Stem cell and organoid based methods for diagnostics and optimization of embryo implantation

Abstract

Methods for producing controlled endometrial organoids using micropatterning technology, as well as methods of using endometrial cells or organoids for diagnostic and predictive purposes, and to optimize IVF and other fertility-related procedures, including embryo transfer and intrauterine insemination (IUI) are disclosed. The organoids grow in a spatially controlled fashion and are less heterogenous than organoids grown directly in 3 dimensional culture. Also described herein are methods of predicting successful embryo transfer or other IVF-related procedures, methods of optimizing embryo transfer, methods of improving embryo implantation in the endometrium, and methods of transferring embryos in a subject. These methods comprise culturing an endometrial organoid or endometrial epithelial cells, and contacting the endometrial organoid or cells with one or a plurality of blastoids. In some embodiments, the endometrial organoid or cells comprises endometrial cells obtained from a subject undergoing or contemplating IVF-related treatments.

Description

STEM CELL AND ORGANOID BASED METHODS FOR DIAGNOSTICS AND OPTIMIZATION OF EMBRYO IMPLANTATION

[0001] This application is related to U.S. application number 63 / 490,244, filed March 14, 2023, and to U.S. application number 63 / 591,059, filed October 17, 2023, the contents of each of which are incorporated herein in their entirety. BACKGROUND

[0002] Organoids can be defined as three dimensional in vitro tissue models that recapitulate many of the physiologically relevant properties and features of the corresponding in vivo tissue. The discovery that human endometrial organoids can be generated from primary endometrial cells has opened up new possibilities to investigate biological processes involved in human pregnancy, disease modeling and testing therapeutic compounds for clinical applications, including in vitro fertilization (IVF) and embryo implantation. Typically, to generate human endometrial organoids, primary endometrial tissue samples are dissociated with enzymatic procedures (collagenase and / or dispase) and resuspended into Matrigel droplets in a defined medium shown to promote organoid formation and maintenance from primary tissue or iPSC-derived cells.

[0003] There remains a need, however, for improved methods of producing endometrial organoids, and also a need to predict and improve IVF outcomes. SUMMARY

[0004] Described herein are methods for producing controlled endometrial organoids using micropatterning technology, as well as methods of using the organoids and cultured endometrial cells for diagnostic and predictive purposes, and to optimize IVF and other fertility-related procedures, including embryo transfer and intrauterine insemination (IUI). The method is advantageous in that the organoids grow in a spatially controlled fashion and are less heterogenous than organoids grown directly in 3 dimensional culture. Also described herein are methods of predicting successful embryo transfer or other IVF-related procedures, methods of optimizing embryo transfer, methods of improving embryo implantation in the endometrium, and methods of transferring embryos in a subject. These methods comprise culturing endometrial cells as an endometrial organoid or layer and contacting the endometrial organoid or cultured cells with one or a plurality of blastoids. In some embodiments, the endometrial organoid or culture comprises endometrial cells obtained from a subject undergoing or contemplating IVF-related treatments. Various parameters can be tested in order to identify optimal conditions for embryo transfer and successful IVF treatment. In some embodiments, these methods are performed using an endometrial organoid produced using micropatterning as described herein.

[0005] In some embodiments, the micropatterning is accomplished through photolithography, or microcontact printing. It is understood to those skilled in the art that micropatterning can be achieved by other means as well.

[0006] In some embodiments, the endometrial cells used to produce the 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 term established organoid cultures. These cells include the epithelial lining of the endometrium grown either with or without endometrial stromal cells.

[0007] The cells are seeded onto the micropatterns which are typically coated with extracellular matrix protein including but not limited to laminin, fibronectin, vitronectin, Matrigel, and Geltrex. This coating could either be a thin molecular layer or a thicker gel.

[0008] The cells then grow into three dimensions, either in a spontaneous fashion or by placing extracellular matrix, including but not limited to the ones listed above, into the growth media, which will typically cause folding in the third dimension.

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

[0010] Also described herein is a method of diagnostics in which artificial embryo-like objects created from human stem cells (this includes but is not limited to the embryo like objects, as described in the following references: 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 28, 1016–1022 (2021); doi.org / 10.1016 / j.stem.2021.04.031; these are referred to hereafter as blastoids) are cultured together with endometrial cells, endometrial organoids, or micropatterned endometrial organoids as described above that have been taken from a patient. In the case of organoids, the three dimensional structure may be disrupted either chemically or mechanically to allow the introduction of the blastoids. Prior to the introduction of the blastoids, the endometrial cells are either cultured in standard media or given a course of hormone treatments.

[0011] The fraction of blastoids that undergo implantation-like events is measured through microscopic observation. This metric is used as an in vitro diagnostic to determine the likelihood of the patient being successful in either implantation of an embryo or successful pregnancy up to a certain point. This microscopic observation could include bright field-based techniques including phase contrast or differential interference contrast (DIC) imaging, as well as performing immunofluorescence for markers specific to the cell populations of the embryo (for example but not limited to, CDX2 for trophectoderm, OCT4 for epiblast, and GATA4 or GATA6 for visceral endoderm) and of the endometrium (for example SOX9 or FOXA2) to observe their relative positions. This imaging includes either live cell imaging of the blastoid during the implantation assay or applying a fixative such as paraformaldehyde or methanol at a certain time point after culturing the blastoids with the endometrium and performing imaging of the fixed samples. This imaging could also include using blastoids derived from human stem cells lines with fluorescent protein based reporters.

[0012] In some embodiments, other parameters beyond the fraction of blastoids that implant are measured and used as a diagnostic, including but not limited to the timing that blastoids implant, the depth of blastoid implantation, and the rate of movement of the blastoids before and after implantation.

[0013] In some embodiments, molecular parameters such as gene expression or chromatin accessibility of particular genes in either the blastoid or the endometrium is measured and used as a diagnostic. 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] Also provided is a method to predict the most likely timings of embryo transfer or other parameters relevant to an in vitro fertilization (IVF) or other fertility-related procedure to produce a successful implantation or pregnancy. Hormone treatments that simulate those produced by the body in preparing the uterus for embryo receptivity are performed on the endometrial cultures and blastoids are introduced at varying times during this course of treatment. The timing of introducing blastoids with these endometrial cultures is varied and for each timing the parameters named above are measured. The outcome of this test in which either the fraction of blastoids that undergo implantation-like events or other parameters are found to be optimal is used to predict the most likely timing of embryo introduction to achieve success in the IVF or other fertility-related procedure.

[0015] In some embodiments, other parameters other than the timing of embryo introduction are varied. An example is simulating in culture varying treatments of luteal phase support such as after the introduction of the blastoids supplementing the culture with molecules meant to optimize implantation-like events including progesterone, progestins, hCG or GnRH agonists. The results of this test are then used to predict the regimen of luteal phase support most likely to produce a successful outcome.

[0016] In some embodiments, the information obtained from the assays described herein is combined with other information obtained from the patient’s biological samples or obtained directly from the patient. This includes but is not limited to the patient’s genome sequence, the transcriptome (for example as measured by RNA sequencing), metabolome, proteome, or epigenome of the patient’s endometrial biopsy sample or a culture of cells from that sample, or information about the patient’s microbiome or health history, demographic information, or current conditions. This information could be used in predicting through any means including rationally designed algorithms or artificial intelligence-based predictions.

[0017] In some embodiments, the blastoid is created from induced pluripotent stem cells created from a sample taken from the patient themselves. This sample could include but is not limited to the patient’s blood or a skin biopsy. The induced pluripotent stem cells are created using methods which include transcription factor-based methods in which the transcription factors as introduced as DNA, RNA, or protein. Examples include, but are not limited to, lentiviral based, adenovirus virus based, and sendai virus based reprogramming and reprogramming based on mRNA transfection. The induced pluripotent stem cells could also be created via somatic cell nuclear transfer.

[0018] In some embodiments, the blastoids are replaced by human embryos donated for this purpose. The embryos could be from the patient themselves or from another donor.

[0019] In some embodiments, the blastoids are created from cells which have been modified to prevent further embryonic development beyond a certain point such as by deleting a gene critical for postimplantation development but not for preimplantation development. This avoids some ethical issues involving the culture of human embryos or blastoids.

[0020] Also provided are methods of predicting successful embryo implantation, which can be performed by assaying for blastoid implantation on cultured endometrial cells or organoids, or by assaying for changes in markers of endometrial receptivity or markers of endometrial pathology. The assays can be performed using various hormonal treatment protocols or varying other conditions and used to select optimal protocols and / or to screen candidates for successful embryo transfer. Additionally provided are computer-implemented methods and computer systems for providing predictions of successful embryo implantation and for selecting optimal protocols and conditions for embryo transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG.1 shows representative photomicroscopic images of human endometrial organoids.

[0022] FIG.2 shows bright field and immunostained photomicroscopic images of open-faced endometrial layer culture. Arrows show representative staining for FOXA2, E-Cadherin (ECAD), and SOX9.

[0023] FIG.3 is a bright field photomicroscopic image of blastoids.

[0024] FIG.4 as an immunostained blastoid photomicroscopic image, in which blue (thin arrow) is CDX2 (TE), yellow (curved arrow) is NANOG (Epi), and magenta (wide arrow) is NR2F2 (polar TE).

[0025] FIG.5 is a series of photomicrographs showing blastoid progression from Day 0 through Day 4.

[0026] FIG.6 is a pair of photomicrographs showing immunofluorescence for NR2F2 (polar trophectoderm) and phalloidin (actin) as used in an assay for blastoid implantation. If a blastoid is implanted, the cells expressing NR2F2 are visible under the endometrial cells (box in right panel).

[0027] FIG.7 is a photograph of six test strips assayed for hCG. The strips show the results from an implantation assay in which the blastoids were transferred onto the endometrial layer in a micropattern plate, in three patients, either not treated (control, strips 1, 3, 5) or treated with hormones (hormones, strips 2, 4, 6). The arrow points to the test area, where appearance of a line indicates a positive result for hCG (as in strips 2, 4, and 6). Negative results (absence of detectable hCG) are shown in strips 1, 3, and 5.

[0028] FIG.8 shows photomicrographs taken at 4x magnification (upper image) and at 10x magnification (lower image) of a blastoid implanted onto the endometrial layer in a micropattern plate. DETAILED DESCRIPTION

[0029] Use of micropatterning technology allows for methods for producing controlled endometrial organoids. The organoids can be used for diagnostic and predictive purposes, and to optimize IVF and other fertility-related procedures, including embryo transfer and intrauterine insemination (IUI). The organoids grow in a spatially controlled fashion and are less heterogenous than organoids grown directly in three dimensional culture. These can be used in methods of predicting successful embryo transfer or other IVF-related procedures, methods of optimizing embryo transfer, methods of improving embryo implantation in the endometrium, and methods of transferring embryos in a subject.

[0030] Definitions

[0031] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0032] As used herein, “stromal cells” refer to the mesenchymal cells that reside adjacent to the epithelial layer of the uterus on the opposite side from the uterine cavity. These cells are capable of differentiating into decidualized stromal fibroblasts naturally (in vivo) and / or upon treatment with hormones. Stromal cells can be obtained as primary samples from patients, from established cell lines, or derived from iPSCs.

[0033] As used herein, a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. The sample can be an actual sample used for testing, or a reference level or range, based on known normal measurements of the corresponding marker.

[0034] As used herein, a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability. In one example, an increase or decrease of 10% relative to a reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference. In yet another example, an increase of two-fold relative to a reference sample is considered significant.

[0035] As used herein, the term "subject" includes any vertebrate animal including, but not limited to, a human, non-human primate, mouse, rat, guinea pig, rabbit, cow, dog, cat, horse, goat, bird, reptile, or fish. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is a domesticated animal, a wild animal, or an agricultural animal.

[0036] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.

[0037] Methods of Producing Endometrial Organoids

[0038] A method of producing endometrial organoids comprises, in some embodiments, seeding endometrial epithelial cells on a surface printed with micropatterns; and culturing the seeded cells under conditions sufficient for growth in three dimensions and organoid formation. In some embodiments, the seeding further comprises seeding stromal cells on the surface. The seeding of stromal cells can occur before, simultaneously with, or after seedingof endometrial epithelial cells. This method produces endometrial assembloids on a culture surface. Endometrial assembloids are combinations of endometrial stromal cells and endometrial epithelial cells. The culture surface could be micropatterned as described herein, or a standard culture surface could 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 the stromal cells is first seeded onto the culture surface and, after the stromal cells adhere, epithelial cells are seeded on top, whereby the basal slide of the epithelial cells attaches to the stromal cells while the apical side faces the media. In some embodiments, the micropatterns are applied via photolithography or microcontact printing. In some embodiments, the micropatterns are coated with extracellular matrix protein. In some embodiments, the culturing conditions comprise culturing the endometrial cells in a culture medium that comprises extracellular matrix protein. In some embodiments, the extracellular matrix protein comprises laminin, fibronectin, vitronectin, Matrigel, and / or Geltrex. This coating could either be a thin molecular layer or a thicker gel. Patterns are typically spaced sufficiently far that they will not influence each other through diffusible 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.

[0039] Also provided are endometrial organoids and assembloids produced in accordance with the methods described herein. Endometrial organoids produced via seeding endometrial cells on a micropatterned surface have been observed to result in more reproducible phenotypes. Endometrial organoids produced by this micropatterning method are thus desirable for use in optimizing conditions for IVF and other fertility-related procedures, predicting successful embryo transfer, as well as for use in toxicology studies.

[0040] In some embodiments, the method further comprises releasing the endometrial cells and resuspending the endometrial cells in growth media or in a gel under conditions sufficient for growth in three dimensions. In some embodiments, the gel comprises an ECM based gel.

[0041] Methods of Producing A Model of Embryo Implantation

[0042] Also described is a method of producing an ex vivo model of implantation of an embryo. In some embodiments, the method comprises culturing endometrial epithelial cells; and contacting a blastoid with endometrial cells of the culture or organoid. The culturing can be performed with or without seeding the endometrial cells on a surface printed with micropatterns. In some embodiments, the endometrial epithelial cells are cultured as anendometrial organoid as described herein. In some embodiments, the cells are cultured as a monolayer of endometrium epithelial cells. In some embodiments, the method further comprises culturing the endometrial epithelial cells together with the blastoid in the presence of implantation support agents. In some embodiments, the blastoid is co-cultured with the endometrial epithelial cells for up to approximately one week with the assay performed at any point during that time. In some embodiments, the implantation support agents are administered to the endometrial epithelial cells prior to contact with the blastoid.

[0043] In some embodiments, the contacting is performed with a plurality of blastoids, and the method further comprises measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid. Implantation can be measured using techniques known in the art, for example, by washing the culture, e.g., with buffer, and detecting whether or how many blastoids remain adhered to the endometrial layer or become suspended in the medium, in which case adherence to the endometrial layer is indicative of implantation.

[0044] The model of embryo implantation can be used in methods of predicting successful embryo implantation. The ability of a subject’s endometrial cells to support implantation can be assayed as described above, using a single blastoid or a plurality of blastoids. The assay can also be performed using a variety of hormonal treatment protocols. One example of a hormonal treatment protocol comprises two days of treatment with estrogen, followed by four days of treatment with estrogen and progesterone. This latter treatment can be performed with or without WNT inhibitor XAV-939 and 8-br-camp in the assay. In some embodiments, the assay further includes a control sample cultured in the same media but not treated with hormones. A comparison is made between the control and the treated sample. Prediction of successful embryo implantation can be based on the relative levels between the samples, the absolute levels in the samples, or a combination of these factors.

[0045] In some embodiments, the endometrial cells are obtained from established endometrial cell lines, patient biopsies, and / or patient biopsies grown as organoids in primary cultures or in long term established organoid cultures. These cells include the epithelial lining of the endometrium grown either with or without endometrial stromal cells. Use of endometrial cells obtained from the patient allows for tailoring IVF, fertility treatment, and embryo transfer conditions to those optimal for the individual patient. Patients can also obtain more informed predictions regarding their likelihood of successful embryo transfer prior to the commitment of scarce resources.

[0046] In some embodiments, the blastoids are replaced by human embryos donated for this purpose. The embryos could be from the patient themselves or from another donor.

[0047] In some embodiments, the culturing comprises culturing the endometrial organoid in the presence of hormones, an activator of cyclic AMP-dependent protein kinase (PKA), or a Wnt inhibitor. In some embodiments, the hormones comprise progesterone, progestins, human chorionic gonadotropin (hCG), and / or gonadotrophin-releasing hormone (GnRH) agonists. In some embodiments, the activator of cyclic AMP-dependent PKA is 8-bromo- cAMP. In some embodiments, the measuring comprises microscopic observation via phase contrast imaging, differential interference contrast imaging, and / or immunofluorescent detection of embryo-specific markers and / or endometrial markers. In some embodiments, the embryo-specific markers comprise CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, GATA4 and / or GATA6. In some embodiments, the endometrial markers comprise SOX9, MUC1, PAEP, OLFM4, acetylated alpha-tubulin, ECAD, vimentin, and / or FOXA2. Markers of endometrial cells have been described, for example, by Hong in Genes & Diseases 10, Issue 3, May 2023, Pages 931-947. In some embodiments, the microscopic observation comprises live cell imaging of the blastoid or imaging of the blastoid after treatment with a fixative.

[0048] In some embodiments, the blastoid is derived from a human stem cell line, and the human stem cell line has been labeled with a fluorescent marker. In some embodiments, the method further comprises measuring the time and / or depth in which the blastoids undergo implantation in the endometrial organoid, and / or measuring a rate of movement of the blastoids before and after implantation in the endometrial organoid. In some embodiments, the method further comprises measuring gene expression and / or chromatin accessibility in the blastoid and / or the endometrial organoid. In some embodiments, the measuring comprises RNA sequencing, Assay for Transposase-Accessible Chromatin (ATAC) sequencing, immunofluorescence, and / or PCR. In some embodiments, the PCR is quantitative real time PCR.

[0049] Methods of Optimizing Embryo Transfer

[0050] Also provided is a method of optimizing embryo transfer in a subject, a method of predicting successful embryo transfer, a method of performing embryo transfer, and likewise methods of improving the outcome of IVF and other fertility-related procedures, including improving the success of embryo transfer. These methods can be employed in the context of treating a patient in need of IVF or other fertility treatment, whereby a method of optimizing or predicting successful embryo transfer is followed by transferring an embryo to the subject. In some embodiments, the timing and selection of treating the subject with implantation support agents or other parameters associated with embryo transfer are based on the outcome of optimizations developed through use of an ex vivo model of embryo implantation as described herein. Such optimization can involve varying the protocol for preparing theendometrium, for example by applying a hormone protocol that mimics what is given to the patient before transfer (or mimics the nature cycle before embryo implantation), as well as optimizing implantation upon embryo transfer.

[0051] In some embodiments, the method comprises producing an ex vivo model comprising an endometrial organoid, wherein the endometrial organoid comprises endometrial cells obtained from the subject, and wherein the culturing of the endometrial organoid is in the presence of implantation support agents. The culturing can be performed with or without stromal cells underneath the epithelial cells. Implantation support agents can be provided to the cultured endometrial organoid prior to the contact with blastoids. The method further comprises contacting a plurality of blastoids with the endometrial organoid at a plurality of timepoints relative to culturing the endometrial organoid in the presence of implantation support agents; and measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid as a function of the timepoints varied, whereby the optimal timepoint for embryo transfer in the subject is indicated by the timepoint that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted.

[0052] In some embodiments, the method comprises producing an ex vivo model comprising a cultured monolayer comprising endometrial epithelial cells, wherein the endometrial epithelial cells have been derived from the subject, and wherein the culturing of the endometrial epithelial cells is in the presence of implantation support agents. The culturing can be performed with or without stromal cells underneath the epithelial cells. Implantation support agents can be provided to the cultured endometrial epithelial cells prior to the contact with blastoids. The method further comprises contacting a plurality of blastoids with the endometrial epithelial cells at a plurality of timepoints relative to culturing the endometrial epithelial cells in the presence of implantation support agents; and measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial epithelial cells as a function of the timepoints varied, whereby the optimal timepoint for embryo transfer in the subject is indicated by the timepoint that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted.

[0053] In some embodiments, the method of optimizing embryo transfer in a subject comprises producing an ex vivo model as described herein, wherein the endometrial organoid (or epithelial cells) comprises endometrial cells obtained from the subject. The method further comprises culturing the endometrial organoid (or epithelial cells) in the presence of a plurality of combinations of implantation support agents, wherein the implantation support agents are selected from progesterone, progestins, human chorionic gonadotropin (hCG), and gonadotrophin-releasing hormone (GnRH) agonists; andmeasuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid (or epithelial cells) as a function of the implantation support agents. The optimal combination of implantation support agents for embryo transfer in the subject is indicated by the combination that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted with the endometrial organoid (or epithelial cells).

[0054] In some embodiments, the method of optimizing embryo transfer is used to vary parameters relevant to an in vitro fertilization (IVF) or other fertility-related procedure to produce a successful implantation or pregnancy. Hormone treatments that simulate those produced by the body in preparing the uterus for embryo receptivity are administered to the endometrial cultures, for example, and blastoids are introduced at varying times during this course of treatment. The timing of introducing blastoids to these endometrial cultures is varied, and for each timing the parameters named above are measured. The outcome of this test in which either the fraction of blastoids that undergo implantation-like events or other parameters are found to be optimal is used to predict the most likely timing of embryo introduction to achieve success in the IVF or other fertility-related procedure.

[0055] In some embodiments, the method of optimizing embryo transfer is used to simulate in culture varying treatments of luteal phase support. One representative example of such a simulation comprises supplementing the culture, after the introduction of the blastoids, with molecules that optimize implantation-like events including progesterone, progestins, hCG or GnRH agonists. The results of this test are then used to predict the regimen of luteal phase support most likely to produce a successful outcome.

[0056] In some embodiments, the information obtained from the assays described herein is combined with other information obtained from the patient’s biological samples or obtained directly from the patient. Representative examples of such information include, but are not limited to, the patient’s genome sequence, the transcriptome (for example, as measured by RNA sequencing), metabolome, proteome, or epigenome of the patient’s endometrial biopsy sample or a culture of cells from that sample, or information about the patient’s microbiome or health history, demographic information, or current conditions. This information could be employed to improve prediction of outcome or successful transfer through use of, for example, computational means, including implementation of rationally designed algorithms, or machine learning-based predictions. One skilled in the art will appreciate how to train a machine-learning-based system by inputting the corresponding information together with data regarding successful embryo transfer associated with the information.

[0057] In some embodiments of the above methods, the plurality of blastoids are obtained from induced pluripotent stem cells created from a sample taken from the subject. In some embodiments, the sample comprises blood or a skin biopsy. In some embodiments, the induced pluripotent stem cells are created using transcription factor-based methods or somatic cell nuclear transfer. In some embodiments, the transcription factor-based methods comprise introducing into a cell from the sample transcription factors comprising DNA, RNA, or protein. In some embodiments, the transcription factor-based methods comprise lentiviral based, adenovirus virus based, or sendai virus based reprogramming, or reprogramming based on mRNA transfection. In some embodiments, the blastoids are created from cells modified to prevent postimplantation embryonic development.

[0058] Methods of Predicting Successful Embryo Implantation

[0059] Also provided are methods of predicting successful implantation of an embryo in a subject, or of evaluating a subject’s likelihood of successful implantation. In some embodiments, the method comprises culturing endometrial cells obtained from the subject. The method further comprises contacting the endometrial cells with a hormonal protocol (for example, estrogen and / or progesterone for varying periods of time). In some embodiments, the contacting comprises contacting the endometrial cells with estrogen for a first period of time, and contacting the endometrial cells estrogen and progesterone for a second period of time. In some embodiments, the first period of time is 2 days, and the second period of time is 4 days.

[0060] In some embodiments, the assay further includes a control sample cultured in the same media but not treated with hormones. A comparison is made between the control and the treated sample. Prediction of successful embryo implantation can be based on the relative levels between the samples, the absolute levels in the samples, or a combination of these factors. The method further comprises assaying a sample of the endometrial cells for markers of receptive endometrium and / or markers of endometrial pathology. In some embodiments, the assaying further comprises comparing the sample of endometrial cells to a control sample that has not been contacted with estrogen, progesterone or other hormone treatment. Successful implantation is detected when markers of receptive endometrium are present (or greater than a reference amount) and / or markers of endometrial pathology are absent (or less than a reference amount), or otherwise differ from a control sample.

[0061] In some embodiments, the markers of receptive endometrium include, but are not limited to, one or more of Progestagen Associated Endometrial Protein (PAEP), acetylated a-tubulin cilia, mucin, CXC motif Chemokine Ligand 14 (CXCL14), and Secreted Phosphoprotein 1 (SPP1). In some embodiments, the markers of endometrial pathologyinclude, but are not limited to, one or more of B-Cell Lymphoma 6 (BCL6), CC motif Chemokine Ligand 19 (CCL19), mucin 5B (MUC5B), Protocadherin 17 (PCDH-17), Protein Tyrosine Phosphatase Receptor type R (PTPRR) and Interleukin 6 cytokine family Signal Transducer (IL6ST).

[0062] In some embodiments, the endometrial cells are cultured as a layer, e.g., in two dimensional culture. In some embodiments, the endometrial cells are cultured as an organoid, e.g., in three dimensional culture. In some embodiments, the assaying comprises RNA sequencing, quantitative polymerase chain reaction (qPCR), immunostaining, or ATAC sequencing. In some embodiments the endometrial cells are obtained from the subject independent of the subject’s hormonal cycle. The method does not require the sample to be obtained while the patient is in a hormone-induced “mock cycle” or in a specific phase of the patient’s natural hormonal cycle.

[0063] The prediction of a subject’s endometrial cells to support successful implantation can be assayed using a variety of hormonal treatment protocols. One example of a hormonal treatment protocol comprises two days of treatment with estrogen, followed by four days of treatment with estrogen and progesterone. This latter treatment can be performed with or without WNT inhibitor XAV-939 and 8-br-camp in the assay.

[0064] Computer-Implemented Methods and Systems

[0065] Also provided is a computer-implemented method for selecting a protocol for embryo implantation in a subject. In some embodiments, the method comprises: a) receiving input comprising at least one parameter selected from: i) percent of implanted blastoids detected in the method of predicting successful embryo impantation described above, optionally in response to treatment with estrogen and / or progesterone; ii) presence of markers of receptive endometrium and / or absence of markers of endometrial pathology detected in the method described above; iii) age, body mass index (BMI), past pregnancy success of the subject, and / orpresence of different bacterial species in a biological sample obtained from the subject (This can be obtained from the microbiome analysis described above, for example, in the context of optimizing embryo transfer.);b) generating, using a machine learning software module, an output comprising an indication of the subject’s likelihood of successful embryo implantation based on the input received in (a).

[0066] In some embodiments, steps (a) and (b) are performed on the basis of a plurality of hormonal treatment conditions, and wherein a protocol for embryo implantation is selected on the basis of the output of step (b).

[0067] Additionally provided is a computer system for selecting a protocol for embryo implantation in a subject. In some embodiments, the computer system comprises: a) a processor; and b) a non-transitory computer readable medium that stores instructions that, when executed by the processor causes the processor to: i) receive input comprising at least one parameter selected from: percent of implanted blastoids detected in the method of predicting successful implantation described above, optionally in response to treatment with estrogen and / or progesterone, and particular hormonal treatment schedules; presence of markers of receptive endometrium and / or absence of markers of endometrial pathology detected in the method of predicting successful embryo implantation described above; age, body mass index (BMI), presence of bacterial species, and / or past pregnancy success of the subject; ii) generate an output comprising an indication of the subject’s likelihood of successful embryo implantation based on the input received in (i). The indication of likelihood of successful embryo implantation can be generated for various treatment protocols, and used for selection of the optimal hormonal treatment and other conditions that can optimize successful embryo transfer.

[0068] Pregnancy success data to be used for inputs can include number of pregnancies, number of miscarriages, number of failed embryo transfers, e.g., as in IVF. Other inputs can include data obtained from the assays described above using the model for embryo implantation and cultured endometrial cells or organoids. For example, the input can be the percent of blastoids implanted for a given hormone treatment protocol, or timing of implant, depth of blastoid implant, rate of movement of blastoids pre / post implant for a given hormone treatment protocol. Additional examples of input data are described in Example 5below. The machine learning module is trained using these inputs and associated outcomes from known patient data and treatment conditions.

[0069] Example Embodiments

[0070] Embodiment 1: A method of producing endometrial organoids, the method comprising: a) seeding endometrial cells on a surface printed with micropatterns; and b) culturing the cells seeded in step (a) under conditions sufficient for growth in three dimensions and organoid formation.

[0071] Embodiment 2: The method of embodiment 1, wherein the seeding of step (a) further comprises seeding stromal cells on the surface.

[0072] Embodiment 3: The method of embodiment 1, wherein the micropatterns are applied via photolithography or microcontact printing.

[0073] Embodiment 4: The method of embodiment 1, wherein the endometrial cells are obtained from established endometrial cell lines, patient biopsies, and / or patient biopsies grown as organoids in primary cultures or in long term established organoid cultures.

[0074] Embodiment 5: The method of embodiment 1, wherein the micropatterns are coated with extracellular matrix (ECM) protein.

[0075] Embodiment 6: The method of embodiment 1, wherein conditions of step (b) comprise culturing the endometrial cells in a culture medium that comprises extracellular matrix protein.

[0076] Embodiment 7: The method of embodiment 5 or 6, wherein the extracellular matrix protein comprises laminin, fibronectin, vitronectin, Matrigel, and / or Geltrex.

[0077] Embodiment 8: The method of any of the preceding embodiments, further comprising releasing the endometrial cells in growth media or in a gel under conditions sufficient for growth in three dimensions.

[0078] Embodiment 9: The method of embodiment 8, wherein the gel comprises an ECM based gel.

[0079] Embodiment 10: A method of producing an ex vivo model of implantation of an embryo, the method comprising: a) culturing an endometrial organoid, wherein the organoid comprises endometrial cells obtained from a human subject; b) contacting a blastoid with endometrial cells of the organoid; andc) culturing the endometrial organoid together with the blastoid in the presence of implantation support agents.

[0080] Embodiment 11: The method of embodiment 10, wherein the contacting of step (b) is performed with a plurality of blastoids, and wherein the method further comprises measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid.

[0081] Embodiment 12: The method of embodiment 10, wherein the culturing of step (a) comprises culturing the endometrial organoid in the presence of hormones, an activator of cyclic AMP-dependent protein kinase (PKA), or a Wnt inhibitor.

[0082] Embodiment 13: The method of embodiment 12, wherein the hormones comprise progesterone, progestins, human chorionic gonadotropin (hCG), and / or gonadotrophin- releasing hormone (GnRH) agonists.

[0083] Embodiment 14: The method of embodiment 12, wherein the activator of cyclic AMP- dependent PKA is 8-bromo-cAMP.

[0084] Embodiment 15: The method of embodiment 11, wherein the measuring comprises microscopic observation via phase contrast imaging, differential interference contrast imaging, and / or immunofluorescent detection of embryo-specific markers and / or endometrial markers.

[0085] Embodiment 16: The method of embodiment 15, wherein the embryo-specific markers comprise CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, and / or GATA6.

[0086] Embodiment 17: The method of embodiment 15, wherein the endometrial markers comprise SOX9, MUC1, PAEP, OLFM4, acetylated alpha-tubulin, ECAD, vimentin, and / or FOXA2.

[0087] Embodiment 18: The method of embodiment 15, wherein the microscopic observation comprises live cell imaging of the blastoid or imaging of the blastoid after treatment with a fixative.

[0088] Embodiment 19: The method of embodiment 15, wherein the blastoid is derived from a human stem cell line, and wherein the human stem cell line has been labeled with a fluorescent marker.

[0089] Embodiment 20: The method of embodiment 11, further comprising: measuring the time and / or depth in which the blastoids undergo implantation in the endometrial organoid, and / or measuring a rate of movement of the blastoids before and after implantation in the endometrial organoid.

[0090] Embodiment 21: The method of embodiment 10, further comprising measuring gene expression and / or chromatin accessibility in the blastoid and / or the endometrial organoid.

[0091] Embodiment 22: The method of embodiment 21, wherein the measuring comprises RNA sequencing, Assay for Transposase-Accessible Chromatin (ATAC) sequencing, immunofluorescence, and / or PCR.

[0092] Embodiment 23: The method of embodiment 22, wherein the PCR is quantitative real time PCR.

[0093] Embodiment 24: A method of optimizing embryo transfer in a subject, the method comprising: a) producing an ex vivo model of embodiment 10, wherein the endometrial organoid comprises endometrial cells obtained from the subject, and wherein the culturing of the endometrial organoid is in the presence of implantation support agents; b) contacting a plurality of blastoids with the endometrial organoid at a plurality of timepoints relative to culturing the endometrial organoid in the presence of implantation support agents; and c) measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid as a function of the timepoints varied in step (b), whereby the optimal timepoint for embryo transfer in the subject is indicated by the timepoint that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted in step (b).

[0094] Embodiment 25: A method of optimizing embryo transfer in a subject, the method comprising: a) producing an ex vivo model of embodiment 11, wherein the endometrial organoid comprises endometrial cells obtained from the subject; b) culturing the endometrial organoid in the presence of a plurality of combinations of implantation support agents, wherein the implantation support agents are selected from progesterone, progestins, human chorionic gonadotropin (hCG), and gonadotrophin-releasing hormone (GnRH) agonists; and c) measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid as a function of the implantation support agents of step (b), whereby the optimal combination of implantation support agents for embryo transfer in the subject is indicated by the combination that results in the largest proportion ofblastoid implantation relative to the total number of blastoids contacted with the endometrial organoid.

[0095] Embodiment 26: The method of embodiment 24 or 25, wherein the plurality of blastoids is obtained from induced pluripotent stem cells created from a sample taken from the subject.

[0096] Embodiment 27: The method of embodiment 26, wherein the sample comprises blood or a skin biopsy.

[0097] Embodiment 28: The method of embodiment 26, wherein induced pluripotent stem cells are created using transcription factor-based methods or somatic cell nuclear transfer.

[0098] Embodiment 29: The method of embodiment 28, wherein the transcription factor- based methods comprise introducing into a cell from the sample transcription factors comprising DNA, RNA, or protein.

[0099] Embodiment 30: The method of embodiment 28, wherein the transcription factor- based methods comprise lentiviral based, adenovirus virus based, or sendai virus based reprogramming, or reprogramming based on mRNA transfection.

[0100] Embodiment 31: The method of embodiment 25 or 26, wherein the blastoids are created from cells modified to prevent postimplantation embryonic development. EXAMPLES

[0101] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

[0102] Example 1: Endometrial Culture

[0103] An endometrial organoid is grown in 3D culture, and treated with a hormone regimen similar to what occurs naturally during the cycle that prepares the uterus for pregnancy or that mimics the course of treatments that a doctor would give an IVF patient to prepare her for embryo transfer. This step enables the blastoids to attach to the endometrium. This course of hormones is varied, and the protocol that works best is identified. The timing of blastoid introduction can be optimized relative to this hormone cycle.

[0104] Endometrial organoids are dissociated and seeded on a Matrigel-coated plate to form an open-faced endometrial layer (OFEL) following the published protocol by Khoei et al. 2023 (doi.org / 10.1038 / s41596-023-00802-1). A representative protocol involved the following steps: (1) Obtained three primary samples (endometrial biopsies); (2) Generatedendometrial organoids from all samples; (3) Generated open-faced endometrial layer (OFEL) culture; OFEL culture is treated with hormone treatment (Estradiol, Progesterone, 8-br- cAMP, XAV939) vs. control (DMSO).

[0105] OFEL cultures are grown to sufficient density to avoid having blastoids touch the culture surface. Blastoids will stick to the culture surface and this can confuse the interpretation.

[0106] Representative images of human endometrial organoids are shown in Fig.1. Bright field and immunostained images of OFEL are shown in Fig.2. FOXA2 has a role in endometrial glandular development and function, ECAD marks glandular epithelium, and SOX9 is a marker of progenitor cells expressed in the base of endometrial glands in vivo.

[0107] Example 2: Blastoid Generation

[0108] Blastoids are introduced and cultured together with an endometrial organoid. The culture could last from a few hours up to a week, and optionally agents are introduced at this stage to help induce or maintain the implantation. These agents are referred to as "implantation support" agents. The timing, combination, and selection of implantation support agents can be varied, and the optimal schedule of implantation support agents identified.

[0109] Blastoids are prepared as exemplified below. Those without cell clumps on the outside are selected to avoid use of blastoids that will stick regardless of endometrial receptivity.

[0110] Blastoids are generated based on a published protocol by Khoei et al.2023 (doi.org / 10.1038 / s41596-023-00802-1) with the following parameters / modifications: Plate type: Aggrewell 24-well plate Incubation on gelatin: 150 minutes (in PXGL + 10 μM Rock inhibitor (RI)) Number of cells: 80,000-90,000 cells per well Aggregation medium: DMEM / F-12 + 0.3% BSA (Sigma) + 10 μM RI Settling time: 20 minutes Centrifugation: 200xg, 2 minutes Total volume on Day -1: 1 mL, comprises: 250 μL Aggregation medium + 250 μL cells in Aggregation medium 500 μL Aggregation medium added after centrifugation PALLY: 2 μM PD0325901, 2 μM A83-01, 2 μM LPA, 10 ng / mL LIF, & 10 μM RI.At Day 0: remove 750 μL of medium and add 1000 μL of PALLY At Day 1-3: remove 900 μL of medium and add 1000 μL of PALLY

[0111] Following this protocol, blastoid formation was observed with efficiency of 70-80% and the presence of correct markers: GATA3 (Trophectoderm), NR2F2 (polar TE), NANOG (Epiblast), SOX17 (Primitive Endoderm).

[0112] A bright field image of blastoids is shown in Fig.3. Fig.4 as an immunostained blastoid image, in which blue (thin arrow) is CDX2 (TE), yellow (curved arrow) is NANOG (Epi), and magenta (wide arrow) is NR2F2 (polar TE). Blastoid progression from Day 0 through Day 4 is depicted in Fig.5.

[0113] Example 3: Implantation assay

[0114] Observation metrics can be used to assess implantation. Bright-field images can be used to differentiate between deflated and cavitated blastoids. A flushing test can be used to differentiate between moving versus sticking. Immunofluorescence for mucin (endometrial glands) and NR2F2 (polar trophectoderm), combined with phalloidin (actin) and DAPI (nuclear) staining is used to assay for blastoid implantation. If a blastoid is implanted, the cells expressing NR2F2 are visible under the endometrial cells (see FIG.6). Representative results using the NR2F2 assay showed that 11 out of 40 blastoids were implanted for Patient #1, 1 out of 23 blastoids were implanted for Patient #2, and 6 out of 38 blastoids were implanted for Patient #3 in hormone-treated OFEL. None of 40, 25, and 35 blastoids for Patient #1, #2, and #3, respectively were implanted in the control-treated OFEL.

[0115] Optionally, one could employ an hCG test kit, for example, at about 4 days after implantation, wherein a positive test for hCG is indicative of successful implantation. Use of an hCG test can provide a more readily ascertainable indication of successful implantation. An example of use of an hCG test kit is depicted in FIG.7, a photograph of six test strips assayed for hCG. These strips show the results from an implantation assay in which the blastoids were transferred onto the endometrial layer in a micropattern plate, in three patients, either not treated (control, strips 1, 3, 5) or treated with hormones (hormones, strips 2, 4, 6). The arrow points to the test area, where appearance of a line indicates a positive result for hCG (as in strips 2, 4, and 6). Negative results (absence of detectable hCG) are shown in strips 1,3, and 5. Representative photomicrographs taken at 4x magnification (upper image) and at 10x magnification (lower image) of a blastoid implanted onto the endometrial layer in a micropattern plate is shown in FIG.8.

[0116] Example 4: Overall assay procedures

[0117] The following illustrates an exemplary protocol for processing samples and preparation of assays.

[0118] SAMPLE CRITERIA

[0119] Endometrial biopsy sample of at least 7 mm3in size is requested at patient’s secretory phase. Alternatively, the sample can be obtained at any phase. Antibiotics should not be taken seven days prior to biopsy for microbiome analysis. Any drugs including antibiotics that are taken during the month prior to the biopsy that may alter microbiota or immunological status should be included in requisition form.

[0120] SAMPLE RECEIPT PROCEDURES

[0121] Number and identities of tubes are checked and verified for each sample received. Tubes are inspected for proper labeling with Sample ID and Collection Date. Unlabeled samples are processed, but stored separately with a temporary label, and reported to the study site coordinator. Unlabeled samples are given permanent labels once its identity is confirmed.

[0122] The following information from each tube is recorded: Sample ID; Date and time of collection; Quality of sample (mostly endometrial tissue, not blood or mucus); Estimated volume of sample (mm3). If sufficient volume of sample is available (larger than 7 mm3), procedure continues to next step.

[0123] SAMPLE SPLITTING AND CRYOPRESERVATION PROCEDURES

[0124] One sample tube is handled at a time in a biological safety cabinet (BSC), and sample ID of the tube being processed is recorded. The tube is sprayed with 70% alcohol prior to bringing it inside the BSC. Samples are sliced into several portions with a dissecting blade, with at least 2 mm3volume each. If a biopsy sample is 2 mm3or less in size, the entire sample is used to generate organoids. If a biopsy sample is between 2 mm3and 4 mm3in size, the sample should be divided into two portions. One portion will be used to generate organoids and the other for metabolomics or transcriptomics-microbiome analysis. If a biopsy sample is between 4 mm3and 6 mm3in size, the sample should be divided into three portions. One portion will be used to generate organoids, one for metabolomics, and one for transcriptomics-microbiome analysis. If a biopsy sample is larger than 6 mm3in size, the sample should be divided into four portions. One portion will be used to generate organoids, one for metabolomics, one for transcriptomics-microbiome analysis, and the last one for cryopreservation.

[0125] All portions except one are placed in cryotubes (one portion in each tube) containing cryopreservation media. The tube is labeled with Sample ID, processing date, function code, and the initial of the person processing the sample. Tubes are stored in a -80°C freezer. The next day, the tubes are transferred into their designated locations in a cryopreservation storage container. Function codes are used to identify endometrial organoids (hEMO); endometrial stromal cells (hESC); primary sample for metabolomics (MET); primary sample for transcriptomics-microbiome analysis (TR-MIC); and primary sample for cryopreservation (PRE). The remaining one part of the sample is placed in phosphate buffer saline (PBS - / -) solution and stored on ice until all samples are processed and ready for the next set of procedures. The number of sample portions or tubes along with the function codes are stored in the database.

[0126] ENDOMETRIAL CULTURE GENERATION AND CRYOPRESERVATION PROCEDURES

[0127] Generation of Organoids

[0128] The entire procedure is performed in a biological safety cabinet. Multiple samples can be processed simultaneously so long as proper labeling of petri dishes and tubes is performed. This protocol follows the standard protocols developed by Turco et al.2017[1] and Boretto et al.2017[2]: 1. Remove sample from the tube onto a 35 mm petri dish. 2. Mince tissue sample into very small pieces (0.5 mm3) with a dissecting blade. 3. Collect minced tissue pieces back into the tube. 4. Centrifuge the tube at 200xg for 5 minutes and discard supernatant. 5. Add 1 mL of DMEM / F-12 to the tube, followed by 1 mL of collagenase IV (2 mg / mL). 6. Transfer everything with a plastic Pasteur pipette onto a 35 mm dish, keep at 37°C. 7. Pipette up and down 50 times with a plastic Pasteur pipette and observe under a microscope every 30 minutes. 8. For the collection of stromal cells, stop digestion by adding medium (DMEM / F-12 or RPMI) when there are enough glandular elements. 9. Transfer everything into a tube, let stand for 2 minutes. 10. Pass the supernatant through a 40 μm strainer into a 50 mL conical tube to collect the flow-through containing the stromal cells. 11. Wash the strainer a couple times with DMEM / F-12 or RPMI. 12. For human endometrial stromal cells (hESC) culture: centrifuge the tube at 600xg for 6 minutes, remove supernatant, and wash pellet with DMEM / F-12. Centrifuge again at 600xg for 6 minutes, remove supernatant, and resuspend with 5 mL ofhESC media. Count the number of cells and plate ~1 million cells per T-25. Change media every 2 days, culture for 3-5 days. The following is for human endometrial organoid (hEMO) culture: 13. From Step 11, backwash the glandular elements from the strainer onto a petri dish, then transfer back to the tube from Step 9. 14. Pass everything through a 100 μm strainer into a 50 mL conical tube to collect the flow-through containing the glandular elements. 15. Wash the strainer a couple times with DMEM / F-12 or RPMI. 16. Centrifuge the tube at 500xg for 5 minutes, discard supernatant. 17. Resuspend in 1-2 mL Advanced DMEM / F-12. 18. Centrifuge the tube at 500xg for 5 minutes, discard supernatant. 19. Estimate pellet volume, add Matrigel solution (70% Matrigel, 30% DMEM / F-12) in the amount of 20x the estimated pellet volume, and place on ice. 20. Pipette up and down to ensure even distribution of glandular elements in the Matrigel solution, and plate 20-25 μL drops of Matrigel / cell suspension into the center of wells of 48-well tissue culture plates. 21. Place the plate in the 37°C incubator to set for 15 minutes. 22. Overlay each drop with 250 μL of Expansion Medium. 23. Change medium every other day and grow for 7-10 days before the next passage.

[0129] hESC and hEMO Cryopreservation

[0130] For hESC culture: Passage when the culture in T-25 flask reaches 90% confluency with TrypLE. Resuspend cells with 3 mL of freezing medium (90% FBS, 10% DMSO). Freeze 3 tubes per sample labeled with sample ID, date, and initial.

[0131] For hEMO culture: Passage organoids when there are plenty with ~200 μm in diameter (follow protocol in Khoei et al.2023, doi.org / 10.1038 / s41596-023-00802-1). Resuspend cells in Recovery Cell Culture Freezing Medium. Freeze 3 tubes per sample labeled with sample ID, date, and initial.

[0132] SAMPLE METABOLOMICS AND MICROBIOME ANALYSIS

[0133] Metabolomics

[0134] A portion of the biopsy sample will be processed for metabolomics analysis[3].20 mg (2 mm3) of tissue section will be homogenized with ceramic beads in methanol, followed by protein precipitation. Supernatant will be dried under vacuum, resuspended in acetonitrile and re-centrifuged. Supernatant will be injected into the HPLC / LC-MS machine.

[0135] Transcriptomics-Microbiome Analysis

[0136] A portion of the primary samples will be processed for both transcriptomics analysis and microbiome analysis. Identification of microbial species in the endometrial biopsy is performed via 16S rRNA sequencing. A protocol that separates bacterial DNA from human RNA will be implemented.25 mg (2.5 mm3)[4] of the biopsy sample will be allocated for this analysis. RNA and DNA extraction from the samples followed by purification of human RNA and bacterial DNA[5] would be performed prior to sending the DNA sample to the vendor (Novogene) for sequencing.

[0137] The presence of bacterial DNA of the following groups will be detected: Lactobacillus group: L. crispatus, L. gasseri, L. iners, and L. jensenii Pathogens of the reproductive 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. Pathogens of the reproductive tract that are related to 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.

[0138] Next endometrial culture can be performed as described in Example 1, blastoid formation as in Example 2, and implantation assay as in Example 3.

[0139] Example 5: Prediction of embryo transfer success

[0140] The protocols for processing samples and preparation of assays can be part of an ongoing refinement process. For example, biopsies are collected, and various assays are performed, obtaining transcriptomic, microbiome, and other medical information, as well as implantation assays. An array of results obtained from these assays and data collection is entered into a classifier and / or regression algorithm. The results are reviewed and reported, and the algorithm is refined as further iterations are completed.

[0141] Specifically, patient data can be entered into a system trained on such information to provide a patient’s overall likelihood of embryo transfer success given a particular type of hormone treatment, or to provide a set of probabilities for a patient’s likelihood of successful embryo transfer given multiple different hormone treatments tested in parallel.

[0142] The input to the algorithm is a set of "features" (values) generated from all of a patient's analyses during the assay. This can include, but is not limited to: In vitro assay output: the percent of attached blastoids for a given hormone treatment protocol; In vitro assay output: other values called out in the application, including timing of implant, depth of blastoid implant, rate of movement of blastoids pre / post implant for a given hormone treatment protocol; Transcriptomics output: the numerical difference in various gene expressions pre / post a given hormone treatment protocol (Examples include, but are not limited to, the difference in BCL6 and SIRT1.); Other omics output from, e.g. Metabolomics / Proteomics / Epigenomics; Microbiome output: of the primary sample - signals may be either the binary presence or lack thereof of certain pathogens as well as the relative amounts of each detected type of pathogen present (i.e. percent of a particular pathogen found); Demographic data: such as, but not limited to, the patient age, body mass index (BMI), past pregnancy success, etc.

[0143] To train the algorithm, labeled training is compiled by taking the previously mentioned features as input and associating them with a known patient outcome for a given hormone treatment protocol. This is accomplished by extracting the aforementioned set of features from a number of patients--including coding the applied hormone treatment as an input categorical variable--and tagging them with an outcome measure: such as a simple measure of success (0: embryo transfer failure.1: embryo transfer success) or a more complex measure like a set of classes that can take on values such as, but not limited to, "successful pregnancy," "unsuccessful (no positive preganancy test)," "biochemical pregnancy," "molar pregnancy," "ectopic pregnancy," "intrauterine clinical pregnancy," "miscarriage," or "live birth."

[0144] This training data can then be provided to a number of different machine learning algorithms such as, but not limited to, a multivariable logistic regression, support vector machine (SVM), or a neural network with an attached classifier (e.g. such as a Gaussian classifier), to fit a predictive model to the training data.

[0145] After the model is trained, these features can be obtained from a new patient and fed into the algorithm, along with the in vitro results for each hormone treatment protocol, to derive predictions for both an overall probability of transfer success as well as the likelihood of success for each hormone treatment protocol.

[0146] In another embodiment, a predictive algorithm of the in vitro assay itself is formed, such that upon training of the algorithm, the in vitro assay involving blastoids is not requied in order to identify a hormone treatment protocol for a patient. Features are obtained from the in vitro assay (e.g. % blastoids attached) as the measurement outcomes for training a machine learning algorithm. Each hormone treatment protocol would correspond to a numerical output from the model, and the model would predict the percent of blastoids that are likely to attach for that hormone treatment protocol. Additional outcome measures that can be predicted include, for example, attachment depth or timing. A clinician can then select the protocol that provides the highest score. For input, the features would either come from the primary sample or in culture (the organoids) such as the microbiome of the primary sample, demographic information, other omics values, etc. as input values for training.

[0147] REFERENCES

[0148] 1. Turco M, et al. Nature cell biology.2017;19(5):568-577.

[0149] 2. Boretto M, et al. Development (Cambridge, England).2017;144(10):1775-1786.

[0150] 3. Li J, et al. Reproductive biology and endocrinology : RB&E.04 / 30 / 2018 2018;16(1):42.

[0151] 4. Moreno I, et al. Microbiome.01 / 04 / 20222022;10(1):1.

[0152] 5. Bruggeling C, et al. MicrobiologyOpen.2021 Jun 2021;10(3):e1191.

[0153] 6. Heidari Khoei H, et al. Nature protocols.2023;18(5):1584-1620.

[0154] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

[0155] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is:

1. A method of producing endometrial organoids, the method comprising: (a) seeding endometrial cells on a surface printed with micropatterns; and (b) culturing the cells seeded in step (a) under conditions sufficient for growth in three dimensions and organoid formation.

2. The method of claim 1, wherein the seeding of step (a) further comprises seeding stromal cells on the surface.

3. The method of claim 1, wherein the micropatterns are applied via photolithography or microcontact printing.

4. The method of claim 1, wherein the endometrial cells are obtained from established endometrial cell lines, patient biopsies, and / or patient biopsies grown as organoids in primary cultures or in long term established organoid cultures.

5. The method of claim 1, wherein the micropatterns are coated with extracellular matrix (ECM) protein.

6. The method of claim 1, wherein conditions of step (b) comprise culturing the endometrial cells in a culture medium that comprises extracellular matrix protein.

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

8. The method of any of the preceding claims, further comprising releasing the endometrial cells in growth media or in a gel under conditions sufficient for growth in three dimensions.

9. The method of claim 8, wherein the gel comprises an ECM based gel.

10. A method of predicting successful implantation of an embryo in a subject, the method comprising: (a) culturing endometrial cells obtained from the subject; (b) contacting a plurality of blastoids with endometrial cells; (c) culturing the endometrial cells together with the blastoids in the presence of implantation support agents; and (d) detecting implantation of at least one blastoid, wherein detection of implantation of the at least one blastoid is indicative of successful implantation of an embryo on the subject.

11. The method of claim 10, wherein the endometrial cells are cultured as a layer of endometrial cells, or as an endometrial organoid.

12. The method of claim 10, wherein the method further comprises measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial organoid.

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

14. The method of claim 13, wherein the hormones comprise progesterone, progestins, human chorionic gonadotropin (hCG), and / or gonadotrophin-releasing hormone (GnRH) agonists.

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

16. The method of claim 12, wherein the measuring comprises microscopic observation via phase contrast imaging, differential interference contrast imaging, and / or immunofluorescent detection of embryo-specific markers and / or endometrial markers.

17. The method of claim 16, wherein the embryo-specific markers comprise CDX2, OCT4, NANOG, GATA3, SOX17, NR2F2, GATA4, and / or GATA6.

18. The method of claim 16, wherein the endometrial markers comprise SOX9, MUC1, PAEP, OLFM4, acetylated alpha-tubulin, ECAD, vimentin, and / or FOXA2.

19. The method of claim 16, wherein the microscopic observation comprises live cell imaging of the blastoid or imaging of the blastoid after treatment with a fixative.

20. The method of claim 16, wherein the blastoid is derived from a human stem cell line, and wherein the human stem cell line has been labeled with a fluorescent marker.

21. The method of claim 12, further comprising: measuring the time and / or depth in which the blastoids undergo implantation in the endometrial organoid, and / or measuring a rate of movement of the blastoids before and after implantation in the endometrial organoid.

22. The method of claim 10, further comprising measuring gene expression and / or chromatin accessibility in the blastoid(s) and / or the endometrial organoid.

23. The method of claim 22, wherein the measuring comprises RNA sequencing, Assay for Transposase-Accessible Chromatin (ATAC) sequencing, immunofluorescence, and / or polymerase chain reaction (PCR).

24. The method of claim 23, wherein the PCR is quantitative real time PCR.

25. A method of optimizing embryo transfer in a subject, the method comprising: (a) culturing endometrial cells obtained from the subject in the presence of implantation support agents; (b) contacting a plurality of blastoids with the endometrial cells at a plurality of timepoints relative to culturing the endometrial cells in the presence of implantation support agents; and (c) measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial cells as a function of the timepoints varied in step (b), whereby the optimal timepoint for embryo transfer in the subject is indicated by the timepoint that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted in step (b).

26. A method of optimizing embryo transfer in a subject, the method comprising: (a) culturing endometrial cells obtained from the subject in the presence of implantation support agents; (b) contacting a plurality of blastoids with the endometrial cells in the presence of a plurality of combinations of implantation support agents, wherein the implantation support agents are selected from progesterone, progestins, human chorionic gonadotropin (hCG), and gonadotrophin-releasing hormone (GnRH) agonists; and (c) measuring the proportion of the plurality of blastoids that undergo implantation in the endometrial cells as a function of the implantation support agents of step (b), whereby the optimal combination of implantation support agents for embryo transfer in the subject is indicated by the combination that results in the largest proportion of blastoid implantation relative to the total number of blastoids contacted with the endometrial cells.

27. The method of claim 25 or 26, wherein the endometrial cells are cultured as a layer or as an organoid.

28. The method of claim 25 or 26, wherein the plurality of blastoids is obtained from induced pluripotent stem cells created from a sample taken from the subject.

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

30. The method of claim 28, wherein induced pluripotent stem cells are created using transcription factor-based methods or somatic cell nuclear transfer.

31. The method of claim 29, wherein the transcription factor-based methods comprise introducing into a cell from the sample transcription factors comprising DNA, RNA, or protein.

32. The method of claim 29, wherein the transcription factor-based methods comprise lentiviral based, adenovirus virus based, or sendai virus based reprogramming, or reprogramming based on mRNA transfection.

33. The method of claim 25 or 26, wherein the blastoids are created from cells modified to prevent postimplantation embryonic development.

34. A method of predicting successful implantation of an embryo in a subject, the method comprising: (a) culturing endometrial cells obtained from the subject; (b) contacting the endometrial cells with estrogen and / or progesterone for a period of time or in accordance with a predetermined schedule of hormonal treatment; (c) assaying a sample of the endometrial cells for one or more markers of receptive endometrium and / or one or more markers of endometrial pathology; (d) predicting successful implantation when the markers of receptive endometrium are present or greater than a reference amount, and / or the markers of endometrial pathology are absent or less than a reference amount; and wherein the markers of receptive endometrium are selected from PAEP, acetylated a-tubulin cilia, mucin, CXCL14, and SPP1, and wherein the markers of endometrial pathology are selected from BCL6, CCL19, MUC5B, PCDH-17, PTPRR and IL6ST.

35. The method of claim 34, wherein the assaying of step (c) further comprises comparing the sample of endometrial cells to a control sample that has not been contacted with estrogen, progesterone or other hormone treatment.

36. The method of claim 34, wherein the endometrial cells are cultured as a layer or as an organoid.

37. The method of claim 34, wherein the assaying comprises RNA sequencing, quantitative polymerase chain reaction (qPCR), immunostaining, or ATAC sequencing.

38. The method of claim 34, wherein the endometrial cells are obtained from the subject independent of the subject’s hormonal cycle.

39. A computer-implemented method for selecting a protocol for embryo implantation in a subject, the method comprising: (a) receiving input comprising at least one parameter selected from: (i) percent of implanted blastoids detected in the method of claim 10, optionally in response to treatment with estrogen and / or progesterone; (ii) presence of markers of receptive endometrium and / or absence of markers of endometrial pathology detected in the method of claim 34; (iii) age, body mass index (BMI), presence of bacterial species, and / or past pregnancy success of the subject; (b) generating, using a machine learning software module, an output comprising an indication of the subject’s likelihood of successful embryo implantation based on the input received in (a).

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

41. A computer system for selecting a protocol for embryo implantation in a subject, the computer system comprising: (a) a processor; and (b) a non-transitory computer readable medium that stores instructions that, when executed by the processor causes the processor to: (i) receive input comprising at least one parameter selected from: percent of implanted blastoids detected in the method of claim 10, optionally in response to treatment with estrogen and / or progesterone; presence of markers of receptive endometrium and / or absence of markers of endometrial pathology detected in the method of claim 34; and age, body mass index (BMI), presence of bacterial species, and / or past pregnancy success of the subject; (ii) generate an output comprising an indication of the subject’s likelihood of successful embryo implantation based on the input received in (i).

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