Establishment and verification method of infertility endometrial organoid

Abstract

The invention relates to an establishment and verification method of an infertility endometrial organoid. The method comprises the steps of endometrial cell extraction, organoid construction, passage and identification. The method comprises the following steps: digesting and dissociating biopsy tissues of a patient to obtain single cells, re-suspending the single cells in matrigel, culturing the single cells in an ExM culture medium for 7 days to form three-dimensional organs, and after passage, detecting specific markers through immunofluorescent staining, detecting immune cell factors through Luminex and analyzing gene expression through qPCR to verify functional differences. The method has the advantages that the organoid constructed by the method can really simulate the endometrial structure function and immune microenvironment, the hereditary heterogeneity of a patient is reserved, the culture period is short, and the cost is low; the method can be clinically used for etiology analysis, embryo implantation potential prediction and medication scheme optimization of IVF-ET repeated failure patients, and an efficient platform is provided for accurate diagnosis and treatment of infertility, drug screening and endometrial injury repair research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and reproduction, and relates to the extraction of primary cells of the endometrium in infertile and non-infertile women, in particular, the establishment and verification method of endometrial organoids in infertile women. BACKGROUND

[0002] Infertility is an important challenge in the field of reproductive medicine, and its pathogenesis is closely related to immune imbalance, such as Th1 / Th2 cytokine imbalance and abnormal NK cell activity, which accounts for 15%-25% of infertile patients and more than 50% of recurrent miscarriage patients. At present, the clinical treatment mainly relies on in vitro fertilization-embryo transfer (IVF-ET), but the success rate is only about 30%, and the problems of immune abnormalities leading to embryo rejection and endometrial receptivity defects have not been effectively solved. Emerging therapies such as mesenchymal stem cells show potential, but there is a significant difference between preclinical research and actual efficacy, and a precise in vitro model is urgently needed to reveal the nature of the disease.

[0003] The existing research models have obvious limitations:

[0004] Traditional cell models: immortalized endometrial cell lines (such as Ishikawa cells) have a large genetic background difference from in vivo, and cannot reflect the true pathological characteristics of patients; primary endometrial cells cultured in two dimensions (2D) are prone to genetic mutations, lose tissue-specific functions after long-term culture, and cannot simulate the complexity of the three-dimensional (3D) microenvironment in vivo.

[0005] Animal models: Although they can be used to verify infertile-related genes, there are significant differences in genetic and immune mechanisms between species, and experimental results are difficult to translate into clinical protocols, especially individual heterogeneity in infertility cannot be reproduced.

[0006] Existing organoid models: Although they can partially simulate tissue structure, specific models for infertility have not been established, and there is a lack of integrated research on immune microenvironment and hormone dynamic regulation, making it difficult to meet the needs of mechanism exploration and drug screening.

[0007] Therefore, constructing a precise 3D in vitro model that retains patient genetic characteristics and can simulate immune imbalance and hormone response is the key to breaking through the bottleneck of infertility research. SUMMARY

[0008] The purpose of the present application is to solve the above-mentioned problems in the prior art and provide a method for establishing and verifying endometrial organoids in infertile women.

[0009] To achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0010] A method for establishing and validating endometrial organoids in infertile patients includes the following steps: S1, endometrial cell extraction: digesting and dissociating endometrial biopsy tissue from infertile and non-infertile patients into single cells;

[0011] S2. Organoid construction: The single cells obtained in step S1 are resuspended in matrix gel, seeded, and cultured in ExM medium to form three-dimensional organoids.

[0012] S3. Organoid passage: Digest, isolate and passage the cultured organoids.

[0013] S4. Organoid identification: Functional differences of organoids are verified by detecting specific markers through immunofluorescence staining, detecting immune cytokines, and analyzing gene expression.

[0014] Preferably, step S1 includes the following sub-steps:

[0015] (1.1) Endometrial tissue samples were stored in DMEM high glucose medium containing 10% penicillin-streptomycin-amphoteric B triple antibody solution, transported and stored at 4°C, and processed within 4 hours;

[0016] (1.2) Wash the tissue with PBS containing 10% triple antibody solution until no foam is present, and cut it into small pieces of 0.5 mm³;

[0017] (1.3) Add 1.25 U / ml dispase, 2.5 mg / ml type IV collagenase and Advanced DMEM / F12 mixture, and digest at 37°C for 1 h;

[0018] (1.4) Digestion was terminated with DMEM high glucose medium + 10% fetal bovine serum. After lysing red blood cells with 1× red blood cell lysis buffer, an equal volume of PBS was added to terminate the reaction. The cells were centrifuged at 1500 rpm for 5 min and single cells were collected.

[0019] Preferably, step S2 includes the following sub-steps:

[0020] (2.1) Single cells were loaded with 2-4 × 10 5 The density of cells / well was resuspended in ice-cold Matrigel, and 300 μl / well was inoculated into a 6-well plate and fixed in a 37°C incubator for 1 h.

[0021] (2.2) Add ExM medium and incubate at 37℃ and 5% CO2 for 7 days, replacing with fresh ExM medium every 48 hours;

[0022] The ExM medium is Advanced DMEM / F12 supplemented with the following final concentrations: 1x Glutamax, 1 mM HEPES, 1x ITS, 1x Trizma, 10 μM Y-27632, 1x N2 supplement, 1x B27 supplement, 1 mM Nicotinamide, 50 ng / ml EGF, 100 ng / ml Noggin, 200 ng / ml Wnt3A, 100 ng / ml FGF-10, 500 nM A8301, 200 ng / ml R-sponding-1, 1.25 mM Acetyl-L-Cysteine, 10 μM SB.

[0023] Preferably, step S3 comprises the following sub-steps:

[0024] (3.1) Soften Matrigel with pre-cooled PBS, pipette to separate organoids;

[0025] (3.2) TryPLE digestion of organoids for 10 min, stop digestion with DMEM high glucose medium + 10% fetal bovine serum;

[0026] (3.3) Resuspend with pre-cooled Matrigel at a ratio of 1:2-1:6, inoculate into preheated culture containers, and temporarily add 10 μM Y27632 to the medium after subculture.

[0027] Preferably, the organoid identification in step S4 comprises:

[0028] (4.1) Immunofluorescence staining to detect endometrial specific markers, including E-Cadherin, FOXA2, PANCK, MUC1, ER, PR, PAEP, Ki67 and acetylated alpha tubulin;

[0029] (4.2) Luminex to detect the expression differences of Th1 type (TNF-α, IFN-γ), Th2 type (IL-4, IL-10), Th17 type (IL-17) and Treg type (TGF-β1) immune cytokines in the supernatant; and the Th1 / Th2 ratio is calculated by (TNF-α, IFN-γ) / (IL-4, IL-10), Th17 / Treg.

[0030] (4.3) qPCR to detect MUC1 and PAEP gene expression, primer sequences are as follows:

[0031] MUC1 gene forward primer: 5'-GCT-GCT-CCT-CAC-AGT-GCT-TA-3',

[0032] Reverse primer: 5'-TAG GTA TCC CGG GCT GGA A-3';

[0033] PAEP gene forward primer: 5'-ACC TGG AGC TCC CAA AGT TG-3',

[0034] Reverse primer: 5'-GCA CGG CTC TTC CAT CTG TT-3';

[0035] Reference β-actin gene forward primer: 5'-CAT GTA CGT TGC TAT CCA GGC-3', reverse primer: 5'-CTC CTT AAT GTC ACG CAC GAT-3'.

[0036] Preferably, the growth factors in the ExM medium avoid repeated freeze-thawing, are stored at -80℃ after preparation, and are used up within 2 weeks.

[0037] Preferably, the digestion system in step (1.3) is placed on a shaker for uniform digestion, and an equal volume of PBS is added to terminate the reaction after red blood cell lysis in step (1.4).

[0038] Preferably, dispase of 1 U is used to incubate at 37℃ for 1h to melt the Matrigel during passaging, and the organoids are fixed with 4% paraformaldehyde at room temperature for 1h before paraffin embedding and sectioning.

[0039] Preferably, in the immunofluorescence staining, the primary antibody is incubated at 4℃ overnight, the fluorescent secondary antibody and DAPI are incubated at room temperature in the dark for 1h, and the specimen is observed after being sealed with an anti-fluorescence quenching mounting medium.

[0040] Preferably, the infertility samples are derived from patients with a history of ≥3 failed IVF-ET and no endocrine, genetic, autoimmune disease or chronic inflammation; and the non-infertility samples are derived from patients with ≤1 IVF-ET due to tubal or male factors.

[0041] As the above technical solutions are adopted, the application has the following beneficial effects:

[0042] 1. The organoids established by the application can truly reproduce the structure, function and immune microenvironment of the patient's endometrium: through immunofluorescence and Luminex detection, it is confirmed that the organoids of the infertility group highly express Th1 type cytokines (TNF-α, IFN-γ), and the Th1 / Th2 ratio is significantly higher than that of the control group, accurately simulating the core pathology of "immune imbalance leading to embryo rejection"; at the same time, the organoids can dynamically respond to hormone stimulation, showing periodic expression changes of ER / PR and Ki67 in the proliferation phase / secretion phase, and providing a "living model" for disease mechanism research;

[0043] 2. Compared with immortalized cell lines (genetic distortion) or 2D culture (short-term degradation), this invention, through optimized ExM culture medium (containing 14 growth factors) and passage technology (1:2-1:6 ratio + Y27632 protection), can preserve the genetic heterogeneity and epigenetic characteristics of patients for a long time, and there is no degradation in morphology and function after passage; it solves the problem of "difficult clinical translation" of animal models, directly reflects the individual differences in infertility, and lays the foundation for personalized medicine;

[0044] 3. The organoids of this invention have multiple functions including "pathological simulation, hormone response, and immune detection": qPCR can verify the defects in glandular secretory function (difference in MUC1 / PAEP expression), which can be used to explore the linkage mechanism between "immune imbalance and glandular dysfunction"; it supports in vitro hormone regulation experiments and screening of immunomodulatory drugs (such as Th1 / Th2 balance regulators), significantly reducing the cost of preclinical research and providing an efficient tool for the discovery of therapeutic targets and efficacy evaluation of infertility. Attached Figure Description

[0045] Figure 1 This is a structural flowchart of an embodiment of the method for establishing and validating endometrial organoids for infertility according to the present invention.

[0046] Figure 2 This is an immunofluorescence staining image of the endometrial organoids of the present invention.

[0047] Figure 3 This describes the expression of ki67, ER, and PR in the proliferative and secretory phases of the control and experimental groups of this invention.

[0048] Figure 4 This describes the expression of MUC1 and PAEP in the proliferative and secretory phases of the control and experimental groups of this invention.

[0049] Figure 5 This is a graph showing the difference in immune cytokine levels of endometrial organoids between the control and experimental groups of this invention. Detailed Implementation

[0050] Please see Figures 1-5 As shown, this invention aims to construct endometrial organoid models for infertile and non-infertile women. This model, through three-dimensional culture technology, preserves the genetic background and heterogeneity of primary patient tissues, simulating the structural functions (such as glandular secretion and hormone receptor expression) and immune imbalance characteristics (such as Th1 cytokine dominance) of the endometrium. This provides a precise in vitro tool for studying the pathogenesis of infertility, drug screening, and developing personalized treatment plans, filling a gap in existing models. Based on this, this invention mainly provides a method for establishing and validating endometrial organoids for infertility, including the following steps:

[0051] Set up an experimental control group:

[0052] The infertile group (experimental group) is patients with IVF-ET≥3, and the non-infertile group (control group) is patients who need to perform IVF-ET (IVF-ET≤1) due to tubal or male factors. All patients have no history of endocrine abnormalities, genetic abnormalities, thrombosis, autoimmune diseases or chronic inflammation; and all patients have signed the informed consent form. S1 Endometrial single cell extraction: primary endometrial single cells are extracted from endometrial biopsy tissues of patients in the experimental group and the control group, which specifically includes:

[0053] S1.1 Sampling: The endometrial tissue samples of the experimental group and the control group are stored in DMEM high-sugar medium containing 10% penicillin-streptomycin-amphotericin B-triple-antibiotic solution in a sterile operating room, transported at 4°C and processed within 4 hours.

[0054] S1.2 Washing: Fresh endometrial tissue is placed in a 35 mm2 culture dish, and the endometrial tissue is washed with PBS containing 10% triple-antibiotic solution until no foam is generated.

[0055] S1.3 Digestion: The endometrial tissue is cut into tissue blocks with a volume of about 0.5 mm³ using sterile surgical scissors and forceps, 1.25 U / ml dispase and Ⅳ collagenase 2.5 mg / ml are added to the culture dish, and the basic medium is additionally supplemented to 3 ml, and the digestion is carried out in a 37°C constant temperature incubator for 1 h, and then the uniform digestion is carried out on a shaker.

[0056] S1.4 Termination of digestion: DMEM high-sugar medium (containing 10% fetal bovine serum) is added to terminate the digestion, and the digested endometrial suspension is transferred to a 15 ml centrifuge tube, and then it is left to stand for 5-10 min, and the undigested endometrial tissue sinks, and the endometrial suspension supernatant is sucked into a new 15 ml centrifuge tube; centrifugation at 1500 rpm for 5 min, and the supernatant is discarded.

[0057] S1.5 Red blood cell lysis: 2-3 ml of 1× red blood cell lysis solution is added, 5 ml of a Pasteur pipette is gently blown for 10-20 times, and then it is left to stand for 5 min to completely lyse the red blood cells; an equal volume of PBS is added to terminate the digestion; centrifugation at 1500 rpm for 5 min, and the supernatant is discarded; about 6-9 ml of PBS is added to wash the cells, and centrifugation is carried out at 1500 rpm for 5 min to obtain the primary endometrial cells.

[0058] S2, organoid construction: the single cells obtained in step S1 are resuspended in Matrigel, and after inoculation, ExM medium is added for culture to form a three-dimensional organoid, and the specific operation is as follows:

[0059] S2.1 Resuspension: After centrifugation, the supernatant was discarded, and the endometrial cells were resuspended in ice-cold Matrigel at a density of 2-4 x 10 5 / well, and mixed well by gently pipetting up and down 5-10 times, taking care not to create bubbles.

[0060] S2.2 Plating: The endometrial suspension was inoculated into a 6-well plate at a volume of 300 μl per well in 4-5 drops.

[0061] S2.3 Fixation: The plated 6-well plate was placed in a 37°C incubator for 1 h.

[0062] S2.4 Liquid addition: 900 μl of preheated ExM medium was added to each well of the 6-well plate.

[0063] S2.5 Liquid exchange: The 6-well plate was cultured in a 37°C, 5% CO2 incubator for 7 days, and fresh ExM medium was replaced every 48 hours.

[0064] The formula of the ExM medium is as follows: the Advanced DMEM / F12 is used as the basic medium, and the following components are added to a final concentration: 1 x Glutamax, 1 mM HEPES, 1 x ITS, 1 x penicillin-streptomycin-amphotericin B triple antibiotic, 10 μM Y-27632, 1 x N2 supplement, 1 x B27 supplement, 1 mM nicotinamide, 50 ng / ml EGF, 100 ng / ml Noggin, 200 ng / ml Wnt3A, 100 ng / ml FGF-10, 500 nM A8301, 200 ng / ml R-sponding-1, 1.25 mM acetyl-L-cysteine, and 10 μM SB; in addition, the ExM medium needs to be stored at -80°C in the dark after preparation, and repeated freezing and thawing should be avoided, and it should be used within 2 weeks from the date of preparation.

[0065] S2.6 In particular, the supernatant of the first generation of endometrial organoids was collected to detect the differences in the expression of six immune cytokines, including Th1-type immune cytokines (TNF-α, IFN-γ), Th2-type immune cytokines (IL-4, IL-10), Th17-type immune cytokines (IL-17), and Treg-type immune cytokines (TGF-β1) by Luminex.

[0066] S3, Organoid passage: The cultured organoids were digested, separated, and subcultured, and the specific operation was as follows:

[0067] After the culture supernatant is discarded, the pre-cooled PBS is added to soften the matrix glue, the organoids and the matrix glue are separated by blowing with a Pasteur pipette, the TryPLE digestion solution is added and digested at 37°C for 10 min, the DMEM high-sugar culture medium (containing 10% fetal bovine serum) is used to terminate the digestion, and after centrifugation at 1500 rpm for 5 min, the organoids are resuspended in pre-cooled Matrigel at a volume ratio of 1:2-1:6 according to the state and density of the organoids and inoculated into a preheated 6-well plate; 24 hours after the subculture, 10 μM of Y27632 is added to the culture medium at a final concentration;

[0068] S4, organoid identification: the functional differences of the organoids are verified by specific marker detection through immunofluorescence staining, immune cytokine detection and gene expression analysis, and the specific operation is as follows:

[0069] S4.1, immunofluorescence staining of endometrial organoids

[0070] (1) The endometrial organoids are incubated with 1U dispase at 37°C for 1 h to melt the matrix glue.

[0071] (2) 4% paraformaldehyde is used for fixation at room temperature for 1 h

[0072] (3) Paraffin embedding and sectioning, baking at 65°C for 4 h, deparaffinization with an environmentally friendly deparaffinization solution, antigen repair with 1x antigen repair solution, and immunohistochemical pen circle.

[0073] (4) OWB (3% BSA + 0.3% Triton X-100 in PBS) is used for room temperature blocking for 30 min.

[0074] (5) Primary antibody 4°C incubation overnight (E-Cadherin, FOXA2, PANCK, MUC1, ER, PR, PAEP, Ki67, acetylated Tubulin) to label proteins.

[0075] (6) 488, 594 and DAPI labeled fluorescent secondary antibodies are incubated at room temperature in the dark for 1 h on a shaking bed.

[0076] (7) OWB is washed 3 times, 10 min each time.

[0077] (8) Add one drop of anti-fluorescence quenching mounting medium to mount the slide, and take pictures under an inverted microscope.

[0078] (9) Experimental results

[0079] (10) The endometrial organoids treated by ExM medium and E2, E2+P4+cAMP were induced into proliferation phase and secretion phase, and the differences between the experimental group and the control group of endometrial organoids were compared. The epithelial cell markers E-cadherin, PCNA and FOXA2 of endometrial organoids were strongly expressed. Endometrial organoids could differentiate into ciliated cells, and acetylated alpha tubulin was positively expressed. Figure 2 Ki67 is a proliferation cell marker, and Ki67 is significantly expressed in the proliferation phase, and the expression of Ki67 decreases in the secretion phase. Under the action of E2, the expression of ER and PR increases in the proliferation phase, and there is a significant difference in the expression of PR between the control group and the experimental group in the proliferation phase, while after adding P4 and cAMP, the expression of ER and PR in endometrial organoids decreases. Figure 3 MUC1 is strongly expressed in the proliferation phase and the secretion phase, which proves whether endometrial organoids can produce functional mucin. Figure 4 After stimulating the decidualization of endometrial organoids, endometrial organoids express secretion phase-specific marker PAEP. There is a significant difference in MUC1 and PAEP between the control group and the experimental group of endometrial organoids in the secretion phase. Figure 4 .

[0080] S4.2, Extraction of RNA from endometrial tissue and organoids of the control group and the experimental group

[0081] (1) Hormone treatment of endometrial organoids in the control group and the experimental group: After inoculating endometrial organoids, the control group and the experimental group of endometrial organoids were cultured in ExM medium for 24 hours, and then 10 nm estrogen (E2) was added to treat the control group and the experimental group of endometrial organoids for 4 days to make them enter the proliferation phase, and ExM medium containing 10 nM estrogen (E2), 1 μM progesterone (P4), and 0.5 mM cAMP was added to induce the organoids to be in the secretion phase. The RNA of the control group and the experimental group of endometrial organoids treated by ExM medium, E2 and E2+P4+cAMP was extracted.

[0082] (2) Digestion of endometrial organoids in the control and experimental groups: Discard the culture medium and add basal culture medium containing 1.25 U / ml dispase. Repeatedly pipette the endometrial organoids 40-50 times. Place the endometrial organoids in a 37℃ incubator for 1 h to digest the pre-cooled PBS containing the matrix gel, thus separating the organoids from the matrix gel. Centrifuge at 1500 rpm for 5 min, and digest the endometrial organoids with 4-5 ml TryPLE for 10-15 min. Neutralize the culture medium to stop the digestion. Centrifuge at 1500 rpm for 5 min and discard the supernatant.

[0083] (3) Add 1 ml of TRIzol to lyse organoids, 200 μl of chloroform, shake vigorously for at least 1 min, and let stand for 10 min to extract RNA.

[0084] (4) Centrifuge at 12000 rpm for 10 min at 4°.

[0085] (5) Transfer the supernatant to a new 1.5 ml EP tube, add 500 μl of pre-cooled isopropanol at -20°C, and mix by inverting the tube.

[0086] (6) Let it stand in a refrigerator at -20°C for 4 hours.

[0087] (7) Centrifuge at 12000 rpm for 10 min at 4°.

[0088] (8) Carefully aspirate the supernatant, and do not touch the RNA precipitate.

[0089] (9) Add 1 ml of 75% ethanol to wash the RNA precipitate.

[0090] (10) Centrifuge at 12000 rpm for 5 min at 4°, and carefully aspirate the supernatant.

[0091] (11) Centrifuge at 2000 rpm for 1 min at 4°, and carefully aspirate the residual supernatant.

[0092] (12) Air dry the RNA precipitate in the EP tube at room temperature.

[0093] (13) Add 15-30 μl of sterile, enzyme-free water according to the amount of RNA.

[0094] (14) Determine RNA concentration and purity using a nanometer spectrophotometer.

[0095] (15) 2 μg RNA was reverse transcribed into cDNA using the HiScript III RT SuperMix kit.

[0096] (16) TB Green Premix Ex Taq II configuration qpcr system (n=3), verify the gene expression content of endometrial protein MUC1, PAEP, and β-actin as the internal reference gene. LightCycler 480 machine detection. −ΔΔCt Quantitative numerical value.

[0097] The primer sequence of qPCR detection is:

[0098] MUC1 forward (gene forward primer): 5-GCT-GCT-CCT-CAC-AGT-GCT-TA-3'

[0099] MUC1 reverse (reverse primer): 5'-TAG GTA TCC CGG GCT GGA A-3'

[0100] PAEP forward (gene forward primer): 5'-ACC TGG AGC TCC CAA AGT TG-3'

[0101] PAEP reverse (reverse primer): 5'-GCA CGG CTC TTC CAT CTG TT-3'

[0102] β-Actin forward (gene forward primer): 5'-CAT GTA CGT TGC TAT CCA GGC-3',

[0103] β-Actin reverse (reverse primer): 5'-CTC CTT AATGTC ACG CAC GAT-3'.

[0104] (17) Freshly biopsied endometrial tissue (taken from the proliferative phase of the menstrual cycle) is cut into small pieces as much as possible and quickly placed into RNA later, which can be stored at 4° for one week. Repeat steps (3)~(16) of S4.2.

[0105] (18) The qpcr results show that the MUC1 gene expression of the control group endometrial tissue is higher than that of the experimental group. The difference is statistically significant (P<0.05). Figure 4 There is a significant difference in PAEP results between the control group and the experimental group of endometrial tissue, and the PAEP of the control group endometrial organoids is higher than that of the experimental group but the difference is not statistically significant. It is speculated that the expression of this gene in organoids between the control group and the experimental group may be related to individual differences, and the secretion phase of some samples is inconsistent during the treatment time in the secretion phase.

[0106] (19) Luminex experiment results show that: control group and experimental group immune cytokines exist differences on TNF-α / IL-4, IFN-γ / IL-4, the Th1 / Th2 ratio of experimental group is higher than that of control group, Figure 5 ), it is suggested that Th1 type is in immune advantage in pregnancy state, which may lead to embryo rejection or interfere with endometrial receptivity. The experimental results are consistent with the pathological characteristics of immune infertility population.

[0107] It should be noted that: the present application successfully constructs the immune and non-immune infertility endometrial organoid model, and the core advantage lies in high simulation and stability; the organoid not only expresses endometrial specific markers (such as E-Cadherin, FOXA2, MUC1, PAEP) and hormone receptors (ER, PR), but also can simulate the dynamic changes of the proliferation and secretion periods, the expression of ER and PR is up-regulated under the stimulation of estrogen, the proliferation activity of Ki67 is enhanced, and the decidualization marker PAEP is highly expressed under the induction of progesterone, which truly restores the structural and functional heterogeneity of in vivo endometrium. At the same time, the long-term culture can be realized through the optimized subculture method, which retains the genetic background and epigenetic characteristics of the patient's primary tissue, and overcomes the limitations of genetic drift of traditional 2D cell lines or species differences of animal models;

[0108] In the research and application level, the model provides an innovative tool for the mechanism research of infertility; through Luminex detection, it is found that the levels of Th1 type cytokines (TNF-α, IFN-γ) in the supernatant of the experimental organoids are significantly higher than those of the control group, the Th1 / Th2 ratio is imbalanced, which directly reflects the pathological state of infertility patients, and provides in vitro evidence for the mechanism research that Th1 type immune advantage leads to embryo rejection. qPCR verification shows that the expression amounts of MUC1 and PAEP genes in the control group are higher than those in the experimental group, which indicates that the gland secretion function defect exists in the organoids of infertility patients, and can be used to explore the infertility related gene regulation network;

[0109] In the clinical value aspect, the present application promotes the development of precise diagnosis and treatment and translational medicine of infertility; the "personalized" organoid library established based on patient biopsy tissue can reflect the individual immune state (such as Th1 / Th2 imbalance) and gland function difference, and provide an etiological analysis tool for IVF-ET repeated failure patients, and guide the optimization of clinical drug regimen. By simulating the endometrial receptivity (such as MUC1 expression level, PAEP secretion ability), the embryo implantation potential can be predicted, the best implantation window period can be selected, and the risk of repeated implantation failure can be reduced; compared with animal models, the organoid culture period is short (7 days to form), the cost is low, and the ethical controversy is avoided, which provides an efficient efficacy evaluation platform for endometrial damage repair (such as mesenchymal stem cell therapy).

[0110] The above description and the accompanying examples are intended to be illustrative of the application and are not intended to limit the scope of the application. Those skilled in the art will readily recognize variations and modifications of the embodiments discussed herein which fall within the scope of the application. Accordingly, the application is limited only by the appended claims.

Claims

1. A method for establishing and validating an infertile endometrial organoid, characterized by, The method comprises the following steps: S1, endometrial cell extraction: endometrial biopsy tissues of infertile patients and non-infertile patients are digested and dissociated into single cells; S2, organoid construction: the single cells obtained in step S1 are resuspended in Matrigel, and after inoculation, ExM medium is added for culture to form three-dimensional organoids; S3, organoid passage: the cultured organoids are digested, separated and subcultured; S4, organoid identification: the functional differences of the organoids are verified by specific marker detection, immune cytokine detection and gene expression analysis.

2. The method of claim 1, wherein, Step S1 comprises the following sub-steps: (1.1) The endometrial tissue sample is stored in DMEM high-sugar medium containing 10% penicillin-streptomycin-amphotericin B three-antibiotic solution, transported and stored at 4°C, and processed within 4 hours; (1.2) The tissue is washed with PBS containing 10% three-antibiotic solution until no foam is generated, and is cut into small pieces of 0.5 mm³; (1.3) 1.25 U / ml dispase, 2.5 mg / ml collagenase type IV and Advanced DMEM / F12 are mixed uniformly, and are digested at 37°C for 1 hour; (1.4) The digestion is terminated with DMEM high-sugar medium + 10% fetal bovine serum, and after red blood cells are lysed by 1× red blood cell lysis solution, an equal volume of PBS is added to terminate the reaction, and single cells are collected by centrifugation at 1500 rpm for 5 minutes.

3. The method of claim 1, wherein, Step S2 comprises the following sub-steps: (2.1) Resuspend single cells in ice-cold Matrigel at a density of 2-4 x 10 5 cells / well, 300 μl / well in a 6-well plate, and incubate at 37°C for 1 h. (2.2) Add ExM medium, and culture at 37°C, 5% CO2 for 7 days, and replace the fresh ExM medium every 48 hours; The ExM medium is Advanced DMEM / F12 added with the following final concentration components: 1×Glutamax, 1 mM HEPES, 1×ITS, 1×three-antibiotic, 10 μM Y-27632, 1×N2 supplement, 1×B27 supplement, 1 mM nicotinamide, 50 ng / ml EGF, 100 ng / ml Noggin, 200 ng / ml Wnt3A, 100 ng / ml FGF-10, 500 nM A8301, 200 ng / ml R-sponding-1, 1.25 mM acetyl-L-cysteine, and 10 μM SB.

4. The method of claim 1, wherein, Step S3 comprises the following sub-steps: (3.1) Soften the Matrigel with pre-cooled PBS, and separate the organoids by blowing with a Pasteur pipette; (3.2) Digest the organoids with TryPLE for 10 minutes, and terminate the digestion with DMEM high-sugar medium + 10% fetal bovine serum; (3.3) Resuspend the organoids in pre-cooled Matrigel at a ratio of 1:2-1:6, inoculate them in a preheated culture container, and temporarily add 10 μM Y27632 to the medium after subculture.

5. The method of claim 1, wherein, The organoid identification in step S4 comprises: (4.1) Immunofluorescence staining to detect endometrial specific markers, including E-Cadherin, FOXA2, PANCK, MUC1, ER, PR, PAEP, Ki67 and acetylated α-tubulin; (4.2) Luminex detection of supernatant Th1 type (TNF-alpha, IFN-gamma), Th2 type (IL-4, IL-10), Th17 type (IL-17) and Treg type (TGF-beta1) immune cell factor expression differences; and the Th1 / Th2 ratio is calculated by (TNF-alpha, IFN-gamma) / (IL-4, IL-10) and Th17 / Treg (IL-17 / TGF-beta1): (4.3) qPCR detection of MUC1 and PAEP gene expression, primer sequences are as follows: MUC1 gene forward primer: 5'-GCT-GCT-CCT-CAC-AGT-GCT-TA-3', Reverse primer: 5'-TAG GTA TCC CGG GCT GGA A-3'; PAEP gene forward primer: 5'-ACC TGG AGC TCC CAA AGT TG-3', Reverse primer: 5'-GCA CGG CTC TTC CAT CTG TT-3'; Beta-actin gene forward primer: 5'-CAT GTA CGT TGC TAT CCA GGC-3', reverse primer: 5'-CTC CTT AAT GTC ACG CAC GAT-3'.

6. The method of claim 3, wherein, The growth factors in the ExM medium avoid repeated freeze-thawing, are stored at -80°C after preparation, and are used up within 2 weeks.

7. The method of claim 2, wherein, The digestion system in step (1.3) is placed on a shaker for uniform digestion, and after red blood cell lysis in step (1.4), an equal volume of PBS is added to terminate the reaction.

8. The method of claim 4, wherein, When passaging, 1 U dispase is used to incubate at 37°C for 1 h to melt the matrix glue, and after 4% paraformaldehyde room temperature fixation of the organoids for 1 h, paraffin embedding sectioning is performed.

9. The method of claim 5, wherein, In the immunofluorescence staining, the primary antibody is incubated at 4°C overnight, the fluorescent secondary antibody and DAPI are incubated at room temperature in the dark for 1 h, and after mounting with anti-fluorescence quenching mounting medium, microscopic examination is performed.

10. The method according to any one of claims 1 to 9, characterized in that, The infertility samples are derived from patients with IVF-ET failure for more than or equal to 3 times and excluding endocrine, genetic, autoimmune diseases and chronic inflammation history; the non-infertility samples are derived from patients with IVF-ET for less than or equal to 1 time due to fallopian tube or male factors.

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