A candidate drug for promoting embryo implantation efficiency of patients with endometrial microcirculation disorder type RIF

Abstract

The application provides a candidate drug for promoting embryo implantation efficiency of a patient with endometrial microcirculation disorder type RIF, and belongs to the technical field of embryo implantation. In order to solve the problem of how to find a potential intervention drug for the pathological mechanism of microcirculation disorder type RIF, a five-step high-efficiency drug screening strategy based on an in-vitro implantation model of endometrial microcirculation disorder type repeated embryo implantation failure is adopted to ensure the reliability and functional correlation of the screening result. The candidate drug screened by the five-step high-efficiency drug screening strategy includes any one of the following drugs: phenylbutazone, ferulic acid, L-proline, indinavir, solfazone, teneligliptin and gemcitabine. Among the candidate drugs, phenylbutazone, ferulic acid and L-proline are the best three drugs for promoting the implantation efficiency of the patient with endometrial microcirculation disorder type RIF.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of embryo implantation, and particularly relates to a candidate drug for promoting embryo implantation efficiency of a patient with endometrial microcirculation disorder type RIF. BACKGROUND

[0002] Embryo implantation is a key event in human development, requiring complex and coordinated interactions between the blastocyst and the maternal endometrium. The link between implantation abnormalities and reproductive failure has been clearly established, and this influence involves both natural pregnancy and assisted reproductive technology (ART).

[0003] Recurrent implantation failure (RIF) is usually defined as a clinical phenomenon that fails to achieve pregnancy after ≥3 times of high-quality embryo transplantation during in vitro fertilization-embryo transfer (IVF-ET). The clinical manifestations and potential causes of RIF patients are highly heterogeneous, involving endometrial abnormalities, immune function imbalance, microthrombosis, embryonic factors, and endocrine environment and other mechanisms. This heterogeneity not only increases the difficulty of diagnosis and treatment of RIF, but also has a significant impact on the effect of drugs or interventions. In clinical practice, we observed that some patients showed obvious etiology specificity, such as endometrial microcirculation disorder type RIF.

[0004] Endometrial microcirculation disorder type recurrent implantation failure refers to that in patients with recurrent embryo implantation failure, although the endometrial morphology or hormone level may be normal, the microvascular perfusion, hemodynamics and repair capacity of the endometrium during the implantation window period are systematically disordered. The core feature of this functional subtype is insufficient local microcirculation, which limits the transport of oxygen and nutrients to the deep tissues of the endometrium, thereby hindering embryo implantation and early development [1-2] .

[0005] In endometrial microcirculation disorder type RIF patients, the density of endometrial microvessels often decreases significantly, the structure and permeability of blood vessels change, and the local perfusion capacity is limited. Insufficient perfusion will lead to local hypoxia in the endometrium, inducing the hypoxia-inducible factor (HIF) pathway to start. Studies have shown that HIF-1α expression is reduced in some RIF cases, and physical endometrial injury can partially restore its expression and angiogenesis signals, suggesting that hypoxia response abnormalities directly affect endometrial receptivity [3] .

[0006] Based on the above observations, the present study will focus on patients with endometrial microcirculation disorder type RIF, simulate the human endometrial-embryo interaction environment by constructing an in vitro model, and systematically analyze its pathological characteristics and drug sensitivity. The model aims to discover potential intervention drugs targeting the pathological mechanisms related to microcirculation disorders, thereby improving the embryo implantation success rate and pregnancy outcome of RIF patients. SUMMARY

[0007] In view of the above problems, in order to overcome the shortcomings and deficiencies existing in the prior art, the blastocyst-endometrial model proposed in the present application can successfully simulate the selective characteristics of implantation, covering all key stages in the embryo implantation process, including embryo proximity, attachment and invasion. Notably, by using endometrial mesenchymal cells and endometrial epithelial organoids derived from RIF patients with endometrial microcirculation disorders, an in vitro implantation model is constructed, and a second round of screening of the 50 drugs preliminarily screened is performed to identify specific drugs that can significantly improve the implantation rate.

[0008] The technical scheme adopted by the present application is:

[0009] The present application protects a candidate drug for promoting the embryo implantation efficiency of RIF patients with endometrial microcirculation disorders in the first aspect, and the candidate drug includes any one of the following drugs:

[0010] Phenylbutazone, Ferulic acid, L-Proline, Indinavir, Sofalcone, Trelagliptin, Gemcitabine.

[0011] Preferably, the candidate drug includes Phenylbutazone, Ferulic acid or L-Proline.

[0012] Further, the physiological characteristics of RIF patients with endometrial microcirculation disorders are the decrease of endometrial microvessel density, the change of vascular structure and permeability, and the limitation of local perfusion capacity.

[0013] Further, the candidate drug includes any one of Phenylbutazone, Ferulic acid, and L-Proline.

[0014] The present application protects a screening method for a candidate drug for promoting the embryo implantation efficiency of RIF patients with endometrial microcirculation disorders in the second aspect, and the method includes the following steps:

[0015] Step 1: Select 1119 drugs from the FDA-approved drug library based on the endometrial transcriptome characteristics of RIF patients with endometrial microcirculation disorders;

[0016] Step 2: Isolate and culture endometrial epithelial organoids and stromal cells from RIF patients with endometrial microcirculation disorder in vitro, and treat the endometrial epithelial organoids or stromal cells with the 1119 drugs screened in Step 1, identify drugs that can promote cell viability and avoid toxicity using CCK-8 experiment and optical density (OD) determination, and select the top 50 drugs;

[0017] Step 3: Prepare an in vitro implantation model of RIF with endometrial microcirculation disorder;

[0018] Step 4: After treating the in vitro implantation model with the 50 drugs screened in Step 2, add blastocysts to the middle region of the in vitro implantation model for co-culture, monitor the invasion and growth of the blastocysts 48 hours after plating, and screen candidate drugs.

[0019] Step 5: Verify the embryo implantation efficiency of the screened candidate drugs using human blastocysts before implantation in an in vitro embryo implantation model.

[0020] Further, the specific operation of Step 2 is as follows:

[0021] Isolate and culture endometrial epithelial organoids and stromal cells from endometrial tissue;

[0022] Inoculation and pre-culture: inoculate 2 μl of Matrigel containing endometrial epithelial organoids or 3000 stromal cells in each well of a 96-well plate;

[0023] Each plate has a blank control group and a non-drug treatment control group. Each well of the blank control group only adds culture medium, and each well of the non-drug treatment control group inoculates 2 μl of Matrigel containing endometrial epithelial organoids or 3000 stromal cells. Each well has three repeated controls, and the experiment is repeated three times according to the experimental design;

[0024] Each drug screened in Step 1 is added to the corresponding well of the 96-well plate in the experimental group at 10 μM. The drug-containing culture medium is changed every 2-3 days during the culture period, and the total treatment culture is 5 days. The non-drug treatment control group is added with culture medium containing an equal amount of DMSO as the drug, and the blank control group only contains culture medium.

[0025] After 5 days of culture, the OD value is measured using the CCK-8 kit, and the cell viability of each drug-treated well relative to the control group is calculated. The cell viability is ranked from high to low, and the top 50 drugs are selected.

[0026] Further, the calculation expression of cell viability is fold change = (OD_treated-OD_blank) / (OD_control_mean-OD_blank); wherein OD_treated represents the OD value measured after drug treatment, OD_blank represents the background OD value of the blank control group, and OD_control_mean represents the OD value of the non-drug treatment control group.

[0027] Further, the in vitro implantation model construction process in step 3 is as follows:

[0028] Prepare an in vitro embryo implantation chip;

[0029] An artificial extracellular matrix is prepared using hydrogel and Matrigel, and the artificial extracellular matrix is prepared on the in vitro embryo implantation chip through the gel / blastocyst loading window of the in vitro embryo implantation chip as a basement layer, and incubated at 37°C for 30 minutes;

[0030] Mix 5 μl of hydrogel and Matrigel mixture with interstitial cells and endometrial epithelial organoids, and incubate at 37°C for 30 minutes to solidify the scaffold as an intermediate layer;

[0031] Coat 1 μl of 50% Matrigel thin layer on the surface of the intermediate layer, and place it in a 37°C incubator for 30 minutes;

[0032] 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;

[0033] Add endometrial epithelial organoid culture medium through the medium reservoir on the in vitro embryo implantation chip to culture the endometrial model.

[0034] Further, after 3 days of pre-treatment of the in vitro implantation model with the 50 selected drugs in step 4, the blastocyst is added to the middle region of the in vitro implantation model through the gel / blastocyst loading window, and the drug is continuously added during the co-culture period.

[0035] Further, after screening the candidate drugs in step 4, the blastocyst is labeled with Q-tracker 655, and the blastocyst is implanted in the in vitro implantation model treated with the top 3 candidate drugs (Phenylbutazone, Ferulic acid (sodium), and L-Proline) selected in step 4 for 3 days, and the implantation of the blastocyst is observed after 48 hours.

[0036] Alternatively, the blastoids are re-implanted into the in vitro implantation model of the top 3 ranked candidate drugs in step 4 screening for 3 days of pre-treatment, and after 7 days of in vitro culture, the embryo structure (pseudo-amniotic cavity-like structure and pseudo-yolk sac-like structure) reaches 18.43%, 19.74% and 11.15%, respectively, and the probability of the appearance of extraembryonic structures such as syncytial trophoblasts (STB) and villous extravillous trophoblasts (EVT) is 39.53%, 46.88% and 41.63%, respectively, after treatment with the most effective drugs (Phenylbutazone, Ferulic acid (sodium) and L-Proline).

[0037] Further, after screening the candidate drugs in step 4, the human blastoids are re-implanted into the in vitro implantation model of the top 3 ranked candidate drugs (Phenylbutazone, Ferulic acid (sodium) and L-Proline) in step 4 screening for 3 days of pre-treatment in the endometrial microcirculation disorder type RIF in step 5, to verify the best candidate drug screened.

[0038] Beneficial effects:

[0039] The present application adopts a five-step high-efficiency drug screening strategy based on an in vitro implantation model of endometrial microcirculation disorder type repeated embryo implantation failure, to ensure the reliability and functional correlation of the screening results. Round I is based on the endometrial transcriptome characteristics and gene ontology pathway analysis of RIF / other endometrial diseases, to perform priority matching screening on the FDA-approved drug library, to achieve classified drug recommendation; Round II is high-throughput screening in endometrial epithelial organoids and stromal cells, to evaluate the candidate drugs through cell activity and toxicity indicators, to narrow the range of effective drugs; the in vitro implantation model is constructed using endometrial epithelial cells, stromal cells and blastoid of patients with endometrial microcirculation disorder type RIF; Round IV uses the in vitro embryo implantation model and blastoids to verify the implantation ability of the effective drugs in the previous round, to observe the regulation of the drugs on the attachment and invasion of endometrial-embryo interaction; Round V uses human pre-implantation blastoids to perform embryo implantation and early development function verification in the in vitro implantation model, to ensure the physiological relevance and effectiveness of the screened drugs.

[0040] The effective drugs screened by the five-step high-efficiency drug screening strategy are Phenylbutazone, Ferulic acid, L-Proline, Indinavir, Sofalcone, Trelagliptin and Gemcitabine. Among these drugs, Phenylbutazone, Ferulic acid and L-Proline are the three drugs with the best implantation promotion effect for patients with endometrial microcirculation disorder type RIF, and after implantation, the blastocyst embryo structure (pseudo-amnion cavity-like structure and pseudo-yolk sac-like structure) reaches 18.43%, 19.74% and 11.15% respectively, and the extraembryonic structure (STB and EVT) appears 39.53%, 46.88% and 41.63% respectively after treatment with the most effective drugs (Phenylbutazone, Ferulic acid (sodium) and L-Proline). Further, it is found that human blastocysts can be implanted in the endometrial microcirculation disorder type RIF patient in vitro implantation model treated with the top three candidate drugs (Phenylbutazone, Ferulic acid (sodium) and L-Proline) in step 4 for 3 days. BRIEF DESCRIPTION OF DRAWINGS

[0041] 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 are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0042] Figure 1 A schematic diagram of the embryo implantation model constituted by the blastocyst-endometrial model.

[0043] Figure 2 A schematic diagram of the in vitro embryo implantation chip.

[0044] Figure 3 An image of the in vitro embryo implantation chip.

[0045] Figure 4 A representative immunofluorescence (IF) image of the endometrial epithelial organoid showing epithelial cell markers CK7 and KI67.

[0046] Figure 5 A representative IF image of the stromal cells showing stromal cell markers N-Cadherin and Vimentin.

[0047] Figure 6Representative IF image of downstream gene IHH for endometrial epithelial organoids after estrogen (E2) treatment during the proliferative phase.

[0048] Figure 7 Representative IF image of downstream gene HSD17β2 for endometrial epithelial organoids after estrogen (E2), progesterone (P4) and cAMP co-treatment during the secretory phase.

[0049] Figure 8 Representative IF image of downstream gene SPP1 for endometrial epithelial organoids after estrogen (E2), progesterone (P4) and cAMP co-treatment during the secretory phase.

[0050] Figure 9 QPCR of downstream gene expression for endometrial epithelial organoids after estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP co-treatment during the proliferative or secretory phase.

[0051] Figure 10 Representative IF image of progesterone receptor (PGR) translocation from cell membrane to nucleus for endometrial epithelial organoids after estrogen (E2), progesterone (P4) and cAMP co-treatment during the secretory phase.

[0052] Figure 11 Representative IF image of cell morphological changes after decidualization for stromal cells after estrogen (E2), progesterone (P4) and cAMP co-treatment during the secretory phase.

[0053] Figure 12 QPCR of secretory phase genes PRL and IGFBP1 expression for stromal cells after estrogen (E2), progesterone (P4) and cAMP co-treatment during the secretory phase.

[0054] Figure 13 CCK-8 OD value for cell proliferation for endometrial epithelial organoids after estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP co-treatment during the proliferative or secretory phase.

[0055] Figure 14 CCK-8 OD value for cell proliferation for stromal cells after estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP co-treatment during the proliferative or secretory phase.

[0056] Figure 15 Cluster plot of endometrial receptivity gene expression for endometrial epithelial organoids and stromal cells after estrogen (E2) or estrogen (E2), progesterone (P4) and cAMP co-treatment during the proliferative or secretory phase.

[0057] Figure 16 Hardness test for three different Matrigel.

[0058] Figure 17 Representative IF images of mesenchymal cell spreading in three different Matrigel.

[0059] Figure 18 Statistical chart of mesenchymal cell spreading area in three different Matrigel.

[0060] Figure 19 Representative IF images of mesenchymal cell spreading in different mixing ratios of Matrigel and hydrogel.

[0061] Figure 20 Statistical chart of mesenchymal cell spreading ratio in different mixing ratios of Matrigel and hydrogel.

[0062] Figure 21 Hardness test in different mixing ratios of Matrigel and hydrogel.

[0063] Figure 22 Representative IF images of endometrial epithelial organoid fragments spreading in different ratios of Matrigel.

[0064] Figure 23 Representative IF images of three-dimensional light sheets of endometrial models. E-Cad: Glandular epithelium (GE) / Luminal epithelium (LE), VIM: Mesenchymal cells.

[0065] Figure 24 Representative IF images of real uterus and endometrial model. E-Cad: E-Cad: Glandular epithelium (GE) / Luminal epithelium (LE), VIM: Mesenchymal cells.

[0066] Figure 25 Representative IF images of luminal epithelium (LE: LGR5) and glandular epithelium (GE: PAEP) of endometrial model.

[0067] Figure 26 Schematic diagram of drug screening process.

[0068] Figure 27 Endometrial cell transcriptome gene ontology analysis chart of RIF patients with endometrial microcirculation disorder type.

[0069] Figure 28 Relative fold change of OD values of RIF endometrial epithelial organoids (top) and mesenchymal cells (bottom) after treatment with 1119 drugs relative to untreated drug (DMSO) control. Red and blue dots represent the top 50 effective drugs for endometrial epithelial organoids (red dots) and mesenchymal cells (blue dots), respectively; green dots represent shared drugs that act on both endometrial epithelial organoids and mesenchymal cells in the top 50 list.

[0070] Figure 29 The mean ± standard deviation of the implantation rate of the blastoids in the endometrial microcirculation disorder type RIF endometrial model treated with the identified effective drug for 48 hours.

[0071] Figure 30 The image of the implantation of Q-tracker 655 labeled blastoids in the implantation model with or without representative drug treatment.

[0072] Figure 31 The development rate statistics of intraembryonic structures (pseudo-amnion cavity-like structures and pseudo-yolk sac-like structures) and extraembryonic structures (STB and EVT) after the implantation of the blastoids in the endometrial microcirculation disorder type RIF endometrial model treated with the drug.

[0073] Figure 32 The implantation of human blastoids in the endometrial microcirculation disorder type RIF in vitro implantation model with or without drug treatment. DETAILED DESCRIPTION

[0074] In order to make the objects, 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.

[0075] The test materials used in the following examples are commercially available from conventional biochemical reagent stores, unless otherwise specified.

[0076] Cell source:

[0077] 1. Culture of human endometrial epithelial organoids and stromal cells

[0078] All human endometrial tissue biopsy samples were collected in the proliferative phase of the menstrual cycle (3-7 days after menstruation, i.e. 7-13 days of the menstrual cycle), including healthy people and patients with endometrial microcirculation disorder type RIF.

[0079] After the endometrial tissue was washed with DMEM / F12 medium, it was digested with a Dispase and collagenase mixture at 37°C for 40 minutes, and the digestion was terminated with DMEM / F12 containing 10% FBS. After the digestion solution was separated by a 40 μm filter, endometrial epithelial cells (greater than 40 μm) and stromal cells (less than 40 μm) were obtained, respectively.

[0080] 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 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.

[0081] The required culture medium is as follows:

[0082] Mesenchymal cell culture medium: DMEM / F-12 + 10% FBS + 1% anti-anti;

[0083] 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.

[0084] 2. Culture of blastocysts:

[0085] Human naïve embryonic stem cells were incubated with Accutase (Stemcell Technologies, 07922) at 37°C.

[0086] Digest for 3 minutes to disperse the cells into single cells. Then terminate digestion and centrifuge for 5 minutes (200 g). Resuspend the cell pellet in 0.1% gelatin (Thermo Scientific, CAS 9000-70-8) coated tissue culture dishes and incubate at 37°C for 30 minutes to remove mouse embryonic fibroblasts. Collect the supernatant containing human naïve stem cells and filter through a 40 μm cell filter (Falcon, 352340). Count the cells using a hemocytometer (MARIENFELD, AP-650010).

[0087] The cells were resuspended in blastosome formation medium-I (BFM-I).

[0088] The BFM-I medium is formulated as follows: DMEM / F12 medium (Gibco, Cat#11320-033) and Neurobasal medium (Invitrogen, Cat#21103049) are mixed at 1:1 by volume and the following components are added:

[0089] 0.25x N2 supplement (Gibco, Cat#17502048);

[0090] 0.25x B27 supplement (Gibco, Cat#12587010);

[0091] 0.5x GlutaMAX (Gibco, Cat#35050-061);

[0092] 0.5x Non-essential amino acids (Gibco, Cat#11140050);

[0093] 0.1 mM beta-mercaptoethanol (Gibco, Cat#21985023);

[0094] 0.5% KnockOut Serum Replacement (KSR) (Gibco, Cat#10-828-010);

[0095] 0.5% Penicillin-Streptomycin (Gibco, Cat#15140122);

[0096] 1 mM PD0325901 (Selleckchem, Cat#S1036);

[0097] 0.5 mM IM-12 (Enzo, BML-WN102-0025);

[0098] 0.5 mM SB590885 (R&D systems, 2650);

[0099] 1 mM WH-4-023 (Selleckchem, S7565);

[0100] 20 ng / ml recombinant human LIF (Peprotech, 300-05);

[0101] 10 ng / ml Activin A (Peprotech, 120-14E);

[0102] 50 nM Chroman 1 (MedChem Express, 1273579-40-0)

[0103] 5 μM Emricasan (Selleckchem, IDN-6556);

[0104] 1x Polyamine Supplement (Sigma, P8483);

[0105] 0.7 μM Trans-ISRIB (Tocris, 5284).

[0106] At the same time, 500 μl of anti-adhesion wash (STEMCELL Technologies, 07010) was added to each well of the AggreWell-400 plate (STEMCELL Technologies, 34415), centrifuged at 1900 g for 5 minutes, and incubated at room temperature for at least 30 minutes. Wash once with BFM-I, re-add 0.7 ml of BFM-I, and centrifuge at 1900 g for 5 minutes again. Add 1 ml of BFM-I to each well, and add 100,000 cells per well, centrifuge at 100 g for 3 minutes after uniform distribution. Incubate at 37 °C, 5% O2 and 5% CO2.

[0107] Day 0 is the cell seeding day. On day 1, replace the medium from BFM-I to BFM-II, replace half of the volume of medium each time, a total of two times. After that, update the BFM-II medium in the form of replacing half of the volume every day. Usually, the formation of the cystic embryo can be observed after 4 to 6 days of culture.

[0108] The formula of the BFM-II medium is: mix DMEM / F12 medium (Gibco, Cat#11320-033) with Neurobasal medium (Invitrogen, Cat#21103049) at a volume ratio of 3:1, and add the following ingredients:

[0109] 0.25x N2 supplement (Gibco, Cat#17502048);

[0110] 0.25x B27 supplement (Gibco, Cat#12587010);

[0111] 0.5x GlutaMAX (Gibco, Cat#35050-061);

[0112] 0.5x Non-Essential Amino Acids (Gibco, Cat#11140050);

[0113] 0.1 mM β-mercaptoethanol (Gibco, Cat#21985023);

[0114] 0.5% KnockOut Serum Replacement (KSR) (Gibco, Cat#10-828-010);

[0115] 0.5% Penicillin-Streptomycin (Gibco, Cat#15140122);

[0116] 1 μΜ PD0325901 (Selleckchem, Cat#S1036);

[0117] 2 μΜ A83-01 (Tocris Bioscience, Cat#2939);

[0118] 0.5 μΜ SB590885 (R&D Systems, Cat#2650);

[0119] 1 μΜ WH-4-023 (Selleckchem, Cat#S7565);

[0120] 10 ng / ml recombinant human LIF (Peprotech, Cat#300-05);

[0121] 0.5 μΜ LPA (MedChem Express, Cat#HY-107614).

[0122] 3. Design and fabrication of in vitro embryo implantation chip

[0123] The in vitro embryo implantation chip adopts a microfluidic chip structure, which is a parallel three-channel structure: the middle is a gel / blastoid loading channel, and the left and right sides are culture medium channels (medium channels); the middle channel size: 6 mm long x 1.5 mm wide x 5 mm deep.

[0124] The middle channel is connected with the other two medium channels, and there is a 0.1 mm long barrier branch at the top of the chip, which separates the middle channel and the medium channel, allowing the gel to be confined in the middle space by surface tension in the middle channel. This design separates the endometrial-like medium in the medium channel from the implantation medium in the top window.

[0125] The medium channel is used to separate the endometrial organoid culture medium and the implantation culture medium.

[0126] The mold of the microfluidic chip was designed in AutoCAD, imported into Fusion 360 to generate a numerical control machining program, and then a plastic mold was generated using micro-NC. The PDMS curing agent and the base polymer were mixed at a ratio of 1:10, poured into the mold, and a polydimethylsiloxane (PDMS, Dow Corning, 01673921) based microfluidic chip was prepared. The PDMS mold was vacuum treated for 4 hours and baked at 65°C for 12 hours. The PDMS cuttings were peeled off, and holes were punched at predetermined positions. The bottom of the chip and a clean cover glass were treated with plasma at 150 w for 3 minutes, then the bottom and the activated cover glass surface were tightly attached for 1 minute, and baked at 65°C for 12 hours. The chip was applied within 1-7 days after ultraviolet irradiation. The schematic diagram of the prepared in vitro embryo implantation chip structure is shown in Figure 2 , and the in vitro embryo implantation chip is shown in Figure 3 .

[0127] 4. Preparation of in vitro implantation model:

[0128] 4.1 Construction of endometrial model:

[0129] Embryo implantation occurs in the endometrium, involving single-layer luminal epithelial cells (LE), glandular epithelium embedded between interstitial cells (GE), interstitial cells, blood vessels, and immune cells. A three-dimensional endometrial model was established, combining epithelial cells and interstitial cells obtained from the endometrial biopsy of a fertile woman. The epithelial cells were cultured in Matrigel to form endometrial epithelial organoids, which could differentiate into LE and GE; while the interstitial cells were maintained as a monolayer culture. The endometrial epithelial organoids expressed CK7 and E-cadherin (E-CAD) markers, while the interstitial cells were positively stained for Vimentin (VIM) and N-cadherin (N-CAD). Both types of cells were in a proliferative state, as evidenced by KI67 and PCNA staining Figure 4 and Figure 5 ).

[0130] Normal implantation occurs during the “implantation window”, which corresponds to the secretory phase of the menstrual cycle. To mimic this stage in vitro, endometrial epithelial organoids and stromal cells were treated with estrogen (E2) alone to mimic the proliferative phase, or with E2, progesterone (P4), and cyclic adenosine monophosphate (cAMP) to mimic the secretory phase. After hormone treatment, endometrial epithelial organoids significantly upregulated E2-responsive genes, including estrogen receptor (ESR), Indian hedgehog signaling molecule (IHH), and olfactomedin 4 (OLFM4), as well as P4-responsive genes, such as progesterone receptor (PGR), 17beta-hydroxysteroid dehydrogenase (HSD17beta2), secreted phosphoprotein 1 (SPP1), and paired box gene 8 (PAX8). These results confirmed that endometrial epithelial organoids could mimic the hormonal responses of the menstrual cycle in vitro Figures 6-9 ). Notably, PGR translocated from membrane localization to the nucleus in endometrial epithelial organoids, marking the beginning of the P4-dominated secretory phase Figure 10 ).

[0131] Stromal cells responded to E2, P4, and cAMP by decidualizing, changing from long strands to round shapes Figure 11 , and upregulating markers such as prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1), suggesting that they entered the receptive state Figure 12 . Hormone treatment also enhanced the proliferative capacity of endometrial epithelial organoids and stromal cells Figure 13 and Figure 14 . In addition, endometrial receptivity assays (ERAs) identified 248 genes associated with uterine receptivity through second-generation sequencing, confirming that endometrial cells treated with E2, P4, and cAMP had entered the receptive state Figure 15 ).

[0132] To optimize the extracellular matrix (ECM) that is suitable for both endometrial cell spreading and blastocyst invasion, we tested three commercially available Matrigel variants with different stiffness. The results showed that Matrigel 354277 was the most suitable for stromal cell spreading and growth in a three-dimensional environment (hereinafter referred to as Matrigel) Figures 16-18 . However, when used alone, it was not conducive to blastocyst invasion, suggesting that the ECM needs to be further optimized.

[0133] During implantation, trophoblast cells infiltrate the endometrium by degrading the extracellular matrix (ECM) through secretion of matrix metalloproteinases (MMPs). To better recapitulate this process in our model, we combined the MMP-degradable hydrogel with Matrigel. We tested different dilutions and mixing ratios to determine the optimal combination that could support both stromal and glandular epithelial (GE) cells for three-dimensional growth within the gel, while allowing luminal epithelial (LE) cells to form a monolayer on the surface. The results found that the optimal mixing ratio was 80% degradable hydrogel (1:3 dilution) with 20% Matrigel, which supported the most efficient spreading and non-aggregation of stromal cells in a three-dimensional environment ( Figure 19 and Figure 20 ). The Young’s modulus of this ECM was approximately 184 Pa, which highly coincided with the stiffness of the uterine tissue during the gestation period in vivo (171-250 Pa) Figure 21 . For the expansion of endometrial epithelial organoids on the surface to form LE, a 50% Matrigel concentration was optimal Figure 22 .

[0134] To construct a three-dimensional endometrial model in the in vitro embryo implantation chip, as shown in Figure 1 , first, mix 1:3 (v / v) hydrogel (THE WELL BIOSCIENCE, TWG010) + 20% Matrigel (Corning, 354277) (v / v) to prepare the artificial extracellular matrix, and precoat 2 μl of the artificial extracellular matrix onto the chip through the gel / blastocyst loading window as the base layer, and incubate at 37°C for 30 minutes. Mix another 5 μl of 1:3 hydrogel (25%) + 20% Matrigel with 6.5x10 3 stromal cells and 2x10 3 endometrial epithelial organoids, and incubate at 37°C for 30 minutes to solidify the scaffold as the middle part. Coat 1 μl of 50% Matrigel thin layer on the surface in the middle of the chip through the gel / blastocyst loading window, and place the chip in a 37°C incubator for 30 minutes. After the Matrigel layer solidifies, spread the fragmented endometrial epithelial organoids (about 3x10 3 ) on the 50% Matrigel through the gel / blastocyst loading window. Add EXM+10% FBS (endometrial epithelial organoid culture medium) through the culture medium reservoir for endometrial-like cell culture. The stromal cells and glands can expand within 1 day, and the luminal epithelium can form a monolayer within 2 days. The final three-dimensional endometrial epithelial organoid model is similar in structure to the in vivo endometrium, containing luminal epithelial cells (E-CAD + , LGR5 + ), glandular cells (E-CAD + , PAEP + ), and stromal cells (VIM+ )( Figure 23 、 Figure 24 and Figure 25 ).

[0135] 4.2 Endometrial microcirculation disorder type RIF implantation model:

[0136] The three-dimensional endometrial model established by 4.1 verification is similar in structure to the in vivo endometrium, and the in vitro implantation model conforming to the endometrial microcirculation disorder type RIF is established in turn.

[0137] First, the endometrial epithelial organoids and stromal cells of the endometrial microcirculation disorder type RIF are isolated and cultured. Then, 1:3 (v / v) hydrogel (THE WELL BIOSCIENCE, TWG010) + 20% Matrigel (Corning, 354277) (v / v) are mixed to prepare an artificial extracellular matrix, 2 μl of which is pre-coated on the chip through the gel / blastocyst loading window as the base layer, and incubated at 37°C for 30 minutes. Another 5 μl of 1:3 hydrogel (25%) + 20% Matrigel is mixed with 6.5x10 3 Stromal cells (stromal cells of human endometrial microcirculation disorder type RIF) and 2x10 3 Endometrial epithelial organoids (human endometrial microcirculation disorder type RIF endometrial epithelial organoids) are mixed and incubated at 37°C for 30 minutes to solidify the scaffold as the middle part. A 1 μl thin layer of 50% Matrigel is coated on the surface of the middle part of the chip through the gel / blastocyst loading window, and the chip is placed in a 37°C incubator for 30 minutes. After the Matrigel layer is solidified, the fragmented endometrial epithelial organoids (about 3 x 10 3 Human endometrial microcirculation disorder type RIF endometrial epithelial organoids) are laid on the 50% Matrigel. EXM + 10% FBS (endometrial epithelial organoid culture medium) is added through the culture medium reservoir to culture the endometrial-like cells. The stromal cells and glands can be expanded within 1 day, and the luminal epithelium can form a monolayer within 2 days. Further, the in vitro implantation model is constituted by placing the blastocyst / blastocyst construct in the gel / blastocyst loading window of the endometrial model.

[0138] The above endometrial epithelial organoids and stromal cells are respectively the first part of the isolated and cultured human endometrial epithelial organoid cells and human stromal cells.

[0139] 5. Drug screening:

[0140] Although there are currently various animal models, in vitro culture systems and organoid models for studying the endometrium and the process of embryo implantation, there are still the following deficiencies and limitations in the field of drug screening and mechanism research:

[0141] (1) Species differences lead to results that do not match the clinic:

[0142] Existing studies rely on mouse or primate models to verify drug efficacy and study disease mechanisms. However, there are significant differences between different species in the regulation of reproductive endocrinology, immune response, and embryo implantation mechanisms, making it difficult for experimental results to accurately reflect the true physiological environment of the human endometrium.

[0143] Therefore, the drug validation results of animal experiments often do not match the clinical efficacy, limiting their application value in drug screening.

[0144] (2) Two-dimensional cell models are too simple in structure:

[0145] Traditional two-dimensional culture models or microfluidic chip models can to some extent achieve cell drug response evaluation, but due to the lack of three-dimensional tissue structure, cell-cell interaction, and dynamic hormone regulation environment, it is difficult to simulate the whole process of endometrial receptivity changes and embryo implantation.

[0146] Therefore, the predictive ability of such models is limited and not suitable for drug screening and mechanism verification of complex diseases such as RIF.

[0147] (3) Clinical sample research is limited by ethics and sample collection:

[0148] Due to the difficulty in obtaining clinical endometrial tissue, the limited sample size, large individual differences, and complex periodic changes, it is impossible to establish a standardized and repeatable drug screening system.

[0149] (4) Existing three-dimensional models have obstacles in repeatability and high-throughput screening:

[0150] Current three-dimensional organoids or co-culture models have reconstructed the endometrial structure to some extent, but still have the following shortcomings:

[0151] The structure is unstable (such as the water droplet-like structure formed by PEG hydrogel models, which lacks a luminal epithelial layer);

[0152] The culture system is complex (such as the ALI model which relies on Transwell devices, which is not conducive to batch operation and imaging analysis);

[0153] The culture cost is high, the operation is complicated, and a large amount of cells and matrix materials are required;

[0154] The repeatability of the model is poor, the standardization is low, and it is difficult to achieve high-throughput drug screening and quantitative comparison.

[0155] (5) Lack of systematic drug screening platform:

[0156] Currently, there is no in vitro model that can stably, repeatedly and efficiently simulate the physiological state of human endometrium and the process of embryo implantation, resulting in that the screening and verification of RIF-related drugs still mainly rely on clinical experience or low-throughput experiments, which is low in efficiency, long in cycle and high in cost.

[0157] Based on this, the application uses the constructed in vitro cell model for drug screening. The drug screening process is as shown in Figure 26 The steps are as follows:

[0158] Step 1: 1119 drugs are selected from the FDA-approved drug library based on the transcriptome characteristics of RIF endometrium;

[0159] Step 2: In vitro culture of endometrial epithelial organoids, and treatment of endometrial epithelial organoids with 1119 drugs screened in step 1, identification of drugs that can promote cell viability and avoid toxicity by CCK-8 experiment and optical density (OD) determination, and selection of top 50 drugs;

[0160] Step 3: Preparation of in vitro implantation model of endometrial microcirculation disorder type of repeated embryo implantation failure;

[0161] Step 4: After treating the in vitro implantation model with the 50 drugs screened in step 2, blastocysts are added to the middle region of the in vitro implantation model for co-culture, and the invasion and growth of the blastocysts are monitored 48 hours after plating to screen candidate drugs;

[0162] Step 5: Verification stage, using human pre-implantation blastocysts to verify whether the best candidate drug screened promotes embryo implantation in patients with endometrial microcirculation disorder type RIF.

[0163] 5.1 Round I—Drug screening based on RIF endometrial transcriptome characteristics (determine 1119 drugs)

[0164] Technical route: bulk RNAseq transcriptome characteristics and gene ontology (GO) related pathway analysis are performed on endometrial cells of 21 RIF patients, and the RIF patients are classified according to the transcriptome characteristics. Different FDA-approved drug libraries are used as the starting point, more than 3000 drugs per library, according to the classification of RIF patients, their transcriptome characteristics, gene ontology and related pathways, pharmacology and related pathway analysis of drugs, the drug library with the most relevant to the RIF endometrial cells is preferentially selected.

[0165] (1) Sample sequencing:

[0166] Endometrial cell samples of RIF patients and controls were collected, total RNA was extracted and bulk RNA-sequencing was performed to obtain the transcriptome expression matrix of each sample.

[0167] (2) Differential expression and functional annotation;

[0168] (3) Differential expression analysis (DEG) was performed on the RIF group (21) and the control group (6 cases), and gene ontology and pathway enrichment analysis was performed on the differential genes to identify the functional annotations and abnormal signaling pathways related to RIF pathology. The results are as follows Figure 27 , Physiological characteristics of endometrial microcirculation disorder type RIF patients: abnormal angiogenesis, abnormal injury repair, etc.

[0169] (4) Pathway and drug association analysis:

[0170] The gene ontology / pathway characteristics of each RIF transcriptome subtype were compared with the target and action pathway information of the drug, and appropriate drugs were selected from the FDA-approved drug library according to the comparison results (as shown in Tables 1-14 below), and the known target, action pathway and pharmacological annotation information of each drug were obtained.

[0171] Table 1 Drug names on the first plate of the drug library (14 plates in total):

[0172] ;

[0173] Table 2 Drug names on the second plate of the drug library:

[0174] ;

[0175] Table 3 Drug names on the third plate of the drug library:

[0176] ;

[0177] Table 4 Drug names on the fourth plate of the drug library:

[0178] ;

[0179] Table 5 Drug names on the fifth plate of the drug library:

[0180] ;

[0181] Table 6 Drug names on the sixth plate of the drug library:

[0182] ;

[0183] Table 7 Drug names on the seventh plate of the drug library:

[0184]

[0185] Table 8: Drug names on the eighth plate of the drug library

[0186]

[0187] Table 9: Drug names on the ninth plate of the drug library

[0188]

[0189] Table 10: Drug names on the tenth plate of the drug library

[0190]

[0191] Table 11: Drug names on the eleventh plate of the drug library

[0192]

[0193] Table 12: Drug names on the twelfth plate of the drug library

[0194]

[0195] Table 13: Drug names on the thirteenth plate of the drug library

[0196]

[0197] Table 14: Drug names on the fourteenth plate of the drug library

[0198]

[0199] 5.2 Round II - High-throughput primary screening of stromal cells / epithelial organoids:

[0200] (1) Inoculation to pre-culture:

[0201] Inoculate each 96-well plate well with 2 μΐ of Matrigel containing endometrial epithelial organoids (endometrial epithelial organoid volume prepared according to routine) or 3,000 initial stromal cells Part 1 mentioned, plated and allowed to stabilize gel solidification / attachment 1 day before inoculation.

[0202] Set up blank controls (only culture medium) for each plate of drugs, carrier controls (only endometrial epithelial organoids or stromal cells, plus an equivalent amount of drug solvent DMSO), three replicates per well and repeated three times according to the experimental design to ensure repeatability.

[0203] (2) Drug treatment:

[0204] ​​​​​​​​Each drug in the library was added to the corresponding well at a concentration of 10 mM (i.e., 10 mM was used as the initial screening starting concentration). The drug-containing medium was changed every 2-3 days during the culture period, and the total treatment culture was 5 days (inoculation / pre-culture on day 0, drug addition on day 1, and detection on day 5).

[0205] (3) CCK-8 activity / toxicity detection:

[0206] On day 5 of the experiment, 10 μl of CCK-8 was added to each well to 100 μl of culture medium according to the CCK-8 kit instructions, and incubated for 2 hours (according to the kit instructions), and then the OD value was measured on an enzyme marker at 450 nm.

[0207] The OD value of each well was first subtracted from the blank control background OD, and then the average OD of the carrier control (DMSO) wells in the same plate was used as the general control value (general control OD). The fold change of each drug-treated well relative to the general control was calculated, fold change = (OD_treated-OD_blank) / (OD_control_mean-OD_blank).

[0208] Screening threshold: 1 as the lower threshold (fold≥1 indicates at least no decrease in overall OD), sorted from high to low. The results are shown in Figure 28 After 1119 drug treatments, the relative fold change in OD values of endometrial microcirculation disorder type RIF endometrial epithelial organoids (top) and stromal cells (bottom) relative to the untreated drug (DMSO) control. Red and blue dots represent the top 50 effective drugs for endometrial epithelial organoids (red dots) and stromal cells (blue dots) in each endometrial microcirculation disorder type RIF. Green dots represent shared drugs that act on both endometrial epithelial organoids and stromal cells in the top 50 list. The gray line indicates a fold change of 1 relative to the untreated drug control.

[0209] 5.3 Round III Preparation of in vitro implantation model of endometrial microcirculation disorder type RIF:

[0210] First, endometrial epithelial organoids and stromal cells from endometrial microcirculation disorder type RIF patients were isolated and cultured. Then, 1:3 (v / v) hydrogel and 20% Matrigel (v / v) were mixed to prepare an artificial extracellular matrix, and 2 μl of the matrix was pre-coated onto the chip through gel / blastocyst loading wells as a substrate layer, and incubated at 37°C for 30 minutes. Another 5 μl of 1:3 hydrogel (25%) + 20% Matrigel was mixed with 6.5x10 3 stromal cells and 2x10 3Endometrial epithelial organoids were mixed, incubated at 37°C for 30 min to allow the scaffold to solidify as an intermediate. The middle surface of the chip was coated with 1 μΐ of 50% Matrigel thin layer through the gel / blastocyst loading window, and the chip was placed in a 37°C incubator for 30 min. After the Matrigel layer solidified, the fragmented endometrial epithelial organoids (about 3 × 10 3 ) were plated on the 50% Matrigel through the gel / blastocyst loading window. Endometrial-like cell culture was performed by adding EXM + 10% FBS (endometrial epithelial organoid culture medium) through the medium reservoir. The mesenchymal cells and glands can expand within 1 day, and the luminal epithelium can form a monolayer within 2 days. Further, the blastocyst / blastocyst assembly constitutes an in vitro implantation model by placing it in the endometrial model gel / blastocyst loading window.

[0211] 5.4 Round IV—Screening in the in vitro implantation model:

[0212] (1) Drug pretreatment and co-culture:

[0213] For each drug to be tested, the in vitro implantation model established in Part 4 was pretreated for 3 days, after which the blastocyst was labeled with Q-tracker 655 and added to the middle area of the chip to form an embryo implantation chip. Each implantation chip was seeded with 30 blastocysts, and 30 blastocysts were added from the loading window. Drug treatment was continued during co-culture (i.e., the drug was continuously added during the co-culture period, maintaining the same treatment conditions as the pretreatment).

[0214] (2) Observation indicators and determination time points:

[0215] Attachment determination (attachment rate): determined after 24 hours. Criteria: the blastocyst / blastocyst adheres firmly to the surface of the endometrial model, and can not be displaced when the slide / chip is shaken slightly (recorded under a microscope).

[0216] Outgrowth determination (outgrowth rate): determined after 48 hours. Criteria: the blastocyst / blastocyst sphere is obviously broken and the trophoblast cells spread outwards, forming a monolayer, and the shape spreads out from a spherical shape.

[0217] The number of attachments and outgrowths of 30 blastocysts in each chip was recorded and calculated, rate = number_positive / number_seeded x 100%.

[0218] (3) Screening criteria and candidate determination:

[0219] The attachment rate of each drug group was compared with the control group (corresponding RIF endometrial model untreated DMSO). The drug with significant improvement in attachment / expansion rate and no obvious toxicity was preferentially selected, such as Figure 29 and Figure 30 As shown, the growth rate of blastocyst in the endometrial microcirculation disorder type RIF endometrial model after treatment with identified effective drugs for 48 hours. Mean ± standard deviation, n = 3 biological replicates. The screened candidate drugs include Phenylbutazone, Ferulic acid, L-Proline, Indinavir, Sofalcone, Trelagliptin, Gemcitabine.

[0220] After the most effective drug treatment on the endometrial microcirculation disorder type RIF endometrial model, the blastocyst achieved successful implantation in the endometrial microcirculation disorder type RIF endometrial model. After 7 days of in vitro culture, the embryonic structures (pseudo-amniotic cavity-like structure and pseudo-yolk sac-like structure) reached 18.43%, 19.74% and 11.15%, respectively, while the extraembryonic structures (STB and EVT) appeared 39.53%, 46.88% and 41.63%, respectively, after treatment with the most effective drugs (Phenylbutazone, Ferulic acid (sodium) and L-Proline) (p < 0.05). Figure 31 ).

[0221] 5.5 Round V—Human pre-implantation blastocyst in vitro embryo implantation function verification:

[0222] 1) Blastocyst preparation:

[0223] Obtain the pre-implantation blastocyst of RIF patients from frozen or fresh culture, maintain its physiological state and short-term viability under suitable culture conditions (ethics: 2024-KY-109-01 and 2024-126).

[0224] Embryo thawing: Before thawing the embryos, we previously developed embryo culture expansion medium (EBC) was equilibrated overnight, EBC composition as follows: 1:1 (v / v) DMEM / F12 and Neurobasal, 1xN2, 1xB27, 1xGlutaMAX, 1xnon-essential amino acids, 0.1 mM beta-mercaptoethanol, 15% FBS, 8 nM estradiol, 200 ng / ml progesterone, 1 mM sodium pyruvate (Gibco, 11360070), 5 mg / ml gentamicin (Corning, 30-005-CR), and CEPT small molecule combination. Human blastocysts (5-6 days after fertilization) were thawed using Kitazato Thawing Medium Kit (Kitazato Corporation, VT102) according to the manufacturer's instructions. Briefly, the thawing solution (TS) was preheated at 37°C, and the dilution solution (DS) and washing solution (WS) were warmed to room temperature. The blastocyst was quickly immersed in TS (37°C), DS (room temperature), WS (room temperature), WS (room temperature) for 1 minute, 3 minutes, 5 minutes, 5 minutes, respectively. Then the embryo was transferred to the acid Tyrode's solution (Sigma, T1788) and the zona pellucida was removed under a microscope. Once the zona pellucida disappeared, the embryo was immediately transferred to the pre-equilibrated EBC medium and washed twice.

[0225] 2) Chip inoculation:

[0226] The blastocyst was placed in the established in vitro embryo implantation chip, the gel / embryo area was added with embryo culture medium EBC, and the two side channels were added with endometrial epithelial organoid culture medium, and cultured at 37°C, 5% CO2, and the drug was continuously added during the co-culture period. The treatment conditions were kept consistent with the pretreatment.

[0227] 3) Functional evaluation:

[0228] The adhesion and expansion of the blastocyst in the implantation chip were observed after 24 hours and 48 hours of placing the blastocyst, respectively. The implantation ability of the blastocyst was detected by microscopic observation, and these indicators were compared between the drug treatment group and the control group to verify the effect of the candidate drug on the function of the embryo implanting the uterus.

[0229] As shown in Figure 32 , it is shown that after Phenylbutazone, Ferulic acid, L-Proline drug treatment, the blastocyst successfully implanted.

[0230] Several points to note during the drug screening process in Section 5:

[0231] First Embryo Substitute:

[0232] To circumvent the ethical restrictions of using human embryos, reduce the application threshold, and improve the experimental throughput, the embryo samples used in the in vitro embryo implantation chip can use trophoblast spheroids or other embryo-mimetic structures to replace human embryo organoids (blastocyst-like) or actual human blastocysts to achieve approximate functional verification effect.

[0233] Second drug dosage and treatment time:

[0234] In the drug screening process, the 10 μM uniform initial concentration used in the Round II stage and the same concentration of Round IV verification method can be adjusted according to the specific drug properties. When necessary, further optimal concentration verification experiments can be carried out, which does not affect the overall screening process.

[0235] Third, the consumables of the drug screening process:

[0236] The 96-well plate screening system used in the Round II stage can be replaced by a 384-well plate, or combined with an automatic liquid handling workstation for operation, to expand the screening throughput and reduce human error. At the same time, the chip model of Round IV and V can use microfluidic design to improve the data volume and repeatability.

[0237] The present application overcomes the many limitations of existing animal models and two-dimensional cell models in drug screening by constructing a drug screening system based on an in vitro embryo implantation model, and achieves the following technical advantages:

[0238] (1) Improve the predictability and efficiency of drug screening:

[0239] The present application combines high-throughput drug primary screening with the application of primary organoids, functional biological model chips, and real human pre-implantation blastocysts, realizing a process from a large number of candidate drugs to accurate, small number of individualized drugs and functional verification. In terms of throughput, this strategy changes the complexity, long cycle, high cost and low throughput mode of the traditional clinical drug screening method of using drugs first and tracking drug effects later, and overcomes the inconvenience of the traditional human-based drug screening mode, the species difference of animal models, and the lack of physiological relevance of two-dimensional culture systems through the five rounds of effective in vitro screening steps.

[0240] (2) Individualized screening potential:

[0241] The method is based on a system constructed by human epithelial organoids and primary mesenchymal cells, which uniquely reproduces the selective implantation process with minimal cell requirements (about 6500 mesenchymal cells and about 5000 epithelial cells per chip), allowing screening studies of thousands of drugs from a single patient biopsy, especially suitable for patient-derived samples with limited clinical availability. This feature provides a technical basis for realizing individual drug response prediction and precision medicine.

[0242] (3) Stability and repeatability:

[0243] The combination of organoids and chip technology makes the model structure more stable and the operation more simplified, avoiding the problems of unstable morphology, high cost and poor repeatability of traditional three-dimensional culture system. Through batchable operation design, standardized and high-throughput drug screening can be realized, significantly improving experimental efficiency.

[0244] (4) Wide application prospect:

[0245] In addition to RIF, the screening system established by the present application is also suitable for the research and drug development of other endometrial related diseases, including endometritis, endometriosis, etc. The model can be used for drug screening and disease research, and has wide scientific research and clinical application value.

[0246] Literature cited in the background section of the present application:

[0247] 1. Franasiak, J. M., Alecsandru, D., Forman, E. J., Gemmell, L. C., Goldberg, J. M., Llarena, N., Margolis, C., Laven, J., Schoenmakers, S., and Seli, E. (2021) A review of the pathophysiology of recurrent implantation failure. Fertil. Steril. 116, 1436-1448. https: / / doi.org / 10.1016 / j.fertnstert.2021.09.014.

[0248] 2. Ma, J., Gao, W., and Li, D. (2022). Recurrent implantation failure: A comprehensive summary from etiology to treatment. Front. Endocrinol. 13, 1061766. https: / / doi.org / 10.3389 / fendo.2022.1061766.

[0249] 3. Makrigiannakis, A., Makrygiannakis, F., and Vrekoussis, T. (2021). Approaches to Improve Endometrial Receptivity in Case of Repeated Implantation Failures. Front. Cell Dev. Biol. 9, 613277. https: / / doi.org / 10.3389 / fcell.2021.613277.

[0250] The above-described embodiments are merely possible implementations of the present application, and the protection scope of the present application is not limited thereto. Any simple change or equivalent replacement within the technical range disclosed in the present application, which can be obviously obtained by those skilled in the art, shall belong to the protection scope of the present application.

Claims

1. Use of one of the following: phenylbutazone, ferulic acid, L-proline, indinavir, solfenoxone, linagliptin, gemcitabine in the preparation of a drug for promoting the embryo implantation efficiency of patients with endometrial microcirculation disorder type RIF.

2. Use according to claim 1, characterized in that, The physiological characteristics of the endometrial microcirculation disorder type RIF patient are that the endometrial microvessel density is decreased, the vascular structure and permeability are changed, and the local perfusion capacity is limited.

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