Stem cell and organoid-based methods for diagnosis and optimization of embryo implantation

Abstract

Disclosed are methods for producing controlled endometrial organoids using micropatterning techniques, and methods for using endometrial cells or organoids for diagnostic and predictive purposes, and for optimizing IVF and other infertility-related treatments, including embryo transfer and intrauterine insemination (IUI). These organoids grow in a spatially controlled manner and exhibit less heterogeneity than organoids grown directly in a three-dimensional culture. The specification also describes methods for predicting the success of embryo transfer or other IVF-related treatments, optimizing embryo transfer, improving embryo implantation in the endometrium, and performing embryo transfer in subjects. These methods include culturing endometrial organoids or endometrial epithelial cells, and contacting the endometrial organoids or cells with one or more blast-like cells. In some embodiments, the endometrial organoids or cells include endometrial cells obtained from subjects undergoing or considering IVF-related treatment.

Description

[Technical Field]

[0001] This application relates to U.S. Application No. 63 / 490,244, filed on 14 March 2023, and U.S. Application No. 63 / 591,059, filed on 17 October 2023, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Organoids can be defined as three-dimensional in vitro tissue models that reproduce many of the physiologically relevant properties and characteristics of their corresponding in vivo tissues. The discovery that human endometrial organoids can be produced from primary endometrial cells has opened up new possibilities for studying the biological processes involved in human pregnancy, modeling diseases, and testing therapeutic compounds for clinical applications, including in vitro fertilization (IVF) and embryo implantation. Typically, to produce human endometrial organoids, primary endometrial tissue samples are dissociated enzymatically (with collagenases and / or dispases) and resuspended in Matrigel droplets in a prescribed medium that has been shown to promote organoid formation and maintenance from primary tissue or iPSC-derived cells.

[0003] However, there remains a need to improve methods for producing endometrial organoids and to predict and improve the outcomes of in vitro fertilization. [Overview of the project]

[0004] This specification describes a method for producing controlled endometrial organoids using micropatterning techniques, and a method for using organoids and cultured endometrial cells for diagnostic and predictive purposes, and for optimizing IVF and other infertility-related treatments, including embryo transfer and intrauterine insemination (IUI). This method is advantageous in that the organoids grow in a spatially controlled manner and exhibit less heterogeneity than organoids grown directly in a three-dimensional culture. This specification also describes methods for predicting the success of embryo transfer or other IVF-related treatments, methods for optimizing embryo transfer, methods for improving embryo implantation in the endometrium, and methods for embryo transfer in subjects. These methods include culturing endometrial cells as endometrial organoids or layers, and contacting the endometrial organoids or cultured cells with one or more blast-like cells. In some embodiments, the endometrial organoids or cultures include endometrial cells obtained from subjects undergoing or considering IVF-related treatment. Various parameters may be tested to identify the optimal conditions for successful embryo transfer and IVF treatment. In some embodiments, these methods are carried out using endometrial organoids fabricated using micropatterning as described herein.

[0005] In some embodiments, micropatterning is achieved by photolithography or microcontact printing. Those skilled in the art will understand that micropatterning can also be achieved by other means.

[0006] In some embodiments, endometrial cells used to produce organoids include, but are not limited to, established endometrial cell lines, patient biopsies, and patient biopsies grown as organoids, either as primary cultures or as long-established organoid cultures. These cells include endometrium of the endometrium grown with or without endometrial stromal cells.

[0007] Cells are seeded onto micropatterns coated with extracellular matrix proteins, typically including but not limited to laminin, fibronectin, vitronectin, Matrigel, and Geltrex. This coating can be either a thin molecular layer or a thick gel.

[0008] The cells then proliferate three-dimensionally, either spontaneously or by arranging extracellular matrix, including but not limited to those listed above, in a growth medium that causes folding into a third dimension.

[0009] These cultures can then be maintained on a micropatterned surface. Optionally, the cultures are released and resuspended under conditions that allow them to grow three-dimensionally in growth medium or a gel. In some embodiments, the gel is an ECM-based gel, such as Matrigel or a synthetic hydrogel.

[0010] This specification also describes a diagnostic method, in which artificial embryoid bodies (hereinafter referred to as blastoid cells) are created from human stem cells; this includes, but is not limited to, the embryoid bodies described in the following literature: Kagawa, H., et al. Human blastoids model blastocyst development and implantation. Nature 601, 600-605 (2022); Yu, L., et al. Blastocyst-like structures generated from human pluripotent stem cells. Nature 591, 620-626 (2021); doi.org / 10.1038 / s41586-021-03356-y; and Yanagida et al., Cell Stem Cell The diagnostic method is also described, in which endometrial cells, endometrial organoids, or micropatterned endometrial organoids collected from the patient are cultured as described above (28,1016-1022(2021);doi.org / 10.1016 / j.stem.2021.04.031). In the case of organoids, the three-dimensional structure may be chemically or mechanically disrupted to allow for the introduction of blast-like cells. Before introducing blast-like cells, endometrial cells are cultured in standard medium or subjected to hormone therapy.

[0011] The percentage of blast-like cells that undergo implantation-like events is measured by microscopic observation. This indicator is used as an in vitro diagnostic tool to determine a patient's likelihood of successful embryo implantation or successful pregnancy up to a certain point in time. This microscopic observation may include bright-field-based techniques, including phase-contrast or differential interference contrast (DIC) imaging, as well as immunofluorescence of embryonic cell population-specific markers (e.g., CDX2 for ectoderm, OCT4 for epiderm, and GATA4 or GATA6 for visceral endoderm) and endometrial markers (e.g., SOX9 or FOXA2), and observation of their relative positions. This imaging may include live imaging of blast-like cells during implantation analysis, or imaging of fixed samples after culturing blast-like cells with endometrium and then applying a fixative such as paraformaldehyde or methanol at some point in time. This imaging may also include the use of blast-like cells derived from human stem cell lines with fluorescent protein-based reporters.

[0012] In some embodiments, parameters other than the percentage of blast-like cells that implant are measured and used for diagnostic purposes. These include, but are not limited to, the timing of implantation of blast-like cells, the depth of implantation of blast-like cells, and the migration rate of blast-like cells before and after implantation.

[0013] In some embodiments, molecular parameters such as gene expression or chromatin reachability of specific genes in either blastoid cells or endometrium are measured and used for diagnostic purposes. These measurements can be performed via sequencing-based methods such as RNA sequencing or ATAC sequencing, microscopy-based methods such as immunofluorescence, or PCR-based methods such as quantitative real-time PCR.

[0014] The study also provides a method to predict the most likely timing for embryo transfer, or other parameters related to in vitro fertilization (IVF) or other fertility treatments, and to increase the chances of successful implantation or pregnancy. Hormone therapy mimicking the hormones produced in the body when the uterus prepares to receive an embryo is administered to endometrial cultures, and blast-like cells are introduced at various stages of this treatment. The timing of introducing blast-like cells into these endometrial cultures varies, and the parameters described above are measured for each timing. The results of this test, which finds the optimal percentage of blast-like cells that induce implantation-like events or any other parameter, are used to predict the most likely timing for embryo transfer to achieve success in IVF or other conception-related procedures.

[0015] In some embodiments, parameters other than the timing of embryo transfer are altered. For example, various luteal phase support measures are simulated in the culture, such as supplementing the culture with molecules to optimize the implantation-like event, including progesterone, luteinizing hormone, hCG, or GnRH agonists, after the introduction of blast-like cells. The results of this test are then used to predict the most likely successful luteal phase support regimen.

[0016] In some embodiments, information obtained from analyses described herein is combined with other information obtained from or directly from the patient's biological samples. This includes, but is not limited to, the patient's genome sequence, the transcriptome (e.g., as measured by RNA sequencing) of the patient's endometrial biopsy sample or cell culture from such sample, metabolome, proteome, or epigenome, or the patient's microbiome or health history, demographic information, or information about their current condition. This information may be used for prediction through any means, including predictions based on reasonably designed algorithms or artificial intelligence.

[0017] In some embodiments, blast-like cells are produced from induced pluripotent stem cells (PPTs) created from samples taken from the patient themselves. These samples may include, but are not limited to, the patient's blood or skin biopsy. Induced pluripotent stem cells are produced using methods that include transcription factor-based methods, which involve introducing transcription factors as DNA, RNA, or proteins. Examples include, but are not limited to, lentivirus-based methods, adenovirus-based methods, Sendai virus-based methods, and mRNA transfection-based methods. Induced pluripotent stem cells can also be produced via somatic cell nuclear transfer.

[0018] In some embodiments, blast-like cells are replaced with human embryos provided for this purpose. The embryos may be from the patient themselves or from another donor.

[0019] In some embodiments, blast-like cells are generated from cells that have been modified to prevent embryonic development beyond a certain point, such as by deleting genes that are important for post-implantation development but not for pre-implantation development. This avoids the ethical issues associated with culturing human embryos or blast-like cells.

[0020] Methods for predicting successful embryo implantation are also provided. These methods can be carried out by analyzing the implantation of blast-like cells on cultured endometrial cells or organoids, or by analyzing changes in markers of endometrial receptivity or endometrial pathology. The analysis can be performed using various hormone therapy protocols or by varying other conditions, and can be used to select the optimal protocol and / or to screen candidates for successful embryo transfer. Computer-based methods and computer systems are also provided for providing predictions of successful embryo implantation and for selecting the optimal protocol and conditions for embryo transfer. [Brief explanation of the drawing]

[0021] [Figure 1]Shows representative microscopic images of human endometrial organoids. [Figure 2] Bright-field and immunostained microscopic images of an open abdominal endometrial layer culture. Arrows indicate representative staining for FOXA2, E-cadherin (ECAD), and SOX9. [Figure 3] Bright-field microscopic image of blastoid cells. [Figure 4] Microscopic image of immunostained blastoid cells, where blue (thin arrow) is CDX2 (TE), yellow (curved arrow) is NANOG (Epi), and magenta (wide arrow) is NR2F2 (polar TE). [Figure 5] A series of micrographs showing the progression of blastoid cells from day 0 to day 4. [Figure 6] A pair of micrographs showing immunofluorescence of NR2F2 (polar trophectoderm) and phalloidin (actin) used for the analysis of blastoid cell implantation. When blastoid cells are implanted, cells expressing NR2F2 can be seen under the endometrial cells (boxed in the right figure). [Figure 7] Photographs of six test specimens analyzed for hCG. The test specimens show the results of an implantation analysis in which blastoid cells untreated (control, test specimens 1, 3, 5) or treated with hormones (hormone, test specimens 2, 4, 6) from three patients were transplanted into the endometrial layer in a micropattern plate. Arrows point to the test sites, where a line appears to be hCG positive (like in test specimens 2, 4, and 6). Negative results (absence of detectable hCG) are shown in test specimens 1, 3, and 5. [Figure 8] Photomicrographs taken at 4x magnification (upper image) and 10x magnification (lower image) of blastoid cells transplanted into the endometrial layer in a micropattern plate.

Modes for Carrying Out the Invention

[0022] Micropatterning technology enables the creation of controlled endometrial organoids. These organoids can be used for diagnostic and predictive purposes, as well as to optimize IVF and other infertility-related procedures, including embryo transfer and intrauterine insemination (IUI). The organoids grow in a spatially controlled manner and exhibit less heterogeneity than organoids grown directly in three-dimensional culture. They can be used in methods for predicting the success of embryo transfer or other in vitro fertilization-related procedures, optimizing embryo transfer, improving embryo implantation in the endometrium, and in embryo transfer procedures in subjects.

[0023] definition

[0024] All scientific and technical terms used in this application have the meanings commonly used in the art unless otherwise specified. When used in this application, the following terms have the specified meanings:

[0025] As used herein, “stromal cells” refers to mesenchymal cells located adjacent to the uterine epithelium on the side opposite the uterine cavity. These cells can differentiate spontaneously (in vivo) and / or by hormonal treatment into decotyledonized stromal fibroblasts. Stromal cells can be obtained as primary samples from patients, from established cell lines, or from iPSCs.

[0026] As used herein, “control” or “reference” sample means a sample that represents a normal measurement of each marker, such as one obtained from a normal, healthy control subject, or a baseline amount of the marker used for comparison. The sample may be the actual sample used for testing, or a reference level or reference range based on known normal measurements of the corresponding marker.

[0027] As used herein, “significant difference” means a difference that can be detected in a manner that a person skilled in the art would consider reliable, such as a statistically significant difference, or a difference of sufficient magnitude to be detected with reasonable confidence under the given circumstances. In one embodiment, a 10% increase or decrease relative to a reference sample is considered significant. In another embodiment, a 20%, 30%, 40%, or 50% increase or decrease relative to a reference sample is considered significant. In yet another embodiment, a doubling of the reference sample is considered significant.

[0028] As used herein, the term “Subject” includes, but is not limited to, any vertebrate, including, humans, non-human primates, mice, rats, guinea pigs, rabbits, cattle, dogs, cats, horses, goats, birds, reptiles, or fish. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is livestock, wild animals, or farm animals.

[0029] As used herein, "a" or "an" means at least one unless otherwise specified.

[0030] Methods for producing endometrial organoids

[0031] Methods for producing endometrial organoids include, in some embodiments, seeding endometrial epithelial cells onto a micropatterned surface; and culturing the seeded cells under conditions sufficient for three-dimensional proliferation and organoid formation. In some embodiments, seeding further includes seeding stromal cells onto the surface. The seeding of stromal cells may occur before, simultaneously with, or after the seeding of endometrial epithelial cells. This method produces endometrial assembly roids on the culture surface. Endometrial assembly roids are a combination of endometrial stromal cells and endometrial epithelial cells. The culture surface may be micropatterned as described herein, or a standard culture surface may be used. In some embodiments, the stromal cells are derived from primary cultures from patient biopsies or from endometrial cell lines. In some embodiments, a layer of stromal cells is first seeded onto the culture surface, and after the stromal cells adhere, epithelial cells are seeded on top, so that the basal slides of epithelial cells adhere to the stromal cells while the apical side faces the culture medium.

[0032] In some embodiments, the micropatterns are applied via photolithography or microcontact printing. In some embodiments, the micropatterns are coated with extracellular matrix proteins. In some embodiments, the culture conditions involve culturing endometrial cells in a culture medium containing extracellular matrix proteins. In some embodiments, the extracellular matrix proteins include laminin, fibronectin, vitronectin, Matrigel, and / or Geltrex. This coating may be either a thin molecular layer or a thick gel. The patterns are usually spaced far enough apart so that they do not influence each other via diffusive molecules. In some embodiments, the spacing between micropatterns is greater than approximately 100 μm. In some embodiments, the spacing between micropatterns is 80–200 μm. In some embodiments, the spacing between micropatterns is 100–300 μm. In some embodiments, the spacing between micropatterns is 150–250 μm.

[0033] Endometrial organoids and assembly broids produced according to the methods described herein are also provided. Endometrial organoids produced by seeding endometrial cells onto a micropattern surface have been observed to yield more reproducible phenotypes. Therefore, endometrial organoids produced by this micropatterning method are desirable for use in optimizing conditions for in vitro fertilization and other infertility-related procedures, predicting embryo transfer success, and in toxicological studies.

[0034] In some embodiments, the method further comprises releasing endometrial cells and resuspending them in a growth medium or gel under conditions sufficient for three-dimensional proliferation. In some embodiments, the gel includes an ECM-based gel.

[0035] Method for creating an embryo implantation model

[0036] Methods for creating an in vitro model of embryo implantation are also described. In some embodiments, the method comprises culturing endometrial epithelial cells; and contacting endometrial cells and blast-like cells in a culture or organoid. Culturing can be performed by seeding endometrial cells on a surface printed with a micropattern, or without seeding. In some embodiments, endometrial epithelial cells are cultured as endometrial organoids as described herein. In some embodiments, cells are cultured as a monolayer of endometrial epithelial cells. In some embodiments, the method further comprises culturing endometrial epithelial cells with blast-like cells in the presence of an implantation support agent. In some embodiments, blast-like cells are co-cultured with endometrial epithelial cells for up to about one week, during which time analysis is performed at any point. In some embodiments, the implantation support agent is administered to the endometrial epithelial cells before contact with the blast-like cells.

[0037] In some embodiments, the contact described above is performed with a plurality of blast-like cells, and the method further includes measuring the proportion of the plurality of blast-like cells that undergo implantation in the endometrial organoid. Implantation can be measured using techniques known in the art, for example, by washing the culture with a buffer and detecting whether or not the blast-like cells remain attached to the endometrial layer, or how many remain attached, or whether or not they have become suspended in the culture medium, or how many have become suspended, in which case attachment to the endometrial layer indicates implantation.

[0038] Models of embryo implantation can be used as a method to predict the success of embryo implantation. The ability of the target endometrial cells to support implantation can be analyzed using a single blast-like cell or multiple blast-like cells, as described above. This analysis can also be carried out using various hormone treatment protocols. One example of a hormone treatment protocol involves a two-day treatment with estrogen, followed by a four-day treatment with estrogen and progesterone. This latter treatment may be carried out in the analysis with or without the WNT inhibitors XAV-939 and 8-br-camp. In some embodiments, the analysis further includes control samples cultured in the same medium but not treated with hormones. A comparison is made between the control samples and the treated samples. Prediction of embryo implantation success can be based on relative levels between samples, absolute levels in the samples, or a combination of these factors.

[0039] In some embodiments, endometrial cells are obtained from established endometrial cell lines, patient biopsies, and / or patient biopsies grown as organoids in primary culture or long-term established organoid culture. These cells include endometrium of the endometrium grown with or without endometrial stromal cells. Using endometrial cells obtained from patients allows for the adjustment of in vitro fertilization, infertility treatment, and embryo transfer conditions to be optimal for individual patients. Patients can also gain more informed predictions about the likelihood of successful embryo transfer before investing limited resources.

[0040] In some embodiments, blast-like cells are replaced with human embryos provided for this purpose. The embryos may be from the patient themselves or from another donor.

[0041] In some embodiments, the culture involves culturing endometrial organoids in the presence of a hormone, a cyclic AMP-dependent protein kinase (PKA) activator, or a Wnt inhibitor. In some embodiments, the hormone includes progesterone, progestin, human chorionic gonadotropin (hCG), and / or gonadotropin-releasing hormone (GnRH) agonists. In some embodiments, the cyclic AMP-dependent PKA activator is 8-bromo-cAMP. In some embodiments, the measurement includes microscopic observation by phase-contrast imaging, differential interference contrast imaging, and / or immunofluorescence detection of embryo-specific markers and / or endometrial markers. In some embodiments, embryo-specific markers include CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, GATA4, and / or GATA6. In some embodiments, endometrial markers include SOX9, MUC1, PAEP, OLFM4, acetylated α-tubulin, ECAD, vimentin, and / or FOXA2. Markers for endometrial cells are described, for example, by Hong in Genes & Diseases 10, Issue 3, May 2023, Pages 931-947. In some embodiments, microscopic observation includes imaging of live spores or imaging of spores after treatment with a fixative.

[0042] In some embodiments, the blast cells are derived from a human stem cell line, and the human stem cell line is labeled with a fluorescent marker. In some embodiments, the method further includes measuring the time and / or depth at which blast-like cells implant in endometrial organoids, and / or measuring the migration rate of blast-like cells before and after implantation in endometrial organoids. In some embodiments, the method further includes measuring gene expression and / or chromatin reachability in blast-like cells and / or endometrial organoids. In some embodiments, the measurements include RNA sequencing, transposase-reachable chromatin (ATAC) sequencing, immunofluorescence, and / or PCR. In some embodiments, PCR is quantitative real-time PCR.

[0043] Methods for optimizing embryo transfer

[0044] Also provided are methods for optimizing embryo transfer in subjects, methods for predicting the success of embryo transfer, methods for performing embryo transfer, and similarly, methods for improving the outcomes of in vitro fertilization and other infertility-related treatments, including improving the success of embryo transfer. These methods can be employed in the context of treating patients requiring IVF or other infertility treatments, thereby optimizing or predicting the success of embryo transfer, following embryo transfer to a subject. In some embodiments, the timing and selection of the subject with implantation support agents or other parameters related to embryo transfer are based on the results of optimizations developed through the use of exovivo models of embryo implantation as described herein. Such optimizations may require modifying protocols for preparing the endometrium by, for example, applying hormonal protocols that mimic those administered to the patient before transfer (or mimic the natural pre-implantation cycle), and by optimizing implantation during embryo transfer.

[0045] In some embodiments, the method comprises preparing an exovivo model containing endometrial organoids, wherein the endometrial organoids contain endometrial cells obtained from a subject, and wherein the culture of the endometrial organoids is carried out in the presence of an implantation support agent. Culture can be carried out with or without stromal cells beneath the epithelial cells. The implantation support agent may be supplied to the cultured endometrial organoids before contact with blastoid-like cells. The method further comprises contacting multiple blastoid-like cells with the endometrial organoids at multiple time points relative to the culture of the endometrial organoids in the presence of the implantation support agent; and measuring the proportion of multiple blastoid-like cells implanting in the endometrial organoids as a function of the changed time points, thereby indicating the optimal time for embryo transfer in the subject as the time point that results in the implantation of the largest proportion of blastoid-like cells relative to the total number of blastoid-like cells contacted.

[0046] In some embodiments, the method comprises preparing an exovivo model comprising a cultured monolayer containing endometrial epithelial cells, wherein the endometrial epithelial cells are derived from the subject, and wherein the culture of the endometrial epithelial cells is carried out in the presence of an implantation support agent. The culture may be carried out with or without stromal cells beneath the epithelial cells. The implantation support agent may be supplied to the cultured endometrial epithelial cells before contact with blastoid cells. The method further comprises contacting multiple blastoid cells with endometrial epithelial cells at multiple time points relative to the culture of endometrial epithelial cells in the presence of the implantation support agent; and measuring the proportion of multiple blastoid cells implanting in the endometrial epithelial cells as a function of the changed time points, thereby indicating the optimal time for embryo transfer in the subject as the time point that results in the implantation of the largest proportion of blastoid cells relative to the total number of blastoid cells contacted.

[0047] In some embodiments, a method for optimizing embryo transfer in a subject comprises constructing an exovivo model described herein, where endometrial organoids (or epithelial cells) include endometrial cells obtained from the subject. The method further comprises culturing endometrial organoids (or epithelial cells) in the presence of multiple combinations of implantation support agents (where the implantation support agents are selected from progesterone, progestin, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH) agonists), and measuring the proportion of multiple blast-like cells that implant in the endometrial organoids (or epithelial cells) as a function of the implantation support agents. The optimal combination of implantation support agents for embryo transfer in a subject is indicated by the combination that maximizes the proportion of implanted blast-like cells relative to the total number of blast-like cells that come into contact with the endometrial organoids (or epithelial cells).

[0048] In some embodiments, methods for optimizing embryo transfer are used to alter parameters related to in vitro fertilization (IVF) or other infertility-related procedures to achieve successful implantation or pregnancy. For example, a hormonal treatment mimicking hormones produced in the body when the uterus prepares to receive an embryo is applied to an endometrial culture, and blast-like cells are introduced at various points in the course of this treatment. The timing of introducing blast-like cells into this endometrial culture is varied, and the above parameters are measured for each timing. The results of this test, in which the percentage of blast-like cells that cause implantation-like events or any of the other parameters are found to be optimal, are used to predict the most likely timing of embryo introduction to achieve success in IVF or other conception-related procedures.

[0049] In some embodiments, methods for optimizing embryo transfer are used to simulate various luteal phase support treatments in culture. A typical example of such simulation involves supplementing the culture with molecules that optimize implantation-like events, including progesterone, progestin, hCG, or GnRH agonists, after the introduction of blast-like cells. The results of this test are then used to predict the luteal phase support regimen most likely to succeed.

[0050] In some embodiments, information obtained from analyses described herein is combined with other information obtained from or directly from the patient's biological samples. Representative examples of such information include, but are not limited to, the patient's genome sequence, the transcriptome (e.g., as measured by RNA sequencing) of the patient's endometrial biopsy sample or cell culture from such sample, the metabolome, proteome, or epigenome, or the patient's microbiome or health history, demographic information, or information about their current condition. This information may be used, for example, to improve predictions of the outcome or success of the transplantation using computational means including the implementation of a reasonably designed algorithm or computational means including predictions based on machine learning. Those skilled in the art will understand how to train a machine learning-based system by inputting the corresponding information along with data on the success of embryo transfer associated with that information.

[0051] In some embodiments of the above method, multiple blast-like cells are obtained from induced pluripotent stem cells prepared from a sample taken from a subject. In some embodiments, the sample includes blood or skin biopsy. In some embodiments, induced pluripotent stem cells are prepared using a transcription factor-based method or somatic cell nuclear transfer. In some embodiments, the transcription factor-based method includes introducing a transcription factor, including DNA, RNA, or protein, from the sample into cells. In some embodiments, the transcription factor-based method includes lentivirus-based, adenovirus-based, or sendai virus-based reprogramming, or reprogramming based on mRNA transfection. In some embodiments, blast-like cells are prepared from cells modified to prevent post-implantation embryonic development.

[0052] Methods for predicting successful embryo implantation

[0053] Methods for predicting the success of embryo implantation in a subject, or for evaluating the likelihood of implantation success in a subject, are also provided. In some embodiments, the method includes culturing endometrial cells obtained from the subject. The method further includes contacting the endometrial cells with a hormone protocol (e.g., estrogen and / or progesterone, for various periods). In some embodiments, the contact includes contacting the endometrial cells with estrogen for a first period, and contacting the endometrial cells with estrogen and progesterone for a second period. In some embodiments, the first period is 2 days and the second period is 4 days.

[0054] In some embodiments, the analysis further includes a control sample cultured in the same medium but not treated with hormones. A comparison is made between the control sample and the treated sample. Prediction of embryo implantation success may be based on relative levels between samples, absolute levels in the samples, or a combination of these factors. The method further includes analyzing endometrial cell samples for receptive endometrial markers and / or endometrial pathology markers. In some embodiments, the analysis further includes comparing endometrial cell samples with a control sample that has not been exposed to estrogen, progesterone, or other hormone treatments. Implantation success is detected if receptive endometrial markers are present (or in higher than normal levels) and / or endometrial pathology markers are absent (or in lower than normal levels), or otherwise different from the control sample.

[0055] In some embodiments, receptive endometrial markers include, but are not limited to, one or more of the following: progesterone-associated endometrial protein (PAEP), acetylated α-tubulin ciliates, mucin, CXC-motif chemokine ligand 14 (CXCL14), and secretory phosphorylated protein 1 (SPP1). In some embodiments, endometrial pathological markers include, but are not limited to, one or more of the following: B-cell lymphoma 6 (BCL6), CC-motif chemokine ligand 19 (CCL19), mucin 5B (MUC5B), protocadherin 17 (PCDH-17), protein tyrosine dephosphate receptor R (PTPRR), and interleukin-6 cytokine signaling molecule (IL6ST).

[0056] In some embodiments, endometrial cells are cultured as layers, for example, in a two-dimensional culture. In some embodiments, endometrial cells are cultured as organoids, for example, in a three-dimensional culture. In some embodiments, the analysis includes RNA sequencing, quantitative polymerase chain reaction (qPCR), immunostaining, or ATAC sequencing. In some embodiments, endometrial cells are obtained from the subject independently of the subject's hormonal cycle. The method does not require the sample to be obtained while the patient is in a hormone-induced "simulated cycle" or at a specific stage of the patient's natural hormonal cycle.

[0057] The likelihood of target endometrial cells supporting successful implantation can be measured using various hormone treatment protocols. One example of a hormone treatment protocol involves a two-day estrogen treatment followed by a four-day treatment with estrogen and progesterone. This latter treatment may be performed with or without the WNT inhibitors XAV-939 and 8-br-camp in the analysis.

[0058] Computer-based methods and systems

[0059] A computer-based method for selecting a protocol for embryo implantation in a subject is also provided. In some embodiments, this method is: a)i) The percentage of implanted blast-like cells detected in response to estrogen and / or progesterone treatment, as optional in the method for predicting embryonic implantation success described above; ii) The presence of receptive endometrial markers and / or the absence of endometrial pathological markers detected by the above method; iii) Receiving an input that includes at least one parameter selected from the subject's age, body mass index (BMI), past pregnancy success, and / or the presence of different bacterial species in a biological sample obtained from the subject (which can be obtained, for example, from the microbiome analysis described above in the context of optimizing embryo transfer). b) Using a machine learning software module, generate an output that includes an indicator of the likelihood of successful embryo implantation based on the input received in (a), Includes.

[0060] In some embodiments, steps (a) and (b) are performed based on multiple hormone treatment conditions, and a protocol for embryo implantation is selected based on the output of step (b).

[0061] A computer system for selecting a protocol for embryo implantation in a subject is also provided. In some embodiments, the computer system has the following configuration: a) Apparatus, and b) When executed by the processing unit, the processing unit will i) In the above method for predicting embryonic implantation success, optionally, the percentage of implanted blast-like cells detected in response to treatment with estrogen and / or progesterone and a specific hormone therapy schedule; The presence of receptive endometrial markers and / or the absence of endometrial pathological markers detected in the methods described above for predicting successful embryo implantation; Age of the subject, degree of obesity (BMI), presence of bacterial species, and / or past pregnancy success; Accepts input containing at least one parameter selected from: ii) Based on the input received in (i), generate an output that includes an indication of the likelihood of successful embryo implantation. A non-transient computer-readable medium containing instructions to perform the following actions: This includes the ability to generate indicators of the likelihood of successful embryo implantation for various treatment protocols, which can be used to select the optimal hormone therapy and other conditions that can optimize the success of embryo transfer.

[0062] The pregnancy success data used as input can include the number of pregnancies, the number of miscarriages, and, for example, the number of embryo transfer failures observed in in vitro fertilization. Other inputs include data obtained from the above analyses using embryo implantation models and cultured endometrial cells or organoids. For example, the percentage of implanted blast-like cells for a given hormone therapy protocol, or the timing of implantation, the depth of implantation of blast-like cells, and the migration rate of blast-like cells before and after implantation for a given hormone therapy protocol can be used as input. An example of additional input data is described in Example 5 below. The machine learning module is trained using these inputs and related results obtained from known patient data and treatment conditions.

[0063] Example of an embodiment

[0064] Embodiment 1: A method for producing endometrial organoids, the method being: a) Seeding endometrial cells onto a surface printed with a micropattern; and b) Culture the cells seeded in step (a) under conditions sufficient for three-dimensional proliferation and organoid formation; Includes.

[0065] Embodiment 2: The method according to Embodiment 1, wherein the seeding in step (a) further comprises seeding stromal cells onto a surface.

[0066] Embodiment 3: The method according to Embodiment 1, wherein the micropattern is applied via photolithography or microcontact printing.

[0067] Embodiment 4: The method according to Embodiment 1, wherein the endometrial cells are obtained from an established endometrial cell line, a patient biopsy, and / or a patient biopsy that has grown as an organoid in a primary culture or a long-established organoid culture.

[0068] Embodiment 5: The method according to Embodiment 1, wherein the micropattern is coated with an extracellular matrix (ECM) protein.

[0069] Embodiment 6: The method according to Embodiment 1, wherein the condition of step (b) includes culturing the endometrial cells in a culture medium containing extracellular matrix proteins.

[0070] Embodiment 7: The method according to Embodiment 5 or 6, wherein the extracellular matrix protein comprises laminin, fibronectin, vitronectin, Matrigel, and / or Geltrex.

[0071] Embodiment 8: The method according to any of the prior embodiments, further comprising releasing the endometrial cells into a growth medium or gel under conditions sufficient for three-dimensional proliferation.

[0072] Embodiment 9: The method according to Embodiment 8, wherein the gel comprises an ECM-based gel.

[0073] Embodiment 10: A method for creating an exovivo model of embryo implantation, the method being: a) Culturing endometrial organoids (wherein the organoids include endometrial cells obtained from human subjects); b) Blast-like cells are brought into contact with endometrial cells of the organoid; and c) Culturing the endometrial organoids together with the blast-like cells in the presence of an implantation support agent; Includes.

[0074] Embodiment 11: The method according to Embodiment 10, wherein the contact in step (b) is performed using a plurality of blast-like cells, and the method further comprises measuring the proportion of the plurality of blast-like cells that experience implantation in the endometrial organoid.

[0075] Embodiment 12: The method according to Embodiment 10, wherein step (a) of culturing comprises culturing the endometrial organoid in the presence of a hormone, a cyclic AMP-dependent protein kinase (PKA) activator, or a Wnt inhibitor.

[0076] Embodiment 13: The method according to Embodiment 12, wherein the hormone comprises progesterone, progestin, human chorionic gonadotropin (hCG), and / or a gonadotropin-releasing hormone (GnRH) agonist.

[0077] Embodiment 14: The method according to Embodiment 12, wherein the activator of the cyclic AMP-dependent PKA is 8-bromo-cAMP.

[0078] Embodiment 15: The method according to Embodiment 11, wherein the measurement described above includes phase contrast imaging, differential interference contrast imaging, and / or microscopic observation by immunofluorescence detection of embryo-specific markers and / or endometrial markers.

[0079] Embodiment 16: The method according to Embodiment 15, wherein the embryo-specific markers include CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, and / or GATA6.

[0080] Embodiment 17: The method according to Embodiment 15, wherein the endometrial markers include SOX9, MUC1, PAEP, OLFM4, acetylated α-tubulin, ECAD, vimentin, and / or FOXA2.

[0081] Embodiment 18: The method according to Embodiment 15, wherein the above microscopic observation includes imaging of live blast cells or imaging of blast cells after treatment with a fixative.

[0082] Embodiment 19: The method according to Embodiment 15, wherein the blast-like cells are derived from a human stem cell line, and the human stem cell line is labeled with a fluorescent marker.

[0083] Embodiment 20: The method according to Embodiment 11, further comprising measuring the time and / or depth at which the blast-like cells implant in the endometrial organoid, and / or measuring the migration rate of the blast-like cells before and after implantation in the endometrial organoid.

[0084] Embodiment 21: The method according to Embodiment 10, further comprising measuring gene expression and / or chromatin reachability in the blast-like cells and / or endometrial organoids.

[0085] Embodiment 22: The method according to Embodiment 21, wherein the measurement described above includes RNA sequencing, transposase-reachable chromatin (ATAC) sequencing, immunofluorescence, and / or PCR.

[0086] Embodiment 23: The method according to Embodiment 22, wherein the PCR is quantitative real-time PCR.

[0087] Embodiment 24: A method for optimizing embryo transfer in a subject, wherein the method is: a) To create the exovivo model described in Embodiment 10, wherein the endometrial organoids include endometrial cells obtained from the subject, and the culture of the endometrial organoids is carried out in the presence of an implantation support agent; b) Contacting multiple blast-like cells with the endometrial organoids at multiple time points relative to culturing the endometrial organoids in the presence of implantation support agents; and c) In step (b), measure the proportion of multiple blast-like cells that implant in the endometrial organoid as a function of the changed time (so that the optimal time for embryo transfer in the above subject is indicated by the time that results in the implantation of the largest proportion of blast-like cells relative to the total number of blast-like cells contacted in step (b)), Includes.

[0088] Embodiment 25: A method for optimizing embryo transfer in a subject, wherein the method is: a) To create an exovivo model of Embodiment 11 in which the endometrial organoids include endometrial cells obtained from the subject; b) culturing endometrial organoids in the presence of multiple combinations of implantation support agents, wherein the implantation support agent is selected from progesterone, progestin, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH) agonists; and c) Measure the proportion of multiple blast-like cells that implant in the endometrial organoid as a function of the implantation support agent in step (b) (the optimal combination of implantation support agents for embryo transfer in the subject is indicated by the combination that maximizes the proportion of implanted blast-like cells relative to the total number of blast-like cells that come into contact with the endometrial organoid), Includes.

[0089] Embodiment 26: The method according to Embodiment 24 or 25, wherein the plurality of blast-like cells are obtained from induced pluripotent stem cells prepared from a sample collected from the subject.

[0090] Embodiment 27: The method according to Embodiment 26, wherein the sample includes blood or a skin biopsy.

[0091] Embodiment 28: The method according to Embodiment 26, wherein induced pluripotent stem cells are produced using a transcription factor-based method or somatic cell nuclear transfer.

[0092] Embodiment 29: The method according to Embodiment 28, wherein the transcription factor-based method comprises introducing a transcription factor, including DNA, RNA, or protein, into cells from the sample.

[0093] Embodiment 30: The method according to Embodiment 28, wherein the transcription factor-based method includes lentivirus-based, adenovirus-based, or sendai virus-based reprogramming, or mRNA transfection-based reprogramming. Embodiment 31: The method according to Embodiment 25 or 26, wherein the blast-like cells are produced from cells modified to prevent post-implantation embryonic development. [Examples]

[0094] The following examples are provided to illustrate the present invention and to assist those skilled in the art in the manufacture and use of the present invention. These examples are not intended to limit the scope of the present invention in any way.

[0095] Example 1: Endometrial culture

[0096] Endometrial organoids are grown in a three-dimensional culture and treated with a hormonal regimen that mimics what naturally occurs during the cycle of preparing the uterus for pregnancy, or the treatment course that a physician uses with an IVF patient in preparation for embryo transfer. This step allows blast-like cells to attach to the endometrium. This hormonal treatment is then varied to identify the most effective protocol. The timing of introducing blast-like cells can be optimized in relation to this hormonal cycle.

[0097] Endometrial organoids were dissociated and seeded onto Matrigel-coated plates according to the protocol published by Khoei et al. 2023 (doi.org / 10.1038 / s41596-023-00802-1) to form open endometrial layers (OFELs). A typical protocol included the following steps: (1) three primary samples (endometrial biopsies) were obtained; (2) endometrial organoids were prepared from all samples; (3) open endometrial layer (OFEL) cultures were prepared; OFEL cultures were treated with hormones (estradiol, progesterone, 8-br-cAMP, XAV939) in contrast to a control (DMSO).

[0098] OFEL cultures should be cultured at a sufficient density to prevent blast-like cells from touching the culture surface. Blast-like cells adhere to the culture surface and can confuse interpretation.

[0099] Representative images of human endometrial organoids are shown in Figure 1. Bright-field and immunohistochemical images of OFEL are shown in Figure 2. FOXA2 is involved in the development and function of endometrial glands, ECAD labels glandular epithelium, and SOX9 is a progenitor cell marker expressed in vivo at the base of endometrial glands.

[0100] Example 2: Preparation of blast-like cells

[0101] Blast-like cells are introduced along with endometrial organoids and cultured. The culture lasts from several hours to up to a week, during which, optionally, drugs to induce or maintain implantation are introduced. These drugs are called "implantation support" drugs. The timing, combination, and selection of implantation support drugs can be varied to determine the optimal schedule.

[0102] Blast-like cells are prepared as illustrated below. To avoid using blast-like cells that adhere regardless of endometrial receptivity, select cells that do not have external cell clumps.

[0103] Blast-like cells are generated based on the protocol published by Khoei et al. 2023 (doi.org / 10.1038 / s41596-023-00802-1) using the following parameters / modifications: Plate type: Aggrewell 24-well plate Culture on gelatin: 150 minutes (in PXGL + 10 μM Rock inhibitor (RI)) Cell count: 80,000-90,000 cells / well Aggregation medium: DMEM / F-12+0.3%BSA(Sigma)+10μM RI Sinking time: 20 minutes Centrifugation: 200 × g, 2 minutes Total volume on day 1: 1 mL, including: 250 μL of agglutination medium + cells in 250 μL of agglutination medium 500 μL of agglutination medium to be added after centrifugation. PALLY: 2μM PD0325901, 2μM A83-01, 2μM LPA, 10ng / mL LIF, and 10μM RI Day 0: Remove 750 μL of medium and add 1000 μL of PALLY. Days 1-3: Remove 900 μL of medium and add 1000 μL of PALLY.

[0104] Following this protocol, blast-like cell formation was observed with an efficiency of 70-80%, and the presence of the correct markers, GATA3 (ectoderm), NR2F2 (polar TE), NANOG (epiderm), and SOX17 (primitive endoderm), was confirmed.

[0105] Figure 3 shows a bright-field image of blast-like cells. Figure 4 shows immunostained images of blast-like cells, where blue (thin arrows) indicates CDX2 (TE), yellow (curved arrows) indicates NANOG (Epi), and magenta (thick arrows) indicates NR2F2 (polar TE). Figure 5 shows the progression of blast-like cells from day 0 to day 4.

[0106] Example 3: Implantation Analysis

[0107] Observational indicators can be used to assess implantation. Bright-field imaging can be used to distinguish between shriveled and cavitated blastoid cells. Washing tests can be used to distinguish between migration and fixation. Immunofluorescence of mucin (endometrial gland) and NR2F2 (polar ectoderm), combined with phalloidin (actin) and DAPI (nuclear) staining, is used to analyze blastoid cell implantation. If blastoid cells implant, cells expressing NR2F2 will be visible beneath the endometrial cells (see Figure 6). Representative results using NR2F2 analysis showed that in hormone-treated OFELs, 11 out of 40 blastoid cells implanted in patient #1, 1 out of 23 implanted in patient #2, and 6 out of 38 implanted in patient #3. In the control-treated OFELs, none of the blast-like cells from patients #1, #2, and #3 (40, 25, and 35 cells respectively) implanted.

[0108] Optionally, an hCG test kit can be used, for example, around 4 days after implantation. A positive hCG test indicates successful implantation. Using an hCG test kit makes it easier to confirm successful implantation. An example of using an hCG test kit is shown in Figure 7, which is a photograph of six test strips on which hCG was measured. These strips show the results of implantation analysis in three patients who were either untreated (control, strips 1, 3, and 5) or treated with hormone therapy (hormone therapy, strips 2, 4, and 6), and blast-like cells were transplanted into the endometrial layer in a micropattern plate. The arrows indicate the test site, and the appearance of a line here indicates a positive hCG result (as in strips 2, 4, and 6). Negative results (absence of detectable hCG) are shown in strips 1, 3, and 5. Representative micrographs of blast-like cells transplanted into the endometrial layer in a micropattern plate are shown at 4x magnification (top) and 10x magnification (bottom).

[0109] Example 4: Overall Analytical Procedure

[0110] The following is an exemplary protocol for sample processing and preparation for analysis.

[0111] Sample criteria

[0112] at least 7mm 3 Endometrial biopsy specimens of a certain size are requested during the patient's secretory phase. Alternatively, the specimen may be collected at any time. Antibiotics must not be taken for 7 days prior to the biopsy for microbiome analysis. Any medications, including antibiotics, taken in the month prior to the biopsy that may alter the microbiome or immunological status must be listed on the request form.

[0113] Sample Receiving Procedure

[0114] For each received sample, check and verify the number and characteristics of the tubes. Inspect whether the sample ID and collection date labels are properly affixed to the tubes. Samples without labels are processed but are temporarily labeled and stored separately and reported to the person in charge of coordination at the test facility. Samples without labels will have permanent labels affixed once their characteristics are confirmed.

[0115] From each tube, the following information is recorded: sample ID; collection date and time; sample quality (mostly endometrial tissue, not blood or mucus); estimated sample volume (mm 3 ). If a sufficient amount (more than 7 mm 3 ) of the sample is available, the procedure proceeds to the next step.

[0116] Procedures for sample division and cryopreservation

[0117] In a biological safety cabinet (BSC), handle one sample tube at a time and record the sample ID of the tube being processed. Spray 70% alcohol on the tube before bringing it into the BSC. The sample is sliced thinly into several parts with a scalpel to a volume of at least 2 mm 3 each. If the biopsy sample size is less than 2 mm 3 , use the entire sample for organoid preparation. If the biopsy sample size is 2 mm 3 ~4 mm 3 , the sample should be divided into two parts. One part is used for organoid preparation and the other for metabolomics or transcriptomics - microbiome analysis. If the biopsy sample size is 4 mm 3 ~6 mm 3 , the sample should be divided into three parts. One part is used for organoid preparation, one for metabolomics analysis, and one for transcriptomics - microbiome analysis. If the biopsy sample size exceeds 6 mm 3 , the sample should be divided into four parts. One part is used for organoid preparation, one for metabolomics analysis, one for transcriptomics - microbiome analysis, and the last one for cryopreservation.

[0118] Place all but one portion into cryopreservation tubes containing cryopreservation culture medium (one portion per tube). Label each tube with the sample ID, processing date, function code, and the initials of the person who processed it. Store the tubes in a -80°C freezer. The next day, transfer the tubes to their designated locations in the cryopreservation container. The function code is used to identify endometrial organoids (hEMOs), endometrial stromal cells (hESCs), primary samples for metabolome analysis (METs), primary samples for transcriptomics and microbiome analysis (TR-MICs), and primary samples for cryopreservation (PREs). The remaining single sample portion is placed in phosphate-buffered saline (PBS - / -) solution and stored on ice until all samples have been processed and are ready for the next step. The number of sample portions or tubes, along with the function codes, are stored in the database.

[0119] Procedure for preparing and cryopreserving endometrial cultures

[0120] Creation of organoids

[0121] All procedures are performed in a biological safety cabinet. Multiple samples may be processed simultaneously, provided that the petri dishes and tubes are properly labeled. This protocol follows the standard protocol developed by Turco et al. 2017[1] and Boretto et al. 2017[2]: 1. Transfer the sample from the tube to a 35mm Petri dish. 2. Cut the tissue sample into tiny pieces (0.5 mm) using a dissecting knife. 3 ) subdivide 3. Return the subdivided tissue pieces to the tube. 4. Centrifuge the tube at 200 g for 5 minutes, then discard the supernatant. 5. Add 1 mL of DMEM / F-12 to the tube, followed by 1 mL of collagenase IV (2 mg / mL). 6. Using a plastic Pasteur pipette, transfer everything to a 35mm Petri dish and keep at 37°C. 7. Using a plastic Pasteur pipette, repeat the aspirate / discharge process 50 times, and observe under a microscope every 30 minutes. 8. To collect stromal cells, add culture medium (DMEM / F-12 or RPMI) when sufficient glandular components have been collected to stop digestion. 9. Transfer everything to the tube and let it sit for 2 minutes. 10. Pass the supernatant through a 40 μm filter and collect the transfusion containing stromal cells in a 50 mL conical tube. 11. Wash the filter several times with DMEM / F-12 or RPMI. 12. For human endometrial stromal cell (hESC) cultures: Centrifuge the tube at 600×g for 6 minutes, remove the supernatant, and wash the pellet with DMEM / F-12. Centrifuge again at 600×g for 6 minutes, remove the supernatant, and suspend in 5 mL of hESC medium. Count the number of cells and plate approximately 1 million cells each onto T-25. Change the medium every 2 days and culture for 3-5 days. The following is the case for human endometrial organoid (hEMO) cultures: 13. From step 11, backwash the glandular elements from the filter into a Petri dish and return them to the tube from step 9. 14. Pass the entire mixture through a 100 μm filter into a 50 mL conical tube and collect the transfluid containing glandular elements. 15. Wash the filter several times with DMEM / F-12 or RPMI. 16. Centrifuge the tube at 500 x g for 5 minutes, then discard the supernatant. Resuspend in 17.1-2 mL of Advanced DMEM / F-12. 18. Centrifuge the tube at 500 x g for 5 minutes, then discard the supernatant. 19. Estimate the volume of the pellet, add 20 times the estimated pellet volume of Matrigel solution (70% Matrigel, 30% DMEM / F-12), and place on ice. 20. Using a pipette, draw in / dispense to ensure uniform distribution of glandular elements in the Matrigel solution, and drop 20-25 μL of Matrigel / cell suspension into the center of each well of a 48-well tissue culture plate. 21. Place the plate in a 37°C incubator and let it stand for 15 minutes. 22. Layer each droplet with 250 μL of Expansion Medium. 23. Change the culture medium every 1 day and culture for 7-10 days before the next subculturing.

[0122] cryopreservation of hESC and hEMO

[0123] For hESC cultures: Passage the culture in the T-25 flask when it reaches 90% confluence using TrypLE. Resuspend the cells in 3 mL of frozen medium (90% FBS, 10% DMSO). Freeze three tubes per sample, labeled with sample ID, date, and initial.

[0124] For hEMO cultures: Once a sufficient quantity of organoids with a diameter of approximately 200 μm is obtained, subculture the organoids (follow the protocol in Khoei et al. 2023, doi.org / 10.1038 / s41596-023-00802-1). Suspend the cells in Recovery Cell Culture Freezing Medium. Freeze three tubes per sample, labeled with sample ID, date, and initial.

[0125] Metabolome and microbiome analysis of samples

[0126] Metabolome analysis

[0127] A portion of the biopsy sample is processed for metabolome analysis. [3] 20 mg (2 mm 3 Tissue sections of the sample are homogenized in methanol using ceramic beads, followed by protein precipitation. The supernatant is vacuum-dried, suspended in acetonitrile, and centrifuged again. The supernatant is then injected into an HPLC / LC-MS instrument.

[0128] Transcriptomics and Microbiome Analysis

[0129] A portion of the primary sample is processed for both transcriptomics and microbiome analysis. Microbial species in endometrial biopsy are identified by 16S rRNA sequencing. A protocol for separating bacterial DNA from human RNA is performed. 25 mg (2.5 mm) of the biopsy sample. 3 )[4] is assigned to this analysis. RNA and NA are extracted from the sample, and human RNA and bacterial DNA are purified[5], after which the DNA sample is sent to a supplier (Novogene) for sequencing.

[0130] The presence of bacterial DNA from the following groups was detected: Lactobacillus group: L.crispatus, L.gasseri, L.iners, and L.jensenii Pathogens of the genital tract: Actinomyces israelii, Atopobium vaginae, Bacteroides fragilis, Bifidobacterium spp, Clostridium sordelii, Dialister spp, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Mycobacterium tuberculosis, Mobiluncus spp, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica, Prevotella bivia, Prevotella disiens, Sneathia spp, and Treponema pallidum Genital tract pathogens associated with chronic endometritis: Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus agalactiae group B / Streptococcus viridans, Ureaplasma urealyticum.

[0131] Next, endometrial culture can be performed as described in Example 1, blast-like cell formation as described in Example 2, and implantation analysis as described in Example 3.

[0132] Example 5: Prediction of successful embryo implantation

[0133] The protocols for sample processing and analysis preparation can be part of an ongoing refinement process. For example, a biopsy is taken, and various analyses are performed, including implantation analysis, to obtain transcriptome, microbiome, and other medical information. The results obtained from these analyses and data collections are input into classifiers and / or regression algorithms. The results are reviewed and reported, and the algorithms are refined as iterations are completed.

[0134] Specifically, patient data may be input into a system trained on such information to provide an overall probability of a patient succeeding in embryo transfer when given a particular type of hormone therapy, or to provide a set of probabilities of a patient succeeding in embryo transfer when given multiple different hormone therapies tested in parallel.

[0135] The input to the algorithm is a set of "features" (values) generated from all analyses of the patient being analyzed. This may include, but is not limited to, the following: In vitro analysis output: Percentage of attached blast-like cells in a given hormone therapy protocol; In vitro analysis output: Other values ​​described in the application, such as implantation timing, implantation depth of blast-like cells, and pre- and post-implantation migration rate of blast-like cells in a given hormone therapy protocol; Transcriptomics output: Numerical differences in various gene expression levels before and after a given hormone therapy protocol (for example, differences in BCL6 and SIRT1, but not limited to these); Other omics outputs from metabolomics / proteomics / epigenomics, etc. Microbiome output: Output of the primary sample (the signal may be either the binary presence or absence of a specific pathogen, as well as the relative abundance of various pathogens detected (i.e., the proportion of a specific pathogen detected)); Demographic data: For example, but not limited to, the patient's age, body mass index (BMI), and past pregnancy success.

[0136] To train the algorithm, labeled training is compiled by taking the aforementioned features as input and associating them with known patient outcomes for a given hormone therapy protocol. This is achieved by extracting the aforementioned set of features from a large number of patients (including coding the applied hormone therapy as an input categorical variable) and assigning them outcome measures, such as a simple success measure (0: embryo transfer failure; 1: embryo transfer success), or a more complex measure such as a set of classifications that may take values ​​such as "successful pregnancy," "failure (no positive pregnancy test)," "biochemical pregnancy," "molar pregnancy," "ectopic pregnancy," "intrauterine clinical pregnancy," "miscarriage," or "live birth," but are not limited to these.

[0137] This training data may then be fed to many different machine learning algorithms, such as, but are not limited to, multivariate logistic regression, support vector machines (SVMs), or neural networks with classifiers (e.g., Gaussian classifiers), to fit predictive models to the training data.

[0138] After the model is trained, these features may be obtained from new patients and fed into the algorithm along with in vitro results for each hormone therapy protocol to derive predictions of both the overall probability of transplant success and the likelihood of success for each hormone therapy protocol.

[0139] In another embodiment, a predictive algorithm for the in vitro analysis itself is formed so that, when the algorithm is trained, in vitro analysis involving blast-like cells is no longer required to identify a patient's hormone therapy protocol. Features are obtained from the in vitro analysis as a measurement result for training the machine learning algorithm (e.g., blast-like cell adhesion rate). Each hormone therapy protocol corresponds to a numerical output from the model, and the model will predict the percentage of blast-like cells that are likely to adhere with that hormone therapy protocol. Other outcome measures that can be predicted include, for example, adhesion depth and timing. The clinician can then select the protocol that yields the highest score. For inputs, features are either obtained from the primary sample or are present as training input values ​​in, for example, cultures (organoids) such as microbiomes of the primary sample, demographic information, or other omics values.

[0140] References

[0141] 1.Turco M, et al.Nature cell biology.2017;19(5):568-577.

[0142] 2. Boretto M, et al. Development (Cambridge, England).2017;144(10):1775-1786.

[0143] 3.Li J,et al.Reproductive biology and endocrinology:RB&E.04 / 30 / 2018 2018;16(1):42.

[0144] 4.Moreno I,et al.Microbiome.01 / 04 / 2022 2022;10(1):1.

[0145] 5.Bruggeling C,et al.MicrobiologyOpen.2021 Jun 2021;10(3):e1191.

[0146] 6. Heidari Khoei H, et al. Nature protocols.2023;18(5):1584-1620.

[0147] Throughout this application, various publications are referenced. The entirety of the disclosures in these publications is incorporated herein by reference to more fully illustrate the state of the art to which the present invention belongs.

[0148] Those skilled in the art will understand that the ideas and specific embodiments disclosed in the foregoing description may readily be used as a basis for modifying or designing other embodiments for accomplishing the same objective of the present invention. Those skilled in the art will also understand that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A method for producing endometrial organoids, (a) seeding endometrial cells onto a surface printed with a micropattern; and (b) Culturing the cells seeded in step (a) under conditions sufficient for three-dimensional proliferation and organoid formation; The method, including the method described above.

2. The method according to claim 1, wherein the seeding in step (a) further comprises seeding stromal cells onto the surface.

3. The method according to claim 1, wherein the micropattern is applied via photolithography or microcontact printing.

4. The method according to claim 1, wherein the endometrial cells are obtained from an established endometrial cell line, a patient biopsy, and / or a patient biopsy that has grown as an organoid in a primary culture or a long-established organoid culture.

5. The method according to claim 1, wherein the micropattern is coated with an extracellular matrix (ECM) protein.

6. The method according to claim 1, wherein the condition of step (b) includes culturing the endometrial cells in a culture medium containing extracellular matrix proteins.

7. The method according to claim 5 or 6, wherein the extracellular matrix protein comprises laminin, fibronectin, vitronectin, Matrigel, and / or Geltrex.

8. The method according to any of the prior claims, further comprising releasing the endometrial cells into a growth medium or gel under conditions sufficient for three-dimensional proliferation.

9. The method according to claim 8, wherein the gel comprises an ECM-based gel.

10. A method for predicting the success of embryo implantation in a subject, (a) Culturing endometrial cells obtained from the subject; (b) Bringing multiple blast-like cells into contact with endometrial cells; (c) culturing the endometrial cells together with the blast-like cells in the presence of an implantation support agent; and (d) detecting the implantation of at least one blast-like cell, wherein the detection of the implantation of at least one blast-like cell indicates successful implantation of the embryo into the subject, The method, including the method described above.

11. The method according to claim 10, wherein the endometrial cells are cultured as a layer of endometrial cells or as endometrial organoids.

12. The method according to claim 10, further comprising measuring the percentage of the plurality of blast-like cells that experience implantation in the endometrial organoid.

13. The method according to claim 10, wherein the culturing in step (a) comprises culturing the endometrial cells in the presence of a hormone, a cyclic AMP-dependent protein kinase (PKA) activator, or a Wnt inhibitor.

14. The method according to claim 13, wherein the hormone comprises progesterone, progestin, human chorionic gonadotropin (hCG), and / or a gonadotropin-releasing hormone (GnRH) agonist.

15. The method according to claim 13, wherein the activator of the cyclic AMP-dependent PKA is 8-bromo-cAMP.

16. The method according to claim 12, wherein the measurement includes microscopic observation by phase contrast imaging, differential interference contrast imaging, and / or immunofluorescence detection of embryo-specific markers and / or endometrial markers.

17. The method according to claim 16, wherein the embryo-specific marker includes CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, GATA4, and / or GATA6.

18. The method according to claim 16, wherein the endometrial marker comprises SOX9, MUC1, PAEP, OLFM4, acetylated α-tubulin, ECAD, vimentin, and / or FOXA2.

19. The method according to claim 16, wherein the microscopic observation includes imaging of the blast cells as living cells or imaging of the blast cells after treatment with a fixative.

20. The method according to claim 16, wherein the blast-like cells are derived from a human stem cell line, and the human stem cell line is labeled with a fluorescent marker.

21. The method according to claim 12, further comprising measuring the time and / or depth at which the blast-like cells implant in the endometrial organoid, and / or measuring the migration rate of the blast-like cells before and after implantation in the endometrial organoid.

22. The method according to claim 10, further comprising measuring gene expression and / or chromatin reachability in the blast-like cells (or more) and / or endometrial organoids.

23. The method according to claim 22, wherein the measurement comprises RNA sequencing, transposase-reachable chromatin (ATAC) sequencing, immunofluorescence, and / or polymerase chain reaction (PCR).

24. The method according to claim 23, wherein the PCR is quantitative real-time PCR.

25. A method for optimizing embryo implantation in a target, (a) Culturing endometrial cells obtained from the subject in the presence of an implantation support agent; (b) Contacting multiple blast-like cells with the endometrial cells at multiple time points relative to culturing the endometrial cells in the presence of an implantation support agent; and (c) Measuring the proportion of the plurality of blast-like cells that implant in the endometrial cells in step (b) as a function of the time of change, wherein the optimal time for embryo transfer in the subject is indicated by the time at which the largest proportion of blast-like cells implant relative to the total number of blast-like cells contacted in step (b), The method, including the method described above.

26. A method for optimizing embryo implantation in a target, (a) Culturing endometrial cells obtained from the subject in the presence of an implantation support agent; (b) Contacting a plurality of blast-like cells with endometrial cells in the presence of a plurality of implantation support agents, wherein the implantation support agents are selected from progesterone, progestin, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH) agonists; and (c) Measuring the proportion of the plurality of blast-like cells that implant in the endometrial cells as a function of the implantation support agent of step (b), wherein the optimal combination of implantation support agents for embryo transfer in the subject is indicated by the combination that maximizes the proportion of implanted blast-like cells relative to the total number of blast-like cells that come into contact with the endometrial cells, The method, including the method described above.

27. The method according to claim 25 or 26, wherein the endometrial cells are cultured as layers or organoids.

28. The method according to claim 25 or 26, wherein the plurality of blast-like cells are obtained from induced pluripotent stem cells prepared from a sample collected from the subject.

29. The method according to claim 28, wherein the sample comprises blood or a skin biopsy.

30. The method according to claim 28, wherein induced pluripotent stem cells are produced using a transcription factor-based method or somatic cell nuclear transfer.

31. The method according to claim 29, wherein the method based on the transcription factor includes introducing a transcription factor comprising DNA, RNA, or protein into cells from the sample.

32. The method according to claim 29, wherein the method based on the transcription factor includes lentivirus-based, adenovirus-based, or sendai virus-based reprogramming, or reprogramming based on mRNA transfection.

33. The method according to claim 25 or 26, wherein the blast-like cells are produced from cells modified to prevent post-implantation embryonic development.

34. A method for predicting the success of embryo implantation in a subject, (a) Culturing endometrial cells obtained from the subject; (b) Exposing the endometrial cells to estrogen and / or progesterone for a certain period of time or according to a predetermined hormone therapy schedule; (c) Analyzing the endometrial cell sample for one or more markers of receptive endometrium and / or one or more markers of endometrial pathology; (d) Predicting implantation success when the markers for receptive endometrium are present or in higher than normal levels, and / or when the markers for endometrial pathology are absent or in lower than normal levels; This includes, where the marker for the receptive endometrium is selected from PAEP, acetylated α-tubulin ciliates, mucin, CXCL14, and SPP1, and the marker for the endometrial pathology is selected from BCL6, CCL19, MUC5B, PCDH-17, PTPRR, and IL6ST. The aforementioned method.

35. The method according to claim 34, further comprising the analysis in step (c) comparing the endometrial cell sample with a control sample that has not been in contact with estrogen, progesterone, or other hormone treatment.

36. The method according to claim 34, wherein the endometrial cells are cultured as layers or organoids.

37. The method according to claim 34, wherein the analysis includes RNA sequencing, quantitative polymerase chain reaction (qPCR), immunostaining, or ATAC sequencing.

38. The method according to claim 34, wherein the endometrial cells are obtained from the subject regardless of the subject's hormonal cycle.

39. A computer-based method for selecting a protocol for embryo implantation in a subject, (a)(i) The percentage of implanted blast-like cells detected in response to treatment with estrogen and / or progesterone, as described in the method of claim 10; (ii) The presence of a receptive endometrial marker and / or the absence of an endometrial pathological marker detected in the method of claim 34; (iii) the age of the subject, body mass index (BMI), presence of bacterial species, and / or past pregnancy success; It accepts an input that includes at least one parameter selected from: (b) Using a machine learning software module, generate an output that includes an indicator of the likelihood of successful embryo implantation based on the input received in (a), The method, including the method described above.

40. The computer-based method according to claim 38, wherein steps (a) and (b) are performed based on a plurality of hormone treatment conditions, and a protocol for embryo implantation is selected based on the output of step (b).

41. A computer system for selecting a protocol for embryo implantation in a subject, (a) Processing apparatus, and (b) When executed by the processing unit, the processing unit (i) the percentage of implanted blast-like cells detected in response to treatment with estrogen and / or progesterone and a specific hormonal treatment schedule, as described in claim 10; The presence of a receptive endometrial marker and / or the absence of an endometrial pathological marker detected in the method of claim 34; and Age of the subject, degree of obesity (BMI), presence of bacterial species, and / or past pregnancy success; It accepts an input that includes at least one parameter selected from: (ii) Based on the input received in (i), generate an output that includes a display of the probability of successful embryo implantation. A non-transient computer-readable medium containing instructions to perform the following actions: The computer system including the computer system.

Images