure 1 ) analysis, we found that endometrial epithelial cells and stromal cells in RIF patients showed type-specific expression, and the expression between endometrial epithelial cells and stromal cells also showed differences, which further reflected the high consistency of RIF patients at the transcriptional level. The genes up-regulated in the endometrial epithelial cells of these RIF patients were related to the following pathways: toxicity tolerance, humoral immune response, resistance to bacterial infection; the genes up-regulated in stromal cells were related to the following pathways: cell polarity, tight junction, ubiquitin ligase activity.

[0066] According to the results of transcriptome differential gene enrichment analysis, we compared the FDA-approved drug library (MCE-HY-L022) with the genes up-regulated in the endometrial epithelial cells of RIF patients. Figure 2 ), including various types of drugs, drug action mechanisms, and drug action pathways, and finally selected the MCE-HY-L022 drug library as the target library for initial drug screening. These drug libraries have the following advantages: these drugs have completed extensive preclinical and clinical studies, have good biological activity, safety, and pharmacokinetic characteristics, shorten the development cycle, and reduce the development cost. Through comparison, we initially screened out 1119 drugs from the MCE-HY-L022 drug library, as shown in Tables 1-14 below.

[0067] Table 1 Drug names on the first plate of the drug library (14 plates in total): ; Table 2 Drug names on the second plate of the drug library: ; Table 3 Drug names on the third plate of the drug library: ; Table 4 Drug names on the fourth plate of the drug library: ; Table 5 Drug names on the fifth plate of the drug library: ; Table 6 Drug names on the sixth plate of the drug library: ; Table 7 Drug names on the seventh plate of the drug library: ; Table 8 Drug names on the eighth plate of the drug library: ; Table 9 Drug names on the ninth plate of the drug library: ; Table 10 Drug names on the tenth plate of the drug library: ; Table 11. Drug names on the eleventh panel of the drug database: ; Table 12. Drug names on the twelfth shelf of the drug database: ; Table 13. Drug names on the thirteenth shelf of the drug database: ; Table 14. Drug names on the fourteenth panel of the drug database: ; 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: 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.

[0068] 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.

[0069] 3. Programmed death and metabolism: Apoptosis, Autophagy, Ferroptosis, Mitochondrial metabolism, Reactive Oxygen Species (ROS).

[0070] 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.

[0071] 5. Ion channels and physiological regulation: Calcium Channel, Potassium Channel, Sodium Channel, TRP Channel, corresponding to cardiovascular, electrophysiology, pain transmission research; COX, PDE, HMG-CoA Reductase, HDAC, Carbonic Anhydrase, related to inflammation, blood lipids, enzyme inhibitors, and other classic targets.

[0072] 6. Cancer and signal transduction targets: Bcr-Abl, FGFR, c-Kit, c-Met / HGFR, PDGFR, EGFR, VEGFR, CDK, MEK, ERK, etc. related to tumor signal pathways and proliferation regulation, including various clinical anti-cancer drugs and research inhibitors. HDAC, Topoisomerase, DNA Alkylator, Antifolate, related to DNA damage repair and epigenetic regulation.

[0073] 7. Nuclear receptors and metabolism: PPAR, Progesterone Receptor, Androgen Receptor, Estrogen Receptor, Glucocorticoid Receptor, corresponding to hormone regulation and metabolic syndrome research; P-glycoprotein, Cytochrome P450, NO Synthase, related to drug metabolism and drug resistance mechanism.

[0074] Round II: Screening of effective drugs in endometrial epithelial organoids and stromal cells, the specific operation steps are as follows: 1. Culture of human endometrial epithelial organoids and stromal cells: 22 cases of RIF patients were collected in the proliferative phase of the menstrual cycle (3-7 days after menstruation, i.e. 7-13 days of the menstrual cycle), and the endometrial tissue biopsy was obtained. Endometrial epithelial organoids and stromal cells were isolated and cultured, a total of 44 experimental groups.

[0075] After the endometrial tissue was washed with DMEM / F12 culture medium, it was digested with a mixture of Dispass and collagenase at 37°C for 40 minutes, and then terminated with DMEM / F12 containing 10% FBS. The digestion solution was separated by a 40 μm filter screen to obtain endometrial epithelial cells (greater than 40 μm) and stromal cells (less than 40 μm).

[0076] The obtained stromal cells were resuspended in culture medium and seeded in culture dishes and cultured at 37°C, 5% CO2. After 15 minutes of incubation, the supernatant was discarded and fresh culture medium was added for continuous culture, obtaining endometrial stromal cells adhering to the wall. Endometrial epithelial cells were resuspended in Matrigel (Corning 356231) and seeded in culture dishes at 20 μl per well. After 15 minutes of incubation at 37°C until the gel solidified, endometrial epithelial organoid culture medium was added for continuous culture, and endometrial epithelial organoids with three-dimensional cavity-like structures were formed.

[0077] The required culture medium is as follows: Stromal cell culture medium: DMEM / F-12 + 10% FBS + 1% anti-anti; 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.

[0078] 2. 1119 drugs treated endometrial epithelial organoids and stromal cells: Seeded in 96-well plates at a density of 2 μl of endometrial epithelial organoids containing Matrigel per well and 3000 stromal cells per well, and then added to the 96-well plates the 1119 drugs (drug final concentration 10 μM), each drug was repeated three times, each plate had 0 drug solvent DMSO control (non-drug treatment control group) and blank control containing only culture medium (no cells and no drugs), as shown in Figure 4 .

[0079] 3. CCK-8 kit (cell counting kit-8) for detecting endometrial cell activity: RIF patients have lower endometrial cell activity and proliferation ability, so we evaluate the standard of whether the drug is effective: whether the cell activity of the endometrium is improved compared with the control group without adding drugs. After 5 days of drug treatment, the endometrial cell activity is detected by CCK-8 kit. The core substrate of CCK-8 is water-soluble tetrazolium salt WST-8, which can be reduced by living cells to generate orange water-soluble material—formazan, and the generation amount is approximately proportional to the intensity of cell metabolic activity. Then use the microplate reader to read the absorbance (OD value) of each well at the visible light absorption peak (450 nm), and the OD value can represent the activity of the cells, so as to evaluate the effectiveness of the drug.

[0080] The OD value data of each experimental group, 0 drug control group and blank control group is exported, and the calculation method of cell activity is shown in the following figure. The ratio greater than 1 indicates that the cell activity has improved after adding drugs.

[0081] Cell viability (%) = [A (drug) - A (blank)] / [A (0 drug) - A (blank)] x 100 A (drug): absorbance of wells with cells, CCK solution and drug solution A (blank): absorbance of wells with medium and CCK solution without cells A (0 drug): absorbance of wells with cells, CCK solution without drug solution Cell activity: cell proliferation activity or cytotoxic activity: The above formula can be simplified as: fold change = (OD_treated-OD_blank) / (OD_control_mean-OD_blank); where OD_treated represents A (drug); OD_blank represents A (blank); OD_control_mean represents A (0 drug).

[0082] Round III: Screen effective drugs by algorithm, specific operation steps are as follows: An algorithm was developed to automatically read and calculate the sum of cell viability of each drug in all groups and the effectiveness coverage range of each drug by computer. According to the detection results of step two, the effectiveness of the drugs was ranked by considering these two indicators, and the following 7 effective drugs were selected: Icotinib, Tenofovir alafenamide, Clopidogrel, Tezacaftor, Osimertinib, Venetoclax, Ribociclib. The sum of cell viability of these 7 effective drugs in each group is the highest, which indicates that they can most improve the viability of endometrial cells, and at the same time cover the most combinations with viability value greater than 1, indicating that they play a role in various RIF patients' epithelial and stromal cells.

[0083] The basic process of the algorithm is as follows: 1. Load the Excel file and parse each worksheet; 2. Process the data, and the fold change of the group with fold change <1 is recorded as 0; 3. Calculate the sum of fold change values of each drug in all experimental groups of endometrial epithelial organoids or stromal cells and rank them; 4. Calculate the number of covered groups for each drug with fold change >1; 5. Integrate the sum of fold change values of each drug in different experimental groups of endometrial epithelial organoids or stromal cells and the number of covered groups, rank them according to the weight of 50% each, and select the top 7.

[0084] The running code corresponding to the above algorithm is as follows: import pandas as pd import numpy as np import matplotlib.pyplot as plt # First step: Load Excel file and parse each worksheet file_path = 'C: / Users / recordarse / Desktop / pythonlearning / data / lq.xlsx'# Excel file path data = pd.ExcelFile(file_path) # Load Excel file sheets_data = {sheet: data.parse(sheet) for sheet in data.sheet_names} # Parse each worksheet # Step 2: Process data and filter out drugs with fold change > 1 combined_matrix = pd.DataFrame() # To store the processed matrix original_combined_matrix = pd.DataFrame() # To store the original fold change values for sheet_name, df in sheets_data.items(): if 'Drug Name' in df.columns and 'fold change' in df.columns: df = df.set_index('Drug Name') # Set drug name as index filtered_df = df['fold change'].apply(lambda x: x if x > 1 else 0) # Set fold change <= 1 to 0 combined_matrix[sheet_name] = filtered_df # Save the processed data original_combined_matrix[sheet_name] = df['fold change'] # Save the original data # Fill missing values with 0 (indicating absence of the drug in certain patient groups) combined_matrix = combined_matrix.fillna(0) original_combined_matrix = original_combined_matrix.fillna(0) # Step 3: Calculate total fold change values and rank combined_matrix['Total Fold Change'] = combined_matrix.sum(axis=1) # Calculate total fold change values for each drug combined_matrix['Rank'] = combined_matrix['Total Fold Change'].rank(ascending=False, method='min') # Rank by total value # Step 4: Calculate the rank of each group, and set the rank of groups with fold change 0 as 0 group_ranks = pd.DataFrame(index=combined_matrix.index) for sheet_name in combined_matrix.columns[:-2]: # Ignore Total FoldChange and Rank columns group_ranks[sheet_name] = combined_matrix[sheet_name].rank(ascending=False, method='min') group_ranks[sheet_name]=group_ranks[sheet_name].where(combined_matrix[sheet_name]>0, 0) # Set the rank of groups with fold change 0 as 0 # Step 5: Generate output output_df = combined_matrix[['Total Fold Change', 'Rank']].copy() output_df['Covered Groups'] = combined_matrix.iloc[:, :-2].astype(bool).sum(axis=1) # Calculate the number of covered groups output_df['Group Fold Changes'] = combined_matrix.iloc[:, :-2].apply(lambda row: row[row>0].to_dict(), axis=1) # Record the fold change values of each group output_df['Group Ranks'] = group_ranks.apply(lambda row: row[row>0].to_dict(), axis=1) # Record the rank of each group Save to CSV file output_file = "Drug_plan1_veison3.csv" output_df.to_csv(output_file, index=True) print(f"Drug total fold change values and ranking have been saved to: {output_file}") The 7 effective drug action targets, mechanisms, uses and related drug / metabolic characteristics screened by the above algorithm are shown in Table 15.

[0085] Table 15 7 drug action targets, mechanisms, uses and related drug / metabolic characteristics: ; Round IV: Verify the improvement of 7 effective drug combinations on RIF patient endometrial cells, as follows: According to the patient transcriptome heat map measured by bulk-RNAseq previously, one patient from each type after classification by expression was selected, a total of 5 patients, to verify the effectiveness of the 7 effective drugs. Mixed at 10 μM per drug, forming a mixture of 7 effective drugs, to treat RIF patient endometrial epithelial organoids and their stromal cells (endometrial epithelial organoid and stromal cell isolation and culture method as described in Step 1). After 48 hours, monitor the proliferation and apoptosis of RIF patient endometrial epithelial organoids and stromal cells. As shown in Figure 5 , it can be seen that with the addition of 7 effective drugs, the growth efficiency of endometrial epithelial organoids and stromal cells is greatly improved.

[0086] Round V: Verify the implantation effect of 7 effective drugs on blastoids in the endometrial model, as follows: 1. The endometrial epithelial organoid and stromal cell isolation and culture method is as described in Step 1.

[0087] 2. Blastoid culture: 2.1 Human pluripotent stem cells (hPSC) in naïve state culture: Starting cells: Primed WIBR3 and RUES2 hESCs; Culture medium: Initially cultured in mTeSR Plus, then replaced with modified 5i / L / A or PXGL medium to reset to naïve state; Culture conditions: Attached to inactivated mouse fibroblasts (MEF), 37°C, 5% O2, 5% CO2; Passage: Using Accutase; 5i / L / A formulation: DMEM / F12: Neurobasal 1:1, N2, B27, GlutaMAX, non-essential amino acids, β-mercaptoethanol, 0.5% KSR, antibiotics; Small molecules / cytokines: PD0325901, IM-12, SB590885, WH-4-023, LIF, Activin A, Y-27632; PXGL formulation: DMEM / F12:Neurobasal 1:1, half-strength N2 / B27, GlutaMAX, β-mercaptoethanol, antibiotics; Small molecules / cytokines: PD0325901, LIF, XAV939, Gö6983, Y-27632; 2.2 Thylakoid formation: Naïve hESCs (gifted free of charge by Ali H. Brivanlou's laboratory) were single-celled (Accutase, 37°C, 3 minutes); MEF removal: 0.1% gelatin-coated petri dishes at 37°C for 30 minutes; hPSCs were collected and filtered to 40 μm. Cells were resuspended in BFM-I (5i / L / A + CEPT cocktail, without Y-27632 and BSA); AggreWell-400 pretreatment: Add anti-adhesion solution, centrifuge at 1900 g for 5 minutes, and incubate at room temperature for ≥30 minutes; Cultivation: 100,000 cells / well, centrifuged at 100 g for 3 minutes, 37°C, 5% O2, 5% CO2; Training process: Day 0: Vaccination; Day 1: Replace BFM-I with BFM-II (half volume, two replacements each time); Days 2-6: Replace BFM-II with half the dose daily; 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.

[0088] 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.

[0089] 3. Design and fabrication of in vitro embryo implantation microarrays: 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.

[0090] 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.

[0091] The left and right channels are used to separate the endometrial epithelial organoid culture medium and the implantation culture medium.

[0092] 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.

[0093] 4. Preparation of in vitro embryo implantation model: 4.1 Construction of the endometrial model (e.g.) Figure 8 (as shown) 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 ).

[0094] 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 ).

[0095] 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 ).

[0096] 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.

[0097] 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 ).

[0098] 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 .

[0099] 4.2 RIF-type in vitro embryo implantation model: 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] (1) Drug pretreatment and co-culture: 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).

[0104] (2) Observation indicators and judgment time points: 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).

[0105] 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.

[0106] 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%.

[0107] 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 ).

[0108] 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 ).

[0109] 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: Finally, we further explored the potential impact of the combination of these seven effective drugs on the implantation ability of blastocysts in RIF patients.

[0110] 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.

[0111] Several points to note during the fifth and sixth rounds of drug screening: First embryo substitute: 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.

[0112] Second drug dosage and treatment time: 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.

[0113] Consumables for the third drug screening process: 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.

[0114] 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.

[0115] 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 the embryo implantation efficiency of RIF patients, characterized in that, The combination drug includes: icotinib, fumaric acid propofol tenofovir tablets, clopidogrel, tizacato, osimertinib, venetoclax, and ribociclib.

2. The combination drug for improving the embryo implantation efficiency of RIF patients according to claim 1, characterized in that, The molar concentration of icotinib, fumaric acid propofol tenofovir tablets, clopidogrel, tizacato, osimertinib, venetoclax, and ribociclib is 10 μM.

3. A method for screening a combination drug for improving embryo implantation efficiency in RIF patients, characterized in that, The method comprises the following steps: Step 1: Bulk RNAseq transcriptome analysis is performed on endometrial epithelial cells and stromal cells of RIF patients, and through analysis of differential genes, 1119 drugs are preliminarily screened out from the MCE-HY-L022 drug library; Step 2: Endometrial epithelial organoids and stromal cells are obtained by isolating and culturing the endometrial tissue of RIF patients, and the endometrial epithelial organoids and stromal cells obtained by isolating and culturing are used as the experimental group, and the 1119 drugs screened in step 1 are used to treat the endometrial epithelial organoids and stromal cells respectively; Step 3: Calculate the sum of cell activity of each drug in all experimental groups, and the effective coverage range of each drug in all experimental groups, and sort the effectiveness of the drugs according to the two indexes, and select 7 effective drugs; Step 4: Preliminary verification, the endometrial epithelial organoids and stromal cells isolated and cultured from RIF patients are co-cultured with the combination of the 7 effective drugs for 48 hours, and the growth efficiency of the endometrial epithelial organoids and stromal cells is observed; Step 5: In vitro embryo implantation model verification, an in vitro embryo implantation model of RIF patients is established, and the combination of the 7 effective drugs screened in step 3 is used to treat the in vitro embryo implantation model, and the blastocyst implantation efficiency is detected; Step 6: Verify the embryo implantation in the in vitro embryo implantation model of RIF patients using human pre-implantation blastocysts.

4. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 3, characterized in that, The specific processing process of step 2 is as follows: According to the density of 2 μl of Matrigel containing endometrial epithelial organoids per well or 3000 stromal cells per well, inoculate into a 96-well plate, add 1119 drugs to the 96-well plate, and each drug is repeated three times; Each 96-well plate is provided with a non-drug treatment control group and a blank control group; wherein the non-drug treatment control group adds an equal amount of DMSO as the drug, and the blank control group only contains the culture medium.

5. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 4, characterized in that, The specific processing process of step 3 is as follows: After 5 days of treatment, the cell activity of the endometrial epithelial organoids and the cell activity of the stromal cells are detected by using the CCK-8 kit; Calculate the sum of cell activity of each drug in all experimental groups and the effective coverage range of each drug, and select the top 7 effective drugs.

6. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 5, characterized in that, The specific operation of calculating the sum of cell activity of each drug in all experimental groups and the effective coverage range of each drug in all experimental groups, and sorting the effectiveness of the drugs according to the two indexes, and selecting 7 effective drugs is as follows: Load the Excel file and parse each worksheet; Process the data, and the fold change of the group with fold change <1 is recorded as 0; Calculate the sum of fold change values of each drug in all experimental groups of endometrial epithelial organoids or stromal cells and the ranking; Count the number of groups covered for each drug with fold change >1; Sum the fold change values of each drug in all experimental groups of endometrial epithelial organoids or stromal cells, and count the number of groups covered, and sort them by each 50% weight, and select the top 7 as effective drugs.

7. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 6, characterized in that, The 7 effective drugs screened include icotinib, fumaric acid propofol tenofovir tablets, clopidogrel, tizacato, osimertinib, venetoclax, and ribociclib.

8. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 7, characterized in that, The process of establishing the in vitro embryo implantation model of RIF patients is as follows: Prepare the in vitro embryo implantation chip; Prepare the artificial extracellular matrix using hydrogel and Matrigel, and pre-pave the artificial extracellular matrix on the in vitro embryo implantation chip through the gel / blastocyst loading window of the in vitro embryo implantation chip as the basement layer, and incubate at 37℃ for 30 minutes; Mix 5 μl of hydrogel and Matrigel mixture with stromal cells and endometrial epithelial organoids, and incubate at 37℃ for 30 minutes to solidify the scaffold as the intermediate layer; Coat 1 μl of 50% Matrigel thin layer on the surface of the intermediate layer, and place it in a 37℃ incubator for 30 minutes; After the Matrigel thin layer solidifies, place the fragmented endometrial epithelial organoids on the Matrigel thin layer through the gel / blastocyst loading window; Add endometrial epithelial organoid culture medium through the medium reservoir to culture the endometrial epithelial organoids.

9. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 8, characterized in that, The stromal cells and endometrial epithelial organoids used in the process of establishing the in vitro embryo implantation model are obtained by isolating and culturing the endometrial tissue of RIF patients.

10. The method for screening combination drugs to improve embryo implantation efficiency in RIF patients according to claim 9, characterized in that, After 3 days of pretreatment with 7 effective drug combinations, the blastocyst / embryo is added to the middle area of the in vitro embryo implantation chip through the gel / blastocyst loading window, and 7 effective drugs are continuously added during the co-culture period.

Images