Candidate medicine for promoting embryo implantation efficiency of uterus redox imbalance type RIF patient

Abstract

The invention provides a candidate medicine for promoting the embryo implantation efficiency of a uterus redox imbalance type RIF patient, and belongs to the technical field of embryo implantation. In order to solve the problem of how to discover a potential intervention drug aiming at a uterine redox imbalance type RIF pathological mechanism, a five-step efficient drug screening strategy based on an in-vitro implantation model of uterine redox imbalance type repeated embryo implantation failure is adopted, so that the reliability and functional correlation of a screening result are ensured. A candidate drug screened by adopting a five-step method efficient drug screening strategy comprises any one of the following drugs: avobenzone, vasopressin, dipotassium ethylene diamine tetraacetate, lalotinib sulfate, citicoline, olprinone, eltrombopag, L-leucine, fulvestrant, malic acid and rucotinib. Among the candidate drugs, avobenzone, vasopressin and dipotassium ethylenediaminetetraacetate are three drugs with optimal implantation promotion efficiency for patients with uterine redox imbalance 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 a redox imbalance 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 multiple mechanisms such as endometrial abnormalities, immune dysfunction, microthrombosis, embryonic factors, and endocrine environment. 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 redox imbalance type RIF.

[0004] Endometrial redox imbalance type RIF refers to a type of recurrent implantation failure in which the balance between oxidative stress and antioxidant defense mechanisms in the endometrium is disrupted, leading to decreased endometrial receptivity and impaired embryo implantation. Numerous studies have shown that the endometrium of RIF patients generally has elevated reactive oxygen species (ROS) levels, down-regulated antioxidant molecule expression, and mitochondrial dysfunction during the implantation window, suggesting that redox homeostasis imbalance plays an important role in the occurrence of RIF. Studies have shown that SIRT1 expression is significantly reduced in the endometrium of RIF patients, leading to ROS accumulation and down-regulation of antioxidant gene expression, thereby inhibiting decidualization [1] . Further studies have found that lipid peroxidation damage is particularly significant in RIF patients, and the presence of GPX4 down-regulation and 4-HNE deposition can cause oxidative damage to the uterine epithelium and weaken the embryo attachment capacity [2] . In addition, transcriptomic-based analysis identified key genes (AXL, SLC7A11, UBQLN1) closely related to oxidative stress, and found that abnormal expression of these genes was closely related to immune cell dysfunction in the endometrium, suggesting that oxidative stress can affect embryo implantation through the immune pathway [3]The transcriptome data further shows that the oxidative phosphorylation and mitochondrial energy metabolism pathway in the endometrium of RIF patients is significantly down-regulated, indicating that impaired cell metabolism and abnormal redox regulation may be an important molecular basis for this type of RIF [4] .

[0005] Based on the above observations, the present study will focus on patients with endometrial redox imbalance type RIF as the key object, and through the construction of an in vitro model to simulate the human endometrial-embryo interaction environment, the pathological characteristics and drug sensitivity thereof will be systematically analyzed. The model aims to discover potential intervention drugs targeting the pathological mechanisms related to endometrial redox imbalance, so as to improve the success rate of embryo implantation and pregnancy outcome of RIF patients. SUMMARY

[0006] 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, the endometrial model is constructed using mesenchymal cells and endometrial epithelial organoids derived from patients with endometrial redox imbalance type RIF, and then the in vitro implantation model is made by combining the blastocyst with the endometrial model. The second round of screening is carried out on the 50 drugs preliminarily screened, in order to identify specific drugs that can significantly improve the implantation rate.

[0007] The technical scheme adopted by the present application is: The present application protects, in a first aspect, a candidate drug for promoting embryo implantation efficiency in patients with endometrial redox imbalance type RIF, wherein the candidate drug comprises any one of the following drugs: Avobenzone, Vasopressin, Potassium Ethylenediaminetetraacetate (K2EDTA), Larotrectinib Sulfate, Citicoline, Olprinone, Eltrombopag, L-Leucine, Fulvestrant, Malic acid, Ruxolitinib.

[0008] Further, the physiological characteristics of patients with endometrial redox imbalance type RIF are that the balance between oxidative stress level and antioxidant defense mechanism in the endometrium is destroyed, which is manifested as a significant decrease in SIRT1 expression, leading to ROS accumulation and decreased expression of antioxidant genes.

[0009] Still further, the candidate drug comprises any one of the following drugs: Avobenzone, Vasopressin, Potassium Ethylenediaminetetraacetate (K2EDTA).

[0010] The second aspect of the application protects a method for screening a candidate drug for promoting embryo implantation efficiency in patients with redox imbalance type RIF, which comprises the following steps: Step 1: Select 1119 drugs from the FDA-approved drug library based on the endometrial transcriptome characteristics of redox imbalance type RIF; Step 2: In vitro isolation and culture of endometrial epithelial organoids and stromal cells of patients with redox imbalance type RIF, and treatment of endometrial epithelial organoids or stromal cells with 1119 drugs screened in step 1, use CCK-8 experiment and optical density (OD) determination to identify drugs that can promote cell viability and avoid toxicity, and select the top 50 drugs; Step 3: Preparation of in vitro implantation model of redox imbalance type RIF; Step 4: After treating the in vitro implantation model with the 50 drugs screened in step 2, add blastocyst to the middle region of the in vitro implantation model for co-culture, monitor the invasion and growth of blastocyst 48 hours after plating, and screen the candidate drug.

[0011] Step 5: Verify the embryo implantation efficiency of the screened candidate drug using human pre-implantation blastocyst in the in vitro embryo implantation model.

[0012] Further, the specific operation of step 2 is as follows: Cultivate endometrial epithelial organoids and stromal cells from endometrial tissue; Inoculation and pre-culture: the isolated and cultured endometrial epithelial organoids and stromal cells are used as the experimental group, 2 μl of Matrigel containing endometrial epithelial organoids or 3000 stromal cells are inoculated in each 96-well plate well; Each 96-well 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, three repeated controls are set in each well, and the experiment is repeated three times according to the experimental design; Each drug screened in step 1 is added to the corresponding well of the experimental group at 10 μM, and the culture medium containing the drug is changed every 2-3 days during the culture period, and the total treatment culture is 5 days; wherein, the non-drug treatment control group adds culture medium containing equal amount of DMSO as the drug, and the blank control group only contains culture medium; After 5 days of culture, the OD value is measured using the CCK-8 kit, the cell viability of each drug treatment well relative to the non-drug treatment control group is calculated, and the cell viability is sorted from high to low.

[0013] Furthermore, the formula for calculating cell viability is: fold change = (OD_treated - OD_blank) / (OD_control_mean - OD_blank); where 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-treated control group.

[0014] Furthermore, the in vitro implantation model construction process in step 3 is as follows: Fabrication of in vitro embryo implantation chips; Artificial extracellular matrix was prepared using hydrogel and matrix gel. The artificial extracellular matrix was then pre-prepared onto the in vitro embryo implantation chip through the gel / blastocyst loading window on the chip as a base layer and incubated at 37°C for 30 minutes. 5 μl of hydrogel and matrix gel mixture was mixed with stromal cells and endometrial epithelial organoids and incubated at 37°C for 30 minutes to solidify the scaffold as an intermediate layer; Coat the surface of the intermediate layer with a 1 μl 50% matrix gel and incubate at 37°C for 30 minutes. After the matrix gel layer solidifies, fragmented endometrial epithelial organoids are laid on the matrix gel layer through the gel / blastocyst loading window; Endometrial models were cultured by adding endometrial epithelial organoid culture medium through the culture medium reservoir.

[0015] Furthermore, in step 4, after pretreating the in vitro implantation model with 50 selected drugs for 3 days, the blastocysts were added to the middle region of the in vitro implantation model through the gel / blastocyst loading window, and the drugs were continuously added during the co-culture period.

[0016] Furthermore, after screening candidate drugs in step 4, the blastocysts were labeled with Q-tracker 655 and implanted into an in vitro implantation model pretreated with the top 3 candidate drugs (Avobenzone, Vasopressin, and K2EDTA) selected in step 4 for 3 days. The implantation status of the blastocysts was observed 48 hours later.

[0017] Alternatively, in an in vitro implantation model where the top three candidate drugs selected in step 4 were treated for 3 days without labeling, after 7 days of in vitro culture, the embryonic structures (amniotic cavity-like structures and yolk sac-like structures) reached 12.54%, 6.63%, and 6.49%, respectively, while the probabilities of the simultaneous occurrence of extraembryonic structures syncytiotrophoblast (STB) and chorionic exotrophoblast (EVT) were 33.53%, 28.02%, and 23.89%, respectively, corresponding to the treatment with the most effective drugs (Avobenzone, Vasopressin, and K2EDTA).

[0018] Furthermore, after screening candidate drugs in step 4, in step 5, the blastocysts that were not implanted into the in vitro implantation model of patients with endometrial redox imbalance RIF who were pretreated with the top 3 candidate drugs (Avobenzone, Vasopressin, and K2EDTA) in step 4 for 3 days, to verify whether the best candidate drugs selected can enable embryo implantation.

[0019] Beneficial effects: This invention employs a five-step efficient drug screening strategy based on an in vitro implantation model of recurrent embryo implantation failure (RIF) with uterine redox imbalance to ensure the reliability and functional relevance of screening results. Round I uses endometrial transcriptomic characteristics and gene ontology pathway analysis based on RIF / other endometrial diseases to prioritize matching against FDA-approved drug libraries for categorized drug recommendations. Round II performs high-throughput initial screening in endometrial epithelial organoids and stromal cells, evaluating candidate drugs through cell activity and toxicity indicators to narrow down the range of effective drugs. Round III utilizes an in vitro implantation model of endometrial epithelial cells, stromal cells, and blastocyst-like constructs from patients with RIF with uterine redox imbalance. Round IV uses the in vitro embryo implantation model and blastocysts to validate the implantation capability of the previously effective drugs, observing the drug's attachment and invasion regulation of the endometrial-embryo interaction. Round V uses human preimplantation blastocysts in the in vitro implantation model to validate embryo implantation and early developmental function, ensuring the physiological relevance and effectiveness of the screened drugs.

[0020] The five-step efficient drug screening strategy employed in this invention selects the following effective drugs: Avobenzone, Vasopressin, K2EDTA, Larotrectinib sulfate, Citicoline, Olprinone, Eltrombopag, L-Leucine, Fulvestrant, Malic acid, and Ruxolitinib. Among these drugs, Avobenzone, Vasopressin, and K2EDTA were the three drugs with the best promoting effect in patients with uterine redox imbalance-type RIF. In the in vitro implantation model where the top three candidate drugs (Avobenzone, Vasopressin, and K2EDTA) were treated for 3 days without labeling, after 7 days of in vitro culture, the embryonic structures (amniotic cavity-like structures and yolk sac-like structures) reached 12.54%, 6.63%, and 6.49%, respectively, while the extraembryonic structures (STB and EVT) appeared at 33.53%, 28.02%, and 23.89%, respectively, corresponding to the treatment with the most effective drugs (Avobenzone, Vasopressin, and K2EDTA). Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the implantation model generated from a blastocyst-endometrial model.

[0023] Figure 2 This is a schematic diagram of an in vitro embryo implantation chip.

[0024] Figure 3 Images of microchip implanted in in vitro embryos.

[0025] Figure 4 This is a representative immunofluorescence (IF) image of endometrial epithelial organoids, showing the epithelial cell markers CK7 and KI67.

[0026] Figure 5 Representative IF images of mesenchymal cells show the mesenchymal cell markers N-Cadherin and Vimentin.

[0027] Figure 6This is a representative IF image of the downstream gene IHH of endometrial epithelial organoids during the proliferative phase after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0028] Figure 7 This is a representative IF image of HSD17β2, a downstream gene of endometrial epithelial organoids in the secretory phase, after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0029] Figure 8 This is a representative IF image of SPP1, a downstream gene of endometrial epithelial organoids during the secretory phase, after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0030] Figure 9 This is a QPCR assay for downstream gene expression in the proliferative or secretory phases of endometrial epithelial organoids after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0031] Figure 10 This is a representative IF image of the translocation of the progesterone receptor (PGR) from the cell membrane to the cell nucleus during the secretory phase of endometrial epithelial organoids after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0032] Figure 11 This is a representative IF image showing the morphological changes of secretory cells after decidualization following co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0033] Figure 12 qPCR was used to express the secretory genes PRL and IGFBP1 in stromal cells after co-treatment with estrogen (E2), progesterone (P4), and cAMP.

[0034] Figure 13 CCK-8 OD values ​​represent the proliferation of endometrial epithelial organoids in the proliferative or secretory phase after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0035] Figure 14 The CCK-8 OD value represents the proliferation of mesenchymal cells in the proliferative or secretory phase after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0036] Figure 15 This is a cluster diagram of endometrial receptivity gene expression in endometrial organoids and stromal cells during the proliferative or secretory phases after treatment with estrogen (E2) or a combination of estrogen (E2), progesterone (P4), and cAMP.

[0037] Figure 16 Hardness tests were conducted on three different base adhesives.

[0038] Figure 17 Representative IF images of the unfolding of three different matrix glial intermediate cells.

[0039] Figure 18 A statistical graph showing the unfolded area of ​​three different matrix glial intermediate cells.

[0040] Figure 19 Representative IF images showing the unfolding of mesenchymal cells under different mixing ratios of matrix gel and hydrogel.

[0041] Figure 20 A statistical graph showing the proportion of mesenchymal cells unfolded under different mixing ratios of matrix gel and hydrogel.

[0042] Figure 21 Hardness tests were conducted for different mixing ratios of the matrix adhesive and hydrogel.

[0043] Figure 22 Representative IF images showing the development of endometrial epithelial organoid fragments in different proportions of Matrices.

[0044] Figure 23 Representative IF images of a 3D radiograph of an endometrial model. E-Cad: glandular epithelium (GE) / luminal epithelium (LE), VIM: Str (stromal cells).

[0045] Figure 24 Representative IF images of a real uterus and endometrial model. E-Cad: glandular epithelium (GE) / luminal epithelium (LE), VIM: Str (stromal cells).

[0046] Figure 25 IF images of the luminal epithelium (LE:LGR5) and glandular epithelium (GE:PAEP) of the endometrial model.

[0047] Figure 26 This is a schematic diagram of the drug screening process.

[0048] Figure 27 Ridge plot for promoting transcriptome gene ontology analysis of endometrial cells in patients with uterine redox imbalance-type RIF.

[0049] Figure 28This represents the relative fold change in OD values ​​of RIF endometrial epithelial organoids (top) and stromal cells (bottom) compared to the untreated drug (DMSO) control after treatment with 1119 drugs. Red and blue dots represent the top 50 effective drugs for endometrial epithelial organoids (red dots) and stromal cells (blue dots), respectively; green dots represent shared drugs in the top 50 list that act on both endometrial epithelial organoids and stromal cells.

[0050] Figure 29 The implantation rate of blastocysts at 48 hours in a uterine redox imbalance RIF endometrial model after treatment with an effective drug was defined as mean ± standard deviation, n=3 biological replicates.

[0051] Figure 30 Images of implantation of Q-tracker 655-labeled blastocysts in a uterine redox imbalance-type RIF in vitro implantation model with or without representative drug treatment.

[0052] Figure 31 Representative intra- and extra-embryonic images (IFs) of blastocyst intra- and extra-embryonic structures after 168 hours of in vitro culture in a blastocyst implantation model of uterine redox imbalance-induced RIF treated with Avobenzone, Vasopressin, and K2EDTA. Green dashed lines represent AC-like structures, yellow dashed lines represent YS-like structures, yellow arrows represent STBs, and blue arrows represent EVTs. GATA3: purple, OCT4 / HLA-G: green, GATA6: yellow, hCG: red.

[0053] Figure 32 After 168 hours of in vitro culture of organoids, the development rates of intraembryonic structures (amniotic cavity-like structures and yolk sac-like structures) and extraembryonic structures (STB and EVT) under different drug treatments were statistically analyzed. W / : present, W / O: absent.

[0054] Figure 33 This describes the implantation status of blastocysts in a redox-disequilibrium RIF implantation model in the uterus with and without drug treatment.

[0055] Figure 34 Human blastocyst development in an endometrial model after Avobenzone treatment. OCT4: green, VIM: yellow, GATA6 / GATA3: purple. Red arrows indicate OCT4+ embryonic epithelium. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0057] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0058] Cell source: 1. Culture of human endometrial epithelial organoids and human cytoplasmic cells: All endometrial tissue biopsy samples were collected during the proliferative phase of the menstrual cycle (days 3–7 after menstruation, i.e., days 7–13 of the menstrual cycle), including healthy individuals and patients with uterine redox imbalance-related recurrent uterine fibroids (RIF).

[0059] Endometrial tissue was washed with DMEM / F12 medium and then digested at 37°C with a mixture of Dispase and collagenase for 40 minutes. Digestion was terminated with DMEM / F12 containing 10% FBS. The digestion solution was separated through a 40 μm filter to obtain endometrial epithelial cells (greater than 40 μm) and stromal cells (less than 40 μm).

[0060] 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.

[0061] The required culture medium is as follows: Mesenchymal 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.

[0062] 2. Culture of blastocysts: Human naïve embryonic stem cells were incubated with Accutase (Stemcell Technologies, 07922) at 37°C. 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 a 0.1% gelatin (Thermo Scientific, CAS 9000-70-8) coated tissue culture dish 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).

[0063] The cells were resuspended in blastoid formation medium-I (BFM-I).

[0064] The formulation of the BFM-I medium is as follows: DMEM / F12 medium (Gibco, Cat#11320-033) and Neurobasal medium (Invitrogen, Cat#21103049) are mixed at a volume ratio of 1:1, and the following components are added: 0.25×N2 additive (Gibco, Cat#17502048); 0.25×B27 additive (Gibco, Cat#12587010); 0.5×GlutaMAX (Gibco, Cat#35050-061); 0.5× non-essential amino acids (Gibco, Cat#11140050); 0.1 mM β-mercaptoethanol (Gibco, Cat#21985023); 0.5% KnockOut Serum Replacement (KSR) (Gibco, Cat#10-828-010); 0.5% penicillin-streptomycin (Gibco, Cat#15140122); 1 μM PD0325901 (Selleckchem, Cat#S1036); 0.5 mM IM-12 (Enzo, BML-WN102-0025); 0.5 μM SB590885 (R&D systems, 2650); 1 μM WH-4-023 (Selleckchem, S7565); 20 ng / ml recombinant human LIF (Peprotech, 300-05); 10 ng / ml Activin A (Peprotech, 120-14E); 50 nM Chroman 1 (MedChem Express, 1273579-40-0) 5 μM Emricasan (Selleckchem, IDN-6556); 1× Polyamine Supplement (Sigma, P8483); 0.7 μM Trans-ISRIB (Tocris, 5284).

[0065] Simultaneously, add 500 μl of anti-adhesion wash buffer (STEMCELL Technologies, 07010) to each well of an AggreWell-400 plate (STEMCELL Technologies, 34415), centrifuge at 1900 g for 5 min, and incubate at room temperature for at least 30 min. Wash once with BFM-I, then add 0.7 ml of BFM-I again, and centrifuge at 1900 g for 5 min again. Add 1 ml of BFM-I to each well, then add 100,000 cells / well, distribute evenly, and centrifuge at 100 g for 3 min. Incubate at 37 ℃, 5% O2, and 5% CO2.

[0066] Day 0 is the cell seeding day. On Day 1, the culture medium is changed from BFM-I to BFM-II, with half the volume of medium replaced each time, for a total of two replacements. Thereafter, the BFM-II medium is replaced daily with half the volume of medium. Thylakoid formation is usually observed after 4 to 6 days of culture.

[0067] The BFM-II medium is formulated as follows: DMEM / F12 medium (Gibco, Cat#11320-033) and Neurobasal medium (Invitrogen, Cat#21103049) are mixed at a volume ratio of 3:1, and the following components are added: 0.25×N2 additive (Gibco, Cat#17502048); 0.25×B27 additive (Gibco, Cat#12587010); 0.5×GlutaMAX (Gibco, Cat#35050-061); 0.5× non-essential amino acids (Gibco, Cat#11140050); 0.1 mM β-mercaptoethanol (Gibco, Cat#21985023); 0.5% KnockOut Serum Replacement (KSR) (Gibco, Cat#10-828-010); 0.5% penicillin-streptomycin (Gibco, Cat#15140122); 1 μM PD0325901 (Selleckchem, Cat#S1036); 2 μM A83-01 (Tocris Bioscience, Cat#2939); 0.5 μM SB590885 (R&D Systems, Cat#2650); 1 μM WH-4-023 (Selleckchem, Cat#S7565); 10 ng / ml recombinant human LIF (Peprotech, Cat#300-05); 0.5 μM LPA (MedChem Express, Cat#HY-107614).

[0068] 3. Design and fabrication of in vitro embryo implantation microarrays: The in vitro embryo implantation chip adopts a microfluidic chip structure, which is a parallel three-channel structure: the middle channel is the gel / blastocyst (blastocyst) loading channel, and the left and right sides are culture medium channels (medium channels); the dimensions of the middle channel are: 6 mm long × 1.5 mm wide × 5 mm deep.

[0069] The central channel is connected to the other two media channels. A 0.1 mm long barrier branch at the top of the chip separates the central channel from the media channels, allowing the gel to be confined within the central space by surface tension. This design separates the endometrial-like culture medium within the media channels from the implantation culture medium within the top window.

[0070] The media channel is used to separate endometrial organoid culture media and implantation culture media.

[0071] The microfluidic chip mold 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 chip bottom 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 prepared in vitro embryo implantation chip structure is shown below. Figure 2 As shown, an in vitro embryo implantation microarray image is as follows. Figure 3 As shown.

[0072] 4. Preparation of in vitro implantation models: 4.1 Construction of the endometrial model: 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. A three-dimensional 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, while stromal cells were positive for Vimentin (VIM) and N-cadherin (N-CAD). Both cell types were in a proliferative state, which was confirmed by KI67 and PCNA staining. Figure 4 and Figure 5 ).

[0073] Normal implantation occurs during the "implantation window," corresponding to the 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, or with the addition of E2, progesterone (P4), and cyclic adenosine monophosphate (cAMP) to simulate 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 6-9 Notably, PGRs shift from membrane localization to the nucleus in endometrial epithelial organoids, marking the beginning of the P4-dominated secretory phase. Figure 10 ).

[0074] Interstitial cells respond to E2, P4, and cAMP, undergoing decidualization and transforming from elongated to round shapes. Figure 11 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 12 Hormone treatment also enhanced the proliferative capacity of endometrial epithelial organoids and stromal cells. Figure 13 and 14 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 15 ).

[0075] 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 16-18 However, when used alone, the matrix gel did not facilitate blastocyst invasion, suggesting the need for further optimization of the ECM.

[0076] 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) to 20% matrix gel, a combination that 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 is approximately 184 Pa, which closely matches the stiffness of uterine tissue during pregnancy (171–250 Pa). Figure 21 For the amplification of endometrial epithelial organoids on the surface to form LE, a 50% Matrigel concentration is most effective. Figure 22 ).

[0077] To construct a three-dimensional endometrial model in an in vitro embryo implantation chip ( Figure 1 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 artificial extracellular 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 and 2x10 3 Endometrial epithelial organoids 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 chip through the gel / blastocyst loading window, and the 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 Endometrial-like cells were spread on 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 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)+ () Figure 23 , Figure 24 and Figure 25 ).

[0078] 4.2 Uterine redox imbalance type RIF in vitro implantation model: The three-dimensional endometrial model established in 4.1 is structurally similar to the in vivo endometrium. Subsequently, an in vitro implantation model conforming to the redox imbalance type RIF of the uterus was established.

[0079] First, endometrial organoids and stromal cells of uterine redox imbalance-induced recurrent fibroblastic infarcts (RIF) were isolated and cultured. Then, an artificial extracellular matrix (ECM) was prepared by mixing 1:3 (v / v) hydrogel (THE WELL BIOSCIENCE, TWG010) + 20% Matrigel (Corning, 354277) (v / v). 2 μl of this artificial ECM 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 of 1:3 hydrogel (25%) + 20% Matrigel was mixed with 6.5 x 10⁻⁶ microarray microarrays. 3 stromal cells (stromal cells of human uterine redox imbalance type RIF) and 2x10 3 Endometrial epithelial organoids (endometrial epithelial organoids from human uterine redox imbalance type RIF) were mixed and incubated at 37°C for 30 minutes to solidify the scaffold, serving as the middle section. A 1 μl layer of 50% matrix gel was coated onto the central surface of the chip through the gel / blastocyst loading window, and the 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 Endometrial epithelial organoids (based on human uterine redox imbalance-type RIF) were seeded 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 one day, and the luminal epithelium formed a monolayer within two days. A blastocyst / blastocyst model was then constructed by placing blastocysts / blastocysts through the endometrial model gel / blastocyst loading window.

[0080] The aforementioned endometrial epithelial organoids and stromal cells are, respectively, the human endometrial epithelial organoids (third generation) and human stromal cells isolated and cultured in Part 1.

[0081] 5. Drug screening: Although various animal models, in vitro culture systems, and organoid models are currently available for studying the endometrium and embryo implantation process, the following shortcomings and limitations still exist in the field of drug screening and mechanism research: (1) Species differences lead to results that do not match clinical findings: Current research largely relies on mouse or primate models to validate drug efficacy and study disease mechanisms. However, significant differences exist among different species in reproductive endocrine regulation, immune responses, and embryo implantation mechanisms, making it difficult for experimental results to accurately reflect the true physiological environment of the human endometrium.

[0082] Therefore, the results of drug validation in animal experiments are often inconsistent with clinical efficacy, which limits their application value in drug screening.

[0083] (2) The two-dimensional cell model structure is too simple: While traditional two-dimensional culture models or microfluidic chip models can achieve cell drug response assessment to some extent, they lack three-dimensional tissue structure, intercellular interactions, and dynamic hormonal regulation environment, making it difficult to simulate the entire process of endometrial receptivity changes and embryo implantation.

[0084] Therefore, such models have limited predictive power and are not suitable for drug screening and mechanism verification for complex diseases such as RIF.

[0085] (3) Clinical sample studies are subject to ethical and material collection restrictions: Due to the difficulty in obtaining clinical endometrial tissue, the limited sample size, large individual differences, and complex cyclical changes, it is impossible to establish a standardized and reproducible drug screening system.

[0086] (4) Existing 3D models face obstacles in terms of repeatability and high-throughput screening: While current three-dimensional organoids or co-culture models have reconstructed the endometrial structure to some extent, they still have the following shortcomings: The structural morphology is unstable (e.g., the teardrop-shaped structure formed by the PEG hydrogel model lacks a luminal epithelial layer). The culture system is complex (e.g., the ALI model requires a Transwell device, which is not conducive to batch operation and imaging analysis). The culture process is costly, complicated, and requires a large amount of cells and matrix materials. The model has poor reproducibility and low standardization, making it difficult to achieve high-throughput drug screening and quantitative comparison.

[0087] (5) Lack of a systematic drug screening platform: Currently, there is no stable, reproducible, and efficient in vitro model that can simulate the physiological state of the human endometrium and the embryo implantation process. As a result, the screening and validation of RIF-related drugs still mainly rely on clinical experience or low-throughput experiments, which are inefficient, time-consuming, and costly.

[0088] Based on this, this application utilizes the constructed in vitro cell model for drug screening. The drug screening process is as follows: Figure 26As shown, it includes the following steps: Step 1: 1119 drugs were selected from the FDA-approved drug library based on the RIF endometrial transcriptome characteristics; Step 2: Endometrial epithelial organoids were cultured in vitro and treated with the 1119 drugs screened in Step 1. The CCK-8 assay and optical density (OD) measurement were used to identify drugs that could promote cell viability while avoiding toxicity, and the top 50 drugs were selected. Step 3: Prepare an in vitro implantation model of uterine redox imbalance type RIF; Step 4: After treating the in vitro implantation model with the 50 drugs initially screened in Step 2, blastocysts were added to the middle region of the in vitro implantation model for co-culture. The invasion and growth of blastocysts were monitored 48 hours after plating to screen for candidate drugs. Step 5: Validation phase, using human preimplantation blastocysts to validate whether the best candidate drug screened promotes embryo implantation in patients with endometrial redox imbalance-type RIF.

[0089] 5.1 Round I—Drug screening based on RIF endometrial transcriptome features (identifying 1119 drugs) Technical approach: Bulk RNAseq transcriptomic characterization and gene ontology (GO) pathway analysis were performed on endometrial cells from 21 patients with recurrent intrauterine febrile inflammatory (RIF) to classify RIF patients based on transcriptomic characteristics. Starting with different FDA-approved drug libraries (each containing over 3000 drugs), and based on the previous classification of RIF patients, transcriptomic characteristics, gene ontology and related pathways, pharmacological and related pathway analyses of the drugs, the drug libraries most relevant to this type of RIF endometrial cell were selected first.

[0090] (1) Sample sequencing: Endometrial cell samples were collected from clinically diagnosed RIF patients and controls. Total RNA was extracted and bulk RNA-sequencing was performed to obtain the transcriptome expression matrix of each sample.

[0091] (2) Differential expression and functional annotation; (3) Differential expression analysis (DEG) was performed on the RIF group (21 cases) and the control group (6 cases), and gene ontology and pathway enrichment analysis was performed on the differentially expressed genes to identify functional annotations and abnormal signaling pathways related to RIF pathology. Based on the transcriptomic characteristics of the samples, RIF patients were clustered or subtyped to generate several transcriptomic subtypes to reflect the intimal abnormalities of different molecular phenotypes. The results are as follows: Figure 27Physiological characteristics of endometrial redox imbalance type RIF patients: DNA damage response and repair mechanisms, response to reactive oxygen species, response to oxidative stress, and alterations in reactive oxygen metabolism.

[0092] (4) Pathway-drug association analysis: The gene ontology / pathway characteristics of each RIF transcriptome subtype were compared with the drug target and pathway information. Based on the comparison results, suitable drugs were selected from the FDA-approved drug library (as shown in Tables 1-14 below), and the known targets, pathways of action, and pharmacological annotation information of each drug were obtained.

[0093] Table 1. Drug names on the first shelf of the drug database (out of 14 shelves): ; Table 2. Drug names on the second panel of the drug database: ; Table 3. Drug names on the third board of the drug database: ; Table 4. Drug names on the fourth panel of the drug database: ; Table 5. Drug names on the fifth panel of the drug database: ; Table 6. Drug names on the sixth panel of the drug database: ; Table 7. Drug Names on the Seventh Board of the Drug Database: ; Table 8. Drug Names on the Eighth Board of the Drug Library: ; Table 9. Drug Names on the Ninth Board of the Drug Library: ; Table 10. Drug names on the tenth panel 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: ; 5.2 Round II—High-throughput primary screening of mesenchymal cells / epithelial organoids: (1) Inoculation and pre-culture: In each 96-well plate, seed 2 μl of matrix gel containing endometrial epithelial organoids (endometrial epithelial organoid volume prepared according to standard) or 3000 initial stromal cells (mentioned in Part 1), plated 1 day prior to seeding and allowed to stabilize and solidify / attach the gel.

[0094] Each plate of drug treatment includes a blank control (culture medium only) and a non-drug treatment control group (containing only endometrial epithelial organoids or stromal cells, with an equal amount of drug solvent DMSO added). Each well has three replicate controls and is repeated three times according to the experimental design to ensure reproducibility.

[0095] (2) Drug treatment: Each drug in the drug library was added to the corresponding well containing culture medium at a concentration of 10 μM (i.e., 10 μM was used as the initial screening concentration). The culture medium containing the drug was changed every 2-3 days during the incubation period, with a total incubation period of 5 days (inoculation / pre-culture on day 0, drug addition on day 1, and detection on day 5).

[0096] (3) CCK-8 activity / toxicity test: 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). Then, the OD value was measured at 450 nm using a microplate reader.

[0097] The OD value of each well is first subtracted from the background OD of the blank control, and then the average OD of the wells of the same plate carrier control (DMSO) is used as the general control OD. The fold change of each drug-treated well relative to the general control is calculated as: fold change = (OD_treated - OD_blank) / (OD_control_mean - OD_blank).

[0098] Screening threshold: A minimum threshold of 1 is used (fold ≥ 1 indicates at least no reduction in overall OD), sorted from highest to lowest. Results are as follows: Figure 28As shown, after treatment with 1119 drugs, the relative fold change of OD values ​​for endometrial epithelial organoids (top) and stromal cells (bottom) in uterine redox-disordered RIF compared 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 uterine redox-disordered RIF, respectively. Green dots represent shared drugs in the top 50 list that act on both endometrial epithelial organoids and stromal cells. The gray line represents a fold change of 1 relative to the untreated drug control.

[0099] 5.3 Round III Preparation of an In Vitro Implantation Model of Endometrial Redox Imbalance-Type RIF: First, endometrial organoids and stromal cells from patients with endometrial redox imbalance-related recurrent fibrosis (RIF) were isolated and cultured. Then, an artificial extracellular matrix was prepared by mixing 1:3 (v / v) hydrogel and 20% Matrigel (v / v). 2 μl of this matrix was pre-coated onto a chip as a base layer through the gel / blastocyst loading wells and incubated at 37°C for 30 minutes. Another 5 μl of 1:3 hydrogel (25%) + 20% Matrigel was then mixed with 6.5 x 10⁻⁶ microarrays. 3 Interstitial cells and 2x10 3 Endometrial epithelial organoids 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 chip through the gel / blastocyst loading window, and the 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 The cells were laid on 50% Matricene. EXM + 10% FBS (Endometrial Epithelial Organoid Culture Medium) was added through the culture medium reservoir for endometrial-like cell culture. Stromal cells and glands expanded within one day, and the luminal epithelium formed a monolayer within two days. A blastocyst / blastocyst model was then constructed by placing blastocysts / blastocysts through the endometrial model gel / blastocyst loading window.

[0100] 5.4 Round IV—Screening in in vitro implantation models: (1) Drug pretreatment and co-culture: For each drug to be tested, the in vitro implantation model established in Part 4 was pretreated for 3 days. After that, blastocysts were added to the central region of the chip to form an embryo implantation chip. 30 blastocysts were placed into each implantation chip, and the 30 blastocysts were added from 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).

[0101] (2) Observation indicators and judgment time points: Attachment determination: Determined after 24 hours. Determination criteria: The blastocyst / blastocyst adheres firmly to the endometrioid surface and does not shift when the slide / chip is slightly shaken (based on microscopic observation records).

[0102] Outgrowth assessment: 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.

[0103] Record the number of attachments and expansions of 30 blastocysts in each chip, and calculate rate = number_positive / number_seeded × 100%.

[0104] (3) Screening criteria and candidate determination: The adhesion rate of each drug group was compared with that of the control group (corresponding to the RIF endometrial model without DMSO treatment). Drugs that significantly improved adhesion / spread rates and had no obvious toxicity were preferentially selected, such as... Figure 29 The figure shows the growth rate of blastocysts in a uterine redox imbalance-induced recurrent embryonic fibrillation (RIF) endometrial model after 48 hours of treatment with identified effective drugs. Mean ± standard deviation, n=3 biological replicates. Candidate drugs screened included Avobenzone, Vasopressin, K2EDTA, Larotrectinib sulfate, Citicoline, Olprinone (Hydrochloride), Eltrombopag, L-Leucine, Fulvestrant, Malic acid, and Ruxolitinib (phosphate).

[0105] After screening candidate drugs in step 4, and labeling blastocysts with Q-tracker 655, successful implantation of blastocysts was achieved in a uterine redox imbalance-induced RIF endometrial model after treatment with the most effective drug. Figures 30-32 As shown, after 7 days of in vitro culture, the embryonic structures (amniotic cavity-like structures and yolk sac-like structures) reached 12.54%, 6.63%, and 6.49%, respectively, while the extraembryonic structures (STB and EVT) appeared at 33.53%, 28.02%, and 23.89%, respectively, corresponding to the treatment with the most effective drugs (Avobenzone, Vasopressin, and K2EDTA).

[0106] The blastocysts were labeled with Q-tracker 655 and implanted into an in vitro implantation model treated with the top three candidate drugs (Avobenzone, Vasopressin, and K2EDTA) selected in step 4 for 3 days. Blastocyst implantation was observed 48 hours later. Figure 33 As shown, it can be seen that blastocysts successfully implanted after treatment with Avobenzone, Vasopressin, and K2EDTA.

[0107] 5.5 Round V—In vitro embryo implantation function verification of human preimplantation blastocysts: 1) Blastocyst preparation: Obtain preimplantation blastocysts from RIF patients, either cryopreserved or freshly cultured, maintain their physiological state, and sustain their viability for a short period under appropriate culture conditions (Ethics: 2024-KY-109-01 and 2024-126).

[0108] Embryo Thawing: Before thawing, the embryos were equilibrated overnight with our previously developed extended embryo culture medium (EBC). The EBC composition was as follows: 1:1 (v / v) DMEM / F12 and Neurobasal, 1xN2, 1xB27, 1xGlutaMAX, 1xNon-essential amino acids, 0.1mM β-mercaptoethanol, 15% FBS, 8nM estradiol, 200 ng / ml progesterone, 1mM sodium pyruvate (Gibco, 11360070), 5 mg / ml gentamicin (Kangning, 30-005-CR), and CEPT small molecule combination. Human blastocysts (5-6 days post-fertilization) were thawed using the Kitazato Thawing Medium Kit (Kitazato Corporation, VT102) according to the manufacturer's instructions. In short, the thawing solution (TS) was preheated to 37°C, and the diluent (DS) and wash buffer (WS) were brought to room temperature. Blastocysts were rapidly immersed in TS (37°C), DS (room temperature), WS (room temperature), and WS (room temperature) for 1 minute, 3 minutes, 5 minutes, and 5 minutes, respectively. The embryos were then transferred to acidic Tyrode's solution (Sigma, T1788) and the zona pellucida was removed under a microscope. Once the zona pellucida disappeared, the embryos were immediately transferred to pre-equilibrated EBC medium and washed twice.

[0109] 2) Microarray implantation: The blastocysts were placed in the established in vitro embryo implantation chip. Embryo culture medium (EBC) was added to the gel / embryo zone, and endometrial epithelial organoid culture medium was added to both sides. The cells were cultured at 37°C and 5% CO2. During the co-culture period, drugs were continuously added to maintain the same treatment conditions as the pretreatment.

[0110] 3) Functional evaluation: The attachment and expansion of blastocysts on the implanted microarray were observed and analyzed 24 and 48 hours after implantation. Early developmental status of the blastocysts after implantation was assessed using microscopic observation and marker staining, including the formation of the amniotic cavity and yolk sac, and the ability of the trophectoderm to differentiate into syncytiotrophectoderm and extraembryonic trophectoderm. These indicators were compared between the drug-treated group and the control group to verify the effect of the candidate drug on embryo implantation function and post-implantation development. Figure 34 ).

[0111] Several points to note during the drug screening process (Part 5): 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 chips can be replaced with trophoblast spheroids or other embryo-mimetic structures to replace human embryonic organoids (blastocysts) or actual human blastocysts (blastocysts) in order to achieve similar functional verification effects.

[0112] Second drug dosage and treatment time: During drug screening, the 10 μM uniform starting concentration used in Round II and the same concentration used in Round IV validation 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 in Round II 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. Meanwhile, the chip models for Rounds IV and V can employ microfluidic design to improve data volume and repeatability.

[0114] This invention, by constructing a drug screening system based on an in vitro embryo implantation model, effectively overcomes several limitations of existing animal models and two-dimensional cell models in drug screening, achieving the following technical advantages: (1) Improve the predictability and efficiency of drug screening: This invention combines high-throughput drug screening with the application of primary organoids, functional biological model chips, and preimplantation blastocysts in real humans, realizing a process from a massive pool of candidate drugs to precise, small-scale, personalized drugs and functional validation. In terms of throughput, this strategy changes the complexity, long cycle, high cost, and low throughput of the traditional clinical drug screening model that involves administering drugs to patients first and then tracking their efficacy. Through five rounds of effective in vitro screening steps, it overcomes the inconvenience of traditional human-based drug screening models, the species differences in animal models, and the lack of physiological relevance in two-dimensional culture systems.

[0115] (2) Possesses the potential for individualized screening: This method, based on a system constructed from human epithelial organoids and primary mesenchymal cells, uniquely reproduces the selective implantation process with minimal cell requirements (approximately 6500 mesenchymal cells and 5000 epithelial cells per chip). It allows for the screening of over a thousand drugs from a single patient biopsy, and is particularly suitable for patient-derived samples with limited clinical availability. This characteristic provides a technological foundation for personalized drug response prediction and precision medicine.

[0116] (3) It has stability and repeatability: The combination of organoid and microarray technology makes the model structure more stable and the operation simpler, avoiding the problems of unstable morphology, high cost, and poor reproducibility of traditional three-dimensional culture systems. Through batch-processing design, standardized and high-throughput drug screening can be achieved, significantly improving experimental efficiency.

[0117] (4) It has broad application prospects: Besides RIF, the screening system established in this invention is also applicable to the research and drug development of other endometrial-related diseases, including endometritis and endometriosis. This model can be used for drug screening and disease research, and has broad scientific and clinical application value.

[0118] References cited in the background section of this invention: 1.Li, J., Qi, J., Yao, G., Zhu, Q., Li, X., Xu, R., Zhu, Z., Zhao,H., Wang, Y., Ding, Y., et al. (2021). Deficiency of Sirtuin 1 ImpedesEndometrial Decidualization in Recurrent Implantation Failure Patients.Front. Cell Dev. Biol. 9, 598364. https: / / doi.org / 10.3389 / fcell.2021.598364. 2. Lu, Y., Shao, Y., Cui, W., Jia, Z., Zhang, Q., Zhao, Q., Chen, Z.-J., Yan, J., Chu, B., and Yuan, J. (2024). Excessive Lipid Peroxidation inUterine Epithelium Causes Implantation Failure and Pregnancy Loss. Adv. Sci.Weinh. Baden-Wurtt. Ger. 11, e2302887. https: / / doi.org / 10.1002 / advs.202302887. 3. Lin, J.-Z., and Lin, N. (2022). Three Oxidative Stress-RelatedGenes That Associate Endometrial Immune Cells Are Considered as PotentialBiomarkers for the Prediction of Unexplained Recurrent Implantation Failure.Front. Immunol. 13, 902268. https: / / doi.org / 10.3389 / fimmu.2022.902268. 4. Liaqat Ali Khan, N., Nafee, T., Shao, T., Hart, AR, Elliott, S., Ola, B., Heath, PR, and Fazeli, A. (2022). Dysregulation in Multiple Transcriptomic Endometrial Pathways Is Associated with Recurrent ImplantationFailure and Recurrent Early Pregnancy Loss. Int. J. Mol. Sci. 23, 16051. https: / / doi.org / 10.3390 / ijms232416051. 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 drug candidate for promoting embryo implantation efficiency in patients with redox imbalance type RIF, characterized in that, The candidate drug includes any one of the following drugs: Avobenzone, vasopressin, ethylenediaminetetraacetic acid dipotassium, larojutine sulfate, citicoline, opiln, icotrapa, L-leucine, fulvestrant, malic acid, and relacorat.

2. The drug candidate for promoting embryo implantation efficiency in patients with redox imbalance type RIF according to claim 1, characterized in that, The physiological characteristics of the redox imbalance type RIF patient is that the balance between the oxidative stress level and the antioxidant defense mechanism in the endometrium is destroyed, which is manifested as a significant decrease in SIRT1 expression, leading to ROS accumulation and a decrease in antioxidant gene expression.

3. The drug candidate for promoting embryo implantation efficiency in patients with redox imbalance type RIF according to claim 2, characterized in that, The candidate drug includes any one of the following drugs: avobenzone, vasopressin, and ethylenediaminetetraacetic acid dipotassium.

4. A method for screening a candidate drug for promoting embryo implantation efficiency in patients with redox imbalance type RIF, characterized in that, The screening method comprises the following steps: Step 1: selecting 1119 drugs from the FDA-approved drug library based on the RIF endometrial transcriptome characteristics; Step 2: in vitro isolation and culture of endometrial epithelial organoids and stromal cells of the redox imbalance type RIF patient, and treatment of the endometrial epithelial organoids or stromal cells with the 1119 drugs screened in step 1, identification of drugs that can promote cell viability and avoid toxicity using CCK-8 experiment and optical density determination, and selection of the top 50 drugs; Step 3: preparation of an in vitro implantation model of the redox imbalance type RIF; Step 4: after the 50 drugs screened in step 2 are used to treat the in vitro implantation model, 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; Step 5: verification of the embryo implantation efficiency of the screened candidate drugs using human pre-implantation blastocysts in the in vitro embryo implantation model.

5. The screening method according to claim 4, characterized in that, The specific operation of step 2 is as follows: Isolation and culture of endometrial epithelial organoids and stromal cells from endometrial tissue; Inoculation and pre-culture: the endometrial epithelial organoids and stromal cells obtained by isolation and culture are used as the experimental group, and 2 μl of Matrigel containing endometrial epithelial organoids or 3000 stromal cells are inoculated in each well of a 96-well plate; Each 96-well plate has a blank control group and a non-drug treatment control group, and each well of the blank control group only has culture medium, and each well of the non-drug treatment control group has 2 μl of Matrigel containing endometrial epithelial organoids or 3000 stromal cells, with three repeated controls in each well, and the experiment is repeated three times according to the experimental design; Each drug screened in step 1 is added to the corresponding well of the experimental group at 10 μM, and the culture medium containing the drug is changed every 2-3 days during the culture period, and the total treatment culture is 5 days; among them, 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; After 5 days of culture, the OD value is measured using a CCK-8 kit, and the cell viability of each drug treatment well relative to the non-drug treatment control group is calculated, and the cell viability is sorted from high to low.

6. The screening method according to claim 4, wherein, The construction process of the in vitro implantation model in step 3 is as follows: Preparation of an in vitro embryo implantation chip; An artificial extracellular matrix is prepared using hydrogel and Matrigel, and the artificial extracellular matrix is preloaded onto the in vitro embryo implantation chip through the gel / blastocyst loading window of the in vitro embryo implantation chip as a basal layer, and incubated at 37°C for 30 minutes; 5 μl of hydrogel and Matrigel mixture is mixed with interstitial cells and endometrial epithelial organoids, and incubated at 37°C for 30 minutes to solidify the scaffold as an intermediate layer; 1 μl of 50% Matrigel thin layer is coated on the surface of the intermediate layer, and placed in a 37°C 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; The endometrial model is cultured by adding endometrial epithelial organoid culture medium through the culture medium reservoir.

7. The screening method according to claim 6, characterized in that, After 3 days of pre-treatment of the in vitro implantation model with the 50 screened 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.

8. The screening method according to claim 7, characterized in that, After screening in step 4, the blastocyst is re-implanted into the in vitro implantation model pre-treated with the top 3 candidate drugs for 3 days, and after 7 days of in vitro culture, the embryo structure reaches 12.54%, 6.63% and 6.49%, respectively, and the probability of simultaneous occurrence of syncytial trophoblast ectoderm and villous trophoblast ectoderm in the extraembryonic structure is 33.53%, 28.02% and 23.89%, respectively. The top 3 candidate drugs are evofaxine, choline and nitrendipine.

9. The screening method according to claim 8, characterized in that, In step 5, the three most effective drugs determined in step 4 are used to pre-treat the in vitro implantation model of uterine oxidative imbalance type RIF for 3 days, and then the human pre-implantation blastocyst is implanted to verify the embryo implantation efficiency.

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