Platform for studying maternal-embryo interactions and implantation

Abstract

Described herein is a system for studying healthy human endometrial development, associated pathologies, embryo implantation, and post-implantation development. The system comprises an endometrial assembloid together with one of the following; a blastoid mimicking a blastocyst, a trophoblast organoid mimicking early placental dynamics, or an embryo model capable of interacting with maternal tissue. Also described herein is a method of using the system to study human endometrial healthy development and pathologies together with embryo implantation and post-implantation development.

Description

[0001] Atorney Docket No. 047162-7518WOl(02693)

[0002] PLATFORM FOR STUDYING MATERNAL-EMBRYO INTERACTIONS AND IMPLANTATION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 706,870, filed October 14, 2024, which is incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006] Implantation failure is a common occurrence in both natural conception and assisted reproductive techniques. It is estimated that between 70% and 75% of embry os generated, either through natural conception or via in vitro fertilization (IVF), fail at some stage in their development before implantation occurs, leading to implantation failure. Recurrent implantation failure, defined as the failure to achieve a clinical pregnancy after several embryo transfers with good-quality embryos, affects about 10% of women undergoing IVF treatments. One of the most common endometrial pathologies is endometriosis, which is a chronic gynecological condition characterized by painful hormone-responsive lesions and abnormal proliferation of uterine tissue. This disease impacts around 10% of women during their reproductive years, and nearly half of those afflicted suffer from infertility.

[0007] Despite the impact of these endometrial pathologies on reproductive health, studies on structural and physiological factors associated with implantation failure remain limited due to the inaccessibility of this process in humans and the technical and ethical constraints in studying human embryo implantation. Furthermore, there is also a lack of suitable animal models due to of physiological, structural and mechanistic disparities between other mammals and humans at this critical stage.

[0008] There is thus a need for platforms, such as cell three-dimensional culture-based models, for studying embryo implantation and infertility, such as in the case of endometriosis. The present study addresses this need.

[0009] SUMMARY

[0010] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0011] System

[0012] 1

[0013] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0014] In some aspects, the present invention is directed to a system.

[0015] In some embodiments, the system comprises an endometrial assembloid and a blastoid mimicking a blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, and / or an embryo model capable of interacting with a maternal tissue or the endometrial assembloid.

[0016] In some embodiments, the endometrial assembloid comprises a plurality of endometrial stromal cells enclosed in a layer of endometrial epithelial cells.

[0017] In some embodiments, the endometrial assembloid further comprises endothelial cells and forms a vascular structure.

[0018] In some embodiments, the embry o model capable of interacting with a maternal tissue or the endometrial assembloid comprises a human extra-embryoid (hEE) representing a postimplantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a peri-implantation embry o model.

[0019] In some embodiments, in the endometrial assembloid. a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0020] In some embodiments, the endometrial assembloid is prepared from human endometrial cells.

[0021] In some embodiments, the endometrial assembloid is prepared using cells from an endometrial sample of a human subject.

[0022] In some embodiments, the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject.

[0023] In some embodiments, the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject.

[0024] In some embodiments, the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

[0025] In some embodiments, the human subject is suffering from endometriosis.

[0026] In some embodiments, the human subject does not suffer from endometriosis.

[0027] In some embodiments, it is unknown whether the human subject is suffering from endometriosis.

[0028] In some embodiments, the human subject is suffering from infertility.

[0029] In some embodiments, it is uncertain if the human subject is suffering from infertility’.

[0030] 2

[0031] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0032] In some embodiments, the blastoid, the trophoblast organoid, or the embryo model capable of implanting is prepared from human pluripotent stem cells (hPSCs), trophoblast stem cells, or induced / transdifferentiated trophoblast stem cells.

[0033] In some embodiments, the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

[0034] In some embodiments, the blastoid, the trophoblast organoid, or the embryo model is prepared from human cells, and the blastoid or the embryo model is incapable of developing into a human organism.

[0035] In some embodiments, the system comprises one or more culture media for suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model.

[0036] In some embodiments, the endometrial assembloid and the blastoid, the trophoblast organoid, or the embry o model are brought in contact with each other to allow apposition, attachment, and / or invasion of the endometrial assembloid.

[0037] Method of studying or evaluating embryo implantation and subsequent post-implantation development

[0038] In some aspects, the present invention is directed to a method of studying or evaluating embryo implantation and subsequent post-implantation development.

[0039] In some embodiments, the method comprises placing an endometrial assembloid and a blastoid mimicking a human blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, or an embry o model embry o model capable of interacting with a maternal tissue or the endometrial assembloid near each other such that the blastoid, the trophoblast organoid, and / or the embryo model is able to appose, attach to, and / or invade the endometrial assembloid.

[0040] In some embodiments, the endometrial assembloid comprises a plurality of endometrial stromal cells, enclosed in a layer of endometrial epithelial cells. In some embodiments, endometrial assembloid further comprises endothelial cell.

[0041] In some embodiments, the blastoid, the trophoblast organoid, and / or the embryo model mimics a blastocyst or a pre- or post-implantation embryo.

[0042] In some embodiments, the embryo model capable of interacting with a maternal tissue or the endometrial assembloid comprises a human extra-embryoid (hEE) representing a postimplantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a peri-implantation

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[0044] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) embry o model.

[0045] In some embodiments, in the endometrial assembloid. a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0046] In some embodiments, the endometrial assembloid is prepared from human endometrial and vascular cells.

[0047] In some embodiments, the endometrial assembloid is prepared using cells from an endometrial sample of a human subject.

[0048] In some embodiments, the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject.

[0049] In some embodiments, the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject.

[0050] In some embodiments, the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

[0051] In some embodiments, the human subject is suffering from endometriosis.

[0052] In some embodiments, the human subject does not suffer from endometriosis.

[0053] In some embodiments, it is unknown whether the human subject is suffering from endometriosis.

[0054] In some embodiments, the human subject is suffering from infertility.

[0055] In some embodiments, it is uncertain if the human subject is suffering from infertility.

[0056] In some embodiments, the blastoid, the trophoblast organoid, or the embryo model capable of implanting, is prepared from human pluripotent stem cells (hPSCs).

[0057] In some embodiments, the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

[0058] In some embodiments, the blastoid is prepared from human cells, and the blastoid, the trophoblast organoid, or the embryo model is incapable of developing into a human organism.

[0059] In some embodiments, the method further comprises suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model is suspended in one or more culture media.

[0060] In some embodiments, the method studies or evaluates the apposition, attachment, and / or invasion of the blastoid, the trophoblast organoid, or the embry o model to the endometrial assembloid.

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[0062] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0063] Endometrial assembloid

[0064] In some aspects, the present invention is directed to an endometrial assembloid.

[0065] In some embodiments, the endometrial assembloid comprises a plurality of endometrial stromal cells; and a layer of endometrial epithelial cells enclosing the plurality of endometrial stromal cells.

[0066] In some embodiments, the endometrial stromal cells and the endometrial epithelial cells are from an eutopic or ectopic endometrial sample of a human subject.

[0067] In some embodiments, the endometrial assembloid further comprises a plurality of endothelial cells.

[0068] In some embodiments, the endothelial cells are from an eutopic or ectopic endometrial sample of a human subject.

[0069] In some embodiments, the endometrial assembloid forms a vascular structures.

[0070] In some embodiments, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid,

[0071] In some embodiments, the plurality of endometrial stromal cells and layer of endometrial epithelial cells, together with the plurality of endothelial cells, forming vascular structures in an inner region of the endometrial assembloid.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0074] Fig. 1 A provides an overview of the platform for studying human embryo-uterine interactions in 3D, in accordance with some embodiments. Top: Schematic of human implantation. Bottom: Stem cell-based human blastocyst model, or blastoid, marked by 3 lineage markers. SOX2 marks inner cell mass (ICM), GATA3 marks trophectoderm, and FOXA2 marks hypoblast. EndoMetrial Assembloid model, or EMA, marked by 2 lineage markers. ECAD marks epithelial cells and CD 10 marks stromal cells. Scale = 20 pm.

[0075] Figs. 1B-1C illustrate certain aspects of the co-culture of the blastoid / TO, as well as the EMA models for healthy and endometriosis, in accordance with some embodiments. Fig. IB: Co-culture with a microfluidic chip. Fig. 1C: Suspension-based co-culture. In this model.

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[0077] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) the EMA represented is vascularized, as seen with the included Human uterine microvascular endothelial cells (HUtMEC) population

[0078] Fig. 2A illustrates some possible EMA structures generated, in accordance with some embodiments. EMA structures can be obtained depending on the scientific questions to be tested. The simplest models include epithelial and stromal cells. While the vascularized versions may include tissue-specific endothelial cells (HUtMEC) or Human vascular endothelial vein cells (HUVECs).

[0079] Fig. 2B provides immunofluorescence max projection images of some non-limiting EMAs, in accordance with some embodiments. Left-side: epithelial (EPCAM) in green, endothelial cells (CD31) in red, and stromal cells (vimentin) in cyan. Middle: a blastoid; Epiblast (sox2) in green, hypoblast (FOXA2) in red, and trophoblast (GATA3) in cyan. Right-side: a trophoblast organoid; membrane (F-Actin) in purple, and trophoblast cells (GAT A3) in cyan.

[0080] Fig. 2C provides illustrations of the main co-culture platform which consists of a high-throughput 96-well ultra-low attachment plate where blastoid / TO and EMA are combined in suspension co-culture.

[0081] Figs. 3 A-3C provide illustrations summarizing the overall steps for the endometrial and implantation platform system, in accordance with some embodiments. Fig. 3A: an endometrial biopsy that is processed into individual cell types. Specifically, epithelial and stromal combined with endothelial cells into a suspension well for aggregation. Eventually they are combined with blastoids for co-culture. Figs. 3B-3C denote the second step which is targeted evaluation of implantation steps, which shows three stages of implantation recapitulated in the system at different time-points. At 24 hrs post co-culture (p.c.c) apposition and adhesion. 48 hrs p.c.c. early invasion and 72hrs p.c.c. later invasion.

[0082] Fig. 4 illustrates certain aspects of a personalized embryo implantation evaluation, diagnosis, and treatment method, in accordance with some embodiments.

[0083] DETAILED DESCRIPTION

[0084] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact and

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[0086] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0087] This study provides, in one aspect endometrial models, for studying embryo implantation and post-implantation development.

[0088] In some embodiments, the cell culture model is suitable for studying the impact of endometrial pathologies on embry o implantation. In certain non-limiting embodiments, the pathology' is endometriosis.

[0089] Specifically, the study described herein (“the present study”) developed a model (sometimes referred to as “implantoid”) in which a model for blastocyst (in certain embodiments constructed from pluripotent human stem cells and termed “blastoid” herein), as well as a model for trophoblast development (in certain embodiments constructed from trophoblast stem cells, or induced / transdifferentiated trophoblast cells, sometimes referred to as “trophoblast organoid” or TO herein). In the models, the blastocyst or the trophoblast is co-cultured with an endometrial assembloid, thus providing a model for implantation and post-implantation development of the early embry o onto the endometrium in 3D.

[0090] It has been hypothesized that in endometriosis, the molecular dysregulation of endometrial receptivity- impedes the proper crosstalk between the embryo and endometrium, leading to implantation failure and thus reduced female reproduction. It is envisaged that the model herein will enable studies and tests relating to such hypotheses, as the implantoid model herein allows for bioengineering, genetics, imaging, and single-cell profiling to map the molecular and cellular events that drive embryo implantation and post-implantation development under healthy or different patient-derived uterine pathological conditions. Furthermore, the implantoid model herein also enables the comprehensive molecular analysis of factors influencing implantation failure in endometriosis, such as through of single-cell transcriptomics, and / or functional and signaling-based assays.

[0091] As will be apparent to one of ordinary skill in the art, the implantoid model herein provides a controllable, efficient, and high-throughput modeling of human implantation and post-implantation development. Further, the implantoid model enables the assessment of endometrial receptivity- and blastocyst implantation through imaging methods, such as high- resolution confocal live-imaging.

[0092] Furthermore, the implantoid model provides a strategy to acquire a much-needed

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[0094] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) mechanistic understanding of human embryo-uterine interactions, as well as an understanding of pregnancy and embryonic health in general.

[0095] Furthermore, the implantoid model provides a strategy to acquire subsequent embryonic developmental stages during post-implantation.

[0096] Accordingly, in some aspects, the present invention is directed to systems and methods for studying embryo implantation and / or post-implantation development.

[0097] In certain non-limiting embodiments, the endometrial assembloid developed in the present study differs from conventional endometrial models in that it can be prepared from eutopic endometrial samples from endometriosis patients, is multicellular, and / or gel-free, and allows the analysis of implantation failure at a molecular level.

[0098] Accordingly, in some aspects, the present invention is directed to an endometrial assembloid model.

[0099] Definitions

[0100] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory' procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0101] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0102] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be

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[0104] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0105] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”

[0106] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0107] System

[0108] In some aspects, the present invention is directed to a system.

[0109] In some embodiments, the system provides a model for studying the embryo implantation and / or post-implantation development of mammalians, such as humans.

[0110] In some embodiments, the system comprises an endometrial assembloid.

[0111] In some embodiments, the system further comprises a stem cell-based embryo model mimicking implanting embryo.

[0112] In some embodiments, the embryo model comprises a blastoid mimicking a blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, and / or an embryo model capable of interacting with maternal tissue endometrial assembloid.

[0113] In some embodiments, the embryo model capable of interacting with maternal tissue endometrial assembloid compirses a human extra-embryoid (hEE) representing a postimplantation stage of an embryo, a mouse blastocyst for cross-species assessments, and / or a human suspension-based epiblast-like spheroid (hSES) representing a peri-implantation embryo model.

[0114] In some embodiments, the endometrial assembloid provides a model of the endometrium of mammals, such as humans.

[0115] In some embodiments, the endometrial assembloid comprises a plurality of endometrial stromal cells. In some embodiments, the endometrial assembloid comprises a layer of endometrial epithelial cells fully or partially enclosing the plurality of endometrial stromal cells. In some embodiments, the endometrial assembloid contains endothelial cells in the inner and / or outer compartments of the assembloid.

[0116] In some embodiments, in the endometrial assembloid, a basal side of the endometrial

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[0118] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) epithelial cells faces the endometrial stromal cells. In some embodiments, in the endometrial assembloid, an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0119] In some embodiments, the endometrial assembloid is prepared from endometrial biopsies processed into human endometrial stromal and epithelial cells.

[0120] In some embodiments, the endometrial assembloid is prepared using cells from an endometrial sample of a human subject.

[0121] In some embodiments, the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject.

[0122] In some embodiments, the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject.

[0123] In some embodiments, the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

[0124] In some embodiments, the human subject is suffering from endometriosis.

[0125] In some embodiments, the human subject is suffering from endometriosis and infertility.

[0126] In some embodiments, the human subject does not suffer from endometriosis.

[0127] In some embodiments, it is unknown whether the human subject is suffering from endometriosis and / or other gynecological disorders and uterine pathologies.

[0128] In some embodiments, the human subject is suffering from infertility.

[0129] In some embodiments, it is uncertain if the human subject is suffering from infertility.

[0130] The method of preparing endometrial assembloid is described herein (see e.g., the “Examples” section). Alternative methods of preparing endometrial assembloids are described in Wiwatpanit et al. (J Clin Endocrinol Metab . 2020 Mar 1; 105(3):769-780) and Song et al. (JCI Insight. 2023 Jun 8; 8(11): el 60815), which can be used for the purposes of the present invention, as well.

[0131] In some embodiments, the blastoid, or the embry o model, is prepared from stem cells, such as pluripotent stem cells, such as human pluripotent stem cells (hPSCs). such as induced pluripotent stem cells (iPSCs).

[0132] In some embodiments, the blastoid, or the human embryo model, is prepared from human stem cells. In some embodiments, the blastoid is incapable of developing into a human organism.

[0133] In some embodiments, the trophoblast organoid is prepared from trophoblast stem cells or induced / transdifferentiated trophoblast cells from pluripotent stem cells, such as

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[0135] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs).

[0136] In some embodiments, at least one marker protein in the blastoid is attached with a tag, such as a fluorescence tag, for imaging purposes.

[0137] Non-limiting examples of the at least one marker protein includes NR2F2, SOX2, GATA3, FOXA2, hCG, KRT7, and the like.

[0138] Non-limiting examples of fluorescence tag include fluorescence proteins, such as GFP, RFP, and the like.

[0139] In some embodiments, the at least one marker protein is attached with the tag by a genome editing method editing the genes encoding the marker proteins.

[0140] The method of preparing the blastoid is described herein, as well as in WO 2022 / 152774 Al, Yu et al. (Cell Stem Cell. 2023 Sep 7;30(9): 1246-1261. e9): Shibata et al. (Sci Adv. 2024 Feb 23; 10(8):eadi4819); Sozen et al. (Nat Commun. 2021 Sep 21 ; 12(1): 5550); Kagawa et al. (Nature. 2022 Jan;601(7894):600-605); Liu et al., (Nature 591, 627-632 (2021)); Yu et al. (Nature 591, 620-626 (2021)); Rivron et al. (Nature. 2018 May;557(7703): 106-111); Zhang et al. (Sci China Life Sci. 2023 Mar;66(3):423-435); and Li et al. (Cell. 2019 Oct 17; 179(3):687-702.el 8.31).

[0141] In some embodiments, the system further comprises one or more culture media for suspending (i) the endometrial assembloid and (ii) the blastoid or trophoblast organoid.

[0142] In some embodiments, the endometrial assembloid is suspended in a first culture media in the first chamber, and the blastoid or the trophoblast organoid is suspended in a second culture media in the second chamber.

[0143] In some embodiments, (i) the endometrial assembloid and (ii) the blastoid or the trophoblast organoid contact each other via the channel.

[0144] In some embodiments, (i) the endometrial assembloid and (ii) the blastoid or the trophoblast organoid are brought in contact with each other to allow the blastoid to appose, attach, and / or invade the endometrial assembloid.

[0145] In some embodiments, the endometrial assembloid is suspended in the same culture as the blastoid or the trophoblast organoid, such as in a U-well.

[0146] In some embodiments, (i) the endometrial assembloid and (ii) the blastoid or the trophoblast organoid contact each due to proximity, such as in a U-well.

[0147] Method of studying or evaluating embryo implantation and / or post-implantation development

[0148] In some aspects, the present invention is directed to a method of studying or

[0149] 1 1

[0150] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) evaluating embry o implantation.

[0151] In some aspects, the present invention is directed to a method of studying or evaluating embry o post-implantation development.

[0152] In some embodiments, the method uses the systems herein, such as systems the same as or similar to described in the “System” section.

[0153] In some embodiments, the method comprises placing (i) the endometrial assembloid and (ii) the blastoid or the trophoblast organoid near each other, and observe the apposition, attachment, and / or invasion of the blastoid to the endometrial assembloid.

[0154] In some embodiments, the method comprises placing (i) the endometrial assembloid and (ii) the blastoid or the embryo model in a same space, such as a roller-bottle culture or synthetic hydrogels, and observe the integrated development of the blastoid and to the endometrial assembloid to the post-implantation stages.

[0155] In some embodiments, the method herein is a high-throughput method.

[0156] In some embodiments, the method herein is a genetically and environmentally editable method.

[0157] EndoMetrial Assembloid (EMA)

[0158] In some aspects, the present invention is directed to an endometrial assembloid.

[0159] In some embodiments, the endometrial assembloid comprises a plurality of endometrial stromal cells. In some embodiments, the endometrial assembloid comprises a layer of endometrial epithelial cells enclosing the plurality of endometrial stromal cells.

[0160] In some embodiments, the endometrial assembloid comprises a layer of endometrial epithelial cells enclosing the plurality of endometrial stromal cells, and endothelial cells intermingle epithelial and stromal cells.

[0161] In some embodiments, the endometrial stromal cells and the endometrial epithelial cells are from an eutopic endometrial sample of a human subject.

[0162] In some embodiments, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0163] EXAMPLES

[0164] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless so specified. Thus, the instant specification should in no

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[0166] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) way be construed as being limited to the following examples, but rather, should be construed to encompass any variations which become evident as a result of the teaching provided herein.

[0167] Example 1:

[0168] Endometriosis is a common and chronic gynecological condition characterized by the emergence of painful lesions and the abnormal proliferation of uterine tissue. This disease impacts around 10-15% of women during their reproductive years, and nearly half of those afflicted suffer from infertility7. Though it has been proposed that conception can occur despite endometriosis and that implantation failure is the cause of infertility, the mechanism by which endometriosis induces infertility remains poorly understood. This is in part due to the absence of suitable animal models, as spontaneous endometriosis develops only in humans and certain primates who undergo menstrual cycles, as well as due to the technical and ethical issues associated with human embryo research.

[0169] Studies have allowed for modeling human embryonic development with embryo- derived stem cells, holding tremendous potential for biomedical applications. In the present study, a stem cell-based human blastocyst model (blastoids) or trophoblast organoid (TOs), as well as endometrial assembloids (EMAs) derived from patient tissue were established. The present study further demonstrated successful co-culture of the blastoids and the EMAs in a 3D system (referred to as the "implantoid” or "human implantoid") to facilitate organic interactions between blastoids or TOs and EMAs. This biomimetic strategy7for 3D modeling of human implantation enables real-time visualization and high-throughput experimentation to systematically dissect mechanisms driving human embryo implantation in a 3D environment.

[0170] Building upon this foundation, the present study aims to study human embryo implantation ex vivo, and decipher potential causes of implantation failure in the context of endometriosis (Fig. 1). The following studies are proposed:

[0171] 1) Characterize structural patterns of human embryo implantation with endometriosis. Embryo implantation into the uterus marks a key structural transition in human development and underscores reproductive success. However, the roles of embryo- uterine interactions in embry o morphogenesis during implantation are poorly understood due to inaccessibility in utero. Here, through the generation of patient-derived EMAs from control and endometriosis samples, combined with human blastoids or trophoblast organoids (TOs) the present study evaluates the three stages of human embry o implantation -apposition.

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[0173] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) atachment, and invasion- using high-resolution confocal imaging and immunolabelling strategies in the implantoid platform. This defines define not only the first detailed real-time visualization of embryo-uterine interaction in a 3D human-specific context but also mechanistic insights into tissue morphogenesis and paterning in both healthy and disease states.

[0174] 2) Define transcriptional changes associated with structural paterns during human embryo implantation with endometriosis. Through single-cell transcriptomics, the study herein reveals the dynamic evolution of gene expression as embryo-uterine interaction occurs, and apply sophisticated computational analyses to define the molecular deviations at play in endometriosis-diseased state on embryo implantation.

[0175] In summary, a novel experimental strategy that recapitulates key physiological properties of human implantation dynamics in 3D is proposed, which focuses on identifying the mechanisms leading to reproductive failure in endometriosis. The model herein allows the study of molecular mechanisms underlying infertility in endometriosis, paving the way for novel therapeutic interventions and personalized treatment strategies for affected individuals.

[0176] Example 2:

[0177] The ex vivo culture of peri-implantation human embry os has been explored in 2D, and in vitro implantation assays have been demonstrated using co-culture with monolayer uterine cells. While these traditional approaches have been valuable, their potential for mimicking the dynamic mechanisms of human implantation is extremely limited. Firstly, the acquisition of surplus human embryos from clinics poses significant challenges; even w hen available, finite resources, qualify concerns, and low cell numbers within each embryo sample severely restrict the types of analyses that can be conducted. Additionally, the co-culture of human embryos on monolayer uterine cells, while inducing embryo adhesion, disrupts embryo morphogenesis on the 2D surface, and these monolayer cultures fail to capture the physical or biochemical complexify of the bona fide uterine environment. Thus, gaining mechanistic insights into human implantation has been hindered until the development of a robust system that faithfully recapitulates the utenne environment and the interactions between the embryo and uterus upon implantation.

[0178] Extensive studies have focused on advanced in vitro stem cell engineering for studying mammalian development, utilizing self-organizing properties of pluripotent stem cells (PSCs) derived from embryos. Stem-cell -based embryology has been explored. These models offer powerful tools for investigating human embryo development with higher

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[0180] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) temporal resolution, controlled experimental conditions, and improved replicate power.

[0181] Example 3: Implantoid as a model for studying implantation

[0182] The present study leverages stem cell-based models of the human blastocyst, called human blastoids (described in, for example, Sozen et al., Nat Commun. 2021 Sep 21;12(l):5550; Kagawa et al. Nature. 2022 Jan;601(7894):600-605; Liu et al., Nature 591. pages627-632 (2021); Yu et al, Nature 591, pages620-626 (2021); Rivron et al.. Nature. 2018 May;557(7703): 106-111 ; Zhang et al., Sci China Life Sci. 2023 Mar;66(3):423-435; and Li et al., Cell. 2019 Oct 17;179(3):687-702.el8.31).

[0183] Furthermore, the present study developed 3D human endometrial assembloids (EMAs) using patient-derived tissue that includes both endometrial and stromal cell types (Figs. 2A and 2B). Importantly, these EMAs grow in suspension culture, and specifically designed to represent epithelial surface facing outwards to allow for organic interactions with human blastoids (Figs. 2C-2D).

[0184] Capitalizing on these major technological and conceptual advances, the present studyfocuses on expanding on these in vitro systems with the application of a U-well-based coculture system to explore endometrial pathologies (Figs. 1C and 2C-2D) termed the “implantoid” (Figs. 1A-1B).

[0185] Example 4: Materials and Methods

[0186] Materials and Reagents:

[0187] • 30 mL DMEM / F 12 (pre- warmed)

[0188] • Heparin, Hydrocortisone

[0189] • Petri dish

[0190] • Razor blade or scalpel

[0191] • 100 pm cell strainer

[0192] • 20 pm cell strainer

[0193] • 10 mL HBSS (pre- warmed)

[0194] • 4 mL RBC lysis buffer (pre-warmed)

[0195] • 10 mL Dispase

[0196] • 25 mg Collagen

[0197] • 1 mg DNase (50 pL of 20 mg / mL stock)

[0198] 15

[0199] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0200] • 10 mL syringe with needle

[0201] • Syringe filter

[0202] • 96- Ultra low attachment plate

[0203] • 50 mL and 15 mL conical tubes

[0204] • Hemocytometer + cell counter Protocols:

[0205] Preparation of Digestion Solution

[0206] Prepare the digestion solution by combining 10 mL Dispase, 25 mg Collagen, and 1 mg DNase (50 pL of 20 mg / mL stock). Filter the solution using a syringe filter into a clean tube.

[0207] Biopsy Tissue Processing

[0208] In a petri dish, finely mince the endometrial biopsy sample using a razor blade or scalpel. If an excisional biopsy is used, scrape off the endometrial tissue from the muscle layer. Transfer the minced tissue into the digestion solution using a cut pipette tip. Rinse the petri dish with digestion solution and scrape any remaining tissue into the solution. Repeat rinsing and scraping multiple times to ensure complete transfer.

[0209] Tissue Digestion

[0210] Seal the tube containing the digestion solution and minced tissue with parafilm. Incubate the tube in a 37°C shaker for 30-60 minutes.

[0211] Straining the Digest

[0212] After digestion, strain the solution through a 100 pm cell strainer into a 50 mL conical tube, follow ed by 10 mL HBSS to wash through. Discard the contents of the strainer as biohazard waste.

[0213] Stromal and Epithelial Cell Isolation

[0214] Filter the 100 pm filtrate through a 20 pm cell strainer into another 50 mL conical tube. The flow-through contains stromal cells. Label the tube accordingly. Wash the 20 pm strainer by running 20 mL of DMEM / F 12 media backwards through the strainer to collect epithelial cells. Label this tube for epithelial cells.

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[0216] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0217] Cell Separation and Centrifugation

[0218] Divide the contents of both 50 mL conical tubes (stromal and epithelial fractions) into two 15 mL conical tubes each. Label all tubes accordingly. Centrifuge the tubes at 500 RCF for 5 minutes.

[0219] Stromal Cell Processing

[0220] After centrifugation, discard the supernatant from the stromal cell tubes. Resuspend the cells in 2 mL RBC lysis buffer per tube, then combine the contents into one tube. Incubate for 5 minutes in a 37°C water bath to lyse red blood cells.

[0221] Epithelial Cell Processing

[0222] Resuspend the epithelial cell pellet in 100 pL of DMEM / F12 with grow th factors.

[0223] Stromal Cell Resuspension and Counting

[0224] After the RBC lysis step, centrifuge the stromal cells again and discard the supernatant. Resuspend the cells in 30 pL of DMEM / F12 with growth factors. Count the stromal cells using a hemocytometer. The resulting epithelial and stromal cells can now be used for further experimentation or assembloid generation. Stromal cells can be expanded on 2D on tissue cultured treated plates, and epithelial cells can be expanded can as organoids such as endometrial epithelial organoids (EEOs) as described in Shibata et al. (Sci Adv. 2024 Feb 23;10(8):eadi4819).

[0225] Example 5: Implantoid as a model for studying post-implantation development

[0226] High frequency of developmental arrest in early pregnancy and the lack of direct access to peri-implantation tissues, results in a significant gap in knowledge of the mechanisms mediating the interconnection of the embryo and the mother. To overcome the limitations, in some embodiments, the method comprises placing the endometrial assembloid and the blastoid or embryo model into a roller-bottle culture or synthetic hydrogel scaffold, enabling dynamic and physiologically relevant interactions between embryonic and endometrial compartments. This arrangement supports prolonged co-culture and allows the observation of integrated development from implantation through early post-implantation stages.

[0227] By incorporating the structural and cellular complexity of endometrial assembloids together with stem-cell-derived embryo models, this system provides a robust and scalable

[0228] 17

[0229] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) platform for investigating the mechanisms of human post-implantation development. Importantly, the roller-botle or hydrogel-based culture environment preserves three- dimensional architecture and mechanical cues, which are essential for maintaining tissue organization and facilitating embryo-endometrium crosstalk. This innovation enables unprecedented opportunities to dissect the molecular, cellular, and metabolic processes underlying early human development, while providing a tractable framework for studying implantation failures and endometnal pathologies in a controlled laboratory seting.

[0230] Procedure: Stromal Cell Processing for EMA Generation

[0231] Stromal cells were isolated from eutopic endometrial biopsy samples and cultured in T75 flasks using 5 mL of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) per flask. Cell confluency was monitored via brightfield microscopy. Cells were detached using pre-warmed 0.25% Trypsin-EDTA. After aspirating the media, 2.5 mL of Trypsin-EDTA was added and incubated at 37°C in 5% CO2 for 10 minutes. The flasks were manually agitated to enhance detachment.

[0232] The Trypsin-EDTA was neutralized by adding 5 mL of pre-warmed DMEM / F-12, and the cells were washed repeatedly to remove all Trypsin. The cell suspension was transferred to 15 mL conical tubes and centrifuged at 1000 rpm for 2 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of DMEM / F-12. A 10 pL aliquot of the cell suspension was taken for cell counting using a hemocytometer.

[0233] Medici Preparation for EMA Generation

[0234] Specialized media was developed for EMA maintenance and culture. It consists of the following components:

[0235] Base Media

[0236] • DMEM / F - 12 supplemented with:

[0237] • 1% ITS-X supplement

[0238] • 0.15% bovine serum albumin (BS A)

[0239] • 1% Knockout Serum Replacement (KSR)

[0240] • Penn-strep

[0241] Complete Media

[0242] 18

[0243] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0244] Base Media supplemented with:

[0245] • 200 mM L-ascorbic acid

[0246] • 50 ng / ml EGF

[0247] • 2 pM CHIR99021

[0248] • 2 pM SB202190

[0249] • 10 pM Y27632

[0250] • 20ng / ml VEGF

[0251] • 50ng / ml FGF2

[0252] • 1 Omg / ml Heparin

[0253] • Ipg / ml Hydrocortisone

[0254] Procedure: Endometrial Epithelial Organoid (EEO) Processing for EMA Generation

[0255] To process the EEOs maintained in Matrigel, begin by adding ice-cold DMEM / F 12 to the cultures. Homogenize the mixture thoroughly by pipetting about 50 times, then transfer the suspension into a 15 mL Falcon tube. Centrifuge the sample for 3 minutes at 500 g and carefully remove the supernatant. Finally, resuspend the pellet in 1 mL of complete Media.

[0256] Procedure: EMA Generation in Suspension

[0257] Stromal cells can be co-cultured with endometrial epithelial organoids (EEOs) and Human Uterine Microvascular Endothelial Cells (HUtMEC) in suspension-based, gel-free conditions.

[0258] 96-Well Preparation and Seeding

[0259] The plate should have media added and be pre-warmed in 37C for at least 30 minutes.

[0260] Cell Seeding:

[0261] Complete media was also added to the suspension culture wells at the time of seeding.

[0262] Endothelial Cell (HuTMEC / HUVEC) Processing for EMA Generation

[0263] HuTMECs / HUVECs were cultured in T75 flasks and detached using pre-warmed 0.04% Tiypsin-EDTA. After aspirating the media, 7.5 mL of Trypsin-EDTA was added, and the flask was incubated at 37°C for 7 minutes. The flask was manually agitated to enhance detachment. Trypsin-EDTA was neutralized with pre-warmed media, and the cells were

[0264] 19

[0265] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) washed thoroughly to ensure full Try psin removal. The cell suspension was transferred to 15 mL conical tubes and centrifuged at 1000 rpm for 3 minutes. After discarding the supernatant, the cell pellet was resuspended in 1 mL of complete media. A 10 pL aliquot was taken for cell counting using a hemocytometer.

[0266] Cells were then seeded together in the U-Wells as a 3:2:1 density, with 3 epithelial to 2 stromal to 1 HUVEC or HuTMEC. Cells yvere spun down in the plate for 5 minutes at 1000 rpm.

[0267] Procedure: EMA Generation in Gel-Embedded Conditions

[0268] Stromal cells can be co-cultured with endometrial epithelial organoids (EEOs) and HUtMECs or HUVECs in gel-based conditions as well.

[0269] Procedure: Decidualization Treatment

[0270] Materials:

[0271] • Decidualization media containing: o Estradiol (E2) o Medroxyprogesterone acetate (MPA) o 8-Bromo-cAMP

[0272] • Culture plates yvith cells

[0273] Protocol:

[0274] Day 0 (DO): Initiation of Decidualization Treatment

[0275] Add 100 pL of freshly prepared decidualization media (containing E2, MPA. and 8- bromo-cAMP) to each well of the cell culture plate. Move plate to shaker.

[0276] Day 1 (DI): Add lOOul of Decidualization Media

[0277] Day 2 (D2): No media change

[0278] Do not disturb the media or cells.

[0279] Day 3 (D3): Remove lOOul of media and add lOOul of fresh decidualization media, replenishing signanling molecules necessary for continued decidualization.

[0280] Day 4 (D4): Fixation and Staining of Implantoid

[0281] Proceed yvith cell fixation using a suitable fixative such as 4% paraformaldehyde. After fixation, stain the cells as required for further analysis.

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[0283] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0284] Procedure: Implantoid Generation - EMA Co-Culture with Blastoids / TOs in Separate Steps

[0285] Materials:

[0286] • Endometrial Assembloids (EMAs)

[0287] • Blastoids

[0288] • 96-well plates (low-attachment)

[0289] • Advanced DMEM / F12 media with growth factors

[0290] • Fine-tipped pipettes

[0291] 1 . Plate Preparation

[0292] • Use Coming U-bottom well plates

[0293] • Culture conditions: o Normoxia o Shaker at 30 rpm

[0294] • Each plate: 96 structures.

[0295] 2. Media Preparation mIVCl media: prepare ^30 rnL (minimum 20 mL per plate; extra needed for washing EMAs / blastoids and resuspension).

[0296] Blcistoid Preparation

[0297] Collect the blastoids into a single droplet (-20-30 pL) of DMEM / F12 media with growth factors on a sterile dish. Using a stereomicroscope or inverted microscope, visually identify and hand-pickindividual blastoids per EMA. Using a fine-tipped pipette, carefully aspirate blastoids and transfer them into each well of the 96-well plate containing an EMA.

[0298] 3. Plate Labeling & Group Assignment

[0299] • Label each U-well plate with: o Patient number (e.g R#) o Date of Experiment o Initials of scientist plating the cells o Experimental conditions

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[0301] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0302] 4. EMA / Blastoid Preparation & Transfer

[0303] 1. Prepare mIVCl

[0304] 2. EMA preparation: o Collect EMAs in a 15 rnL Falcon tube. o Let them sink (~2 min), remove media. o Wash: add 1 mL room temp PBS, let sink, remove PBS. o Resuspend in 1 mL IVC1.

[0305] 3. Plating: o Add 200 pL mIVCl per well (only in wells to be used). o Transfer EMAs to 96-well plate with minimal media carryover. o Wash blastoids in mlV C 1. o Transfer blastoids to the same wells (with EMAs), minimal media carryover. o Place plate in bottom incubator under normoxia, on 30 rpm shaker.

[0306] 5. Media Recipes

[0307] Modified IVC1 (mIVCl)

[0308] • Base: Advanced DMEM / F12

[0309] • Supplements: o 20% FBS o 2 mM GlutaMAX o 1% Penicillin / Streptomycin o 1% ITS-X o 0.22% Sodium lactate o 8 nM P-estradiol o Progesterone (200 ng / mL) o 25 pM N-acetyl-L-cysteine

[0310] For 40 mL mIVCl base:

[0311] • Advanced DMEM / F12: 31 mL

[0312] • FBS (20%): 8 mL

[0313] • -estradiol (stock 10 pM): 0.8 pL / mL 32 pL total

[0314] • Pen / Strep (1%): 400 pL

[0315] • ITS-X (1%): 400 pL

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[0317] 56504398 2 Atorney Docket No. 047162-7518WOl(02693)

[0318] • Sodium lactate (0.22%); 88 pL

[0319] • Progesterone (stock 1 mg / mL): 8 pL

[0320] • NAC (stock 50 mM): 20 pL

[0321] • GlutaMAX: 10 pL / mL

[0322] With added factors:

[0323] • EGF (50 ng / mL): 0.5 pL / mL

[0324] • FGF2 (50 ng / mL): 0.25 pL / mL

[0325] • Heparin (1 pg / mL): 0.1 pL / mL

[0326] • Y27632 (10 pM): 0.5 pL / mL mIVC2

[0327] For 40 mL mIVC2 base:

[0328] • Advanced DMEM / F12: 28 mL

[0329] • KSR (30%): 12 mL

[0330] • 0-estradiol (stock 10 pM): 32 pL

[0331] • Pen / Strep: 400 pL

[0332] • ITS-X: 400 pL

[0333] • Sodium lactate: 88 pL

[0334] • Progesterone (stock 1 mg / mL): 8 pL

[0335] • NAC (stock 50 mM): 20 pL

[0336] • GlutaMAX: 10 pL / mL

[0337] With added factors:

[0338] • EGF (50 ng / mL; stock 100 pg / mL): 0.5 pL / mL

[0339] • FGF2 (50 ng / mL; stock 200 pg / mL): 0.25 pL / mL

[0340] • IGF-1 (50 ng / mL): 0.5 pL / mL

[0341] • Heparin (1 pg / mL; Wako): 0.1 pL / mL

[0342] 6. Culture Timeline

[0343] • Day 1 (DI): Start co-culture with 200 pL mIVCL

[0344] • Day 2 (D2, 24h): Leave untouched.

[0345] • Day 3 (D3, 48h):

[0346] 23

[0347] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) o Collect structures pre-IVC2 (optional). o Remove 100 pL mIVCl, replace with 100 pL mIVC2.

[0348] • Day 4 (D4, 72h): o Remove 100 pL mIVCl, replace with 100 pL mIVC2.

[0349] • Day 5 (D5, 96h): Collect all structures.

[0350] Co-Culture Incubation

[0351] After placing the blastoids / TOs into each well, incubate the 96-well plate at 37°C in 5% CO2. Monitor the interaction between EMAs and blastoids / TOs under a microscope. The co-culture can be maintained for up to 5 days for downstream analysis, such as fixation, staining, or functional assays.

[0352] Example 5: Characterization of structural patterns of human embryo implantation with endometriosis

[0353] Without wishing to be bound by theory, it is hypothesized that the molecular deregulation affecting endometrial receptivity disrupts the intricate communication between the embryo and endometrium, leading to implantation failure and consequent infertility'.

[0354] To address this, the present study uses the 3D biomimetic co-culture system to model embryo-uterine developmental dynamics across spatial and temporal scales, and to reveal mechanisms governing the human implantation during endometriosis, a process that cannot be captured either in vivo or faithfully ex vivo. This research elucidates the molecular mechanisms that control human implantation and the ways in which this can go awry in health and disease.

[0355] To begin, the present study utilizes the 3D co-culture system to define the multidimensional 3D features of the implanting blastoid on the EMA with and without endometriosis. Human blastoids are generated from hPSCs, incorporating a trophectoderm- and epiblast-like lineage fluorescent tag (NR2F2-RFP / SOX2-GFP, respectively, for example) for visualization during the multiple stages of implantation. Trophoblast organoids are generated using human trophoblast stem cells (TSCs). EMAs are created with both major cell populations of the endometrium - epithelial and stromal - using primary' patient primary' cells. The EMAs are designed to be flipped outward such that the luminal epithelium is facing outwards to allow for full contact of the blastoid / TO to recapitulate physiological implantation more accurately. Each experiment involves a minimum of 20 human

[0356] 24

[0357] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) blastoids / TOs, repeated at least three times, and a minimum of five patient samples for each condition, which have already been shown to be feasible for in terms of patient sample number. Preliminary evidence has confirmed the success in producing human blastoids at high-throughput and novel EMAs that are epithelial (apical)-out in gel-free conditions.

[0358] The present study evaluates the three stages of human embryo implantation - apposition, attachment, and invasion- using high-resolution immunofluorescence confocal microscopy, a routine method in my lab. For apposition, the present study assays for the presence of Mucin- 1 and Leptin receptors on the blastoid. The attachment to the endometrial epithelium are visualized through the co-localization of NR2F2-GFP or GAT A3 (trophoblast) with EPCAM (EMA epithelia). The present study examines invasion by staining for markers of trophoblast differentiation and basement membrane and also stains for specific markers of cytotrophoblasts (e.g. TEAD4 and Cytokeratin-7), STBs (e.g. HCG), and EVTs (e.g. HLA-G) as well as basement membrane (e.g. Laminin and Collagen IV). The present study scores apposition, attachment, and invasion efficiencies in each condition.

[0359] Example 6: Define metabolic and transcriptional changes associated with molecular patterns during human embryo implantation with endometriosis

[0360] To unravel the molecular crosstalk between the blastoid / TO and endometrial tissue during implantation and any defects therein, the present study uses an unbiased metabolomics assays and lOx single-cell RNA sequencing (scRNAseq) to identify differential cell composition and transcriptional changes between infertile endometriosis, fertile endometriosis, and healthy control implantoids. Using candidates identified in scRNAseq, the present study then performs functional perturbation experiments to test putative molecular regulators (e.g. metabolic alternations, gene regulation) of failed implantation in endometriosis. The approach herein uncovers new molecular and cellular determinants of infertility in patients with endometriosis. Several patient samples per condition are needed to make meaningful conclusions. The present study further cross-references the results with published transcriptional and structural data.

[0361] Example 7: Statistical Analysis

[0362] The present study uses a One-sample t-test to compare replicates and unpaired t-test with Welch’s correction between controls and experimental groups (e.g. healty and endometriosis patient EMOs). To achieve statistical power to detect significant differences with <5% probability of Type I error, the present study uses >35 blastoids / TOs per

[0363] 25

[0364] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) experiment. This is easily achievable, as at least 100 blastoids / TOs are produced in each experiment. To ensure rigor, experiments are: (i) validated with appropriate statistical analyses, (ii) are reproduced in >3 independent experiments (iii) include both negative and positive controls, and (iv) validation of all reagents and equipment (e.g. antibodies). The present study accounts for biological covariates including sex-specific features through the use of both female and male hPSC lines across >2 genetic backgrounds of each.

[0365] Enumerated embodiments

[0366] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0367] Embodiment 1 : A system, comprising: an endometrial assembloid, comprising a plurality of endometrial stromal cells enclosed in a layer of endometrial epithelial cells; and a blastoid mimicking a blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, and / or an embryo model capable of interacting with a maternal tissue or the endometrial assembloid, optionally wherein the endometrial assembloid further comprises endothelial cells and forms a vascular structure, optionally wherein the embryo model capable of interacting with a maternal tissue or the endometrial assembloid comprises a human extra-embryoid (hEE) representing a postimplantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a peri-implantation embry o model.

[0368] Embodiment 2: The system of Embodiment 1. wherein, in the endometrial assembloid, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0369] Embodiment 3: The system of any one of Embodiments 1-2, wherein the endometrial assembloid is prepared from human endometrial cells.

[0370] Embodiment 4: The system of Embodiment 3, wherein at least one of the following applies:

[0371] (a) the endometrial assembloid is prepared using cells from an endometrial sample of a human subject;

[0372] (b) the endometrial assembloid is prepared using cells from an eutopic

[0373] 26

[0374] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) endometrial sample of a human subject;

[0375] (c) the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject, or

[0376] (d) the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

[0377] Embodiment 5: The system of Embodiment 4, wherein:

[0378] (a) the human subject is suffering from endometriosis;

[0379] (b) the human subject does not suffer from endometriosis;

[0380] (c) it is unknown whether the human subject is suffering from endometriosis;

[0381] (d) the human subject is suffering from infertility; or

[0382] (e) it is uncertain if the human subject is suffering from infertility.

[0383] Embodiment 6: The system of any one of Embodiments 1-5, wherein the blastoid, the trophoblast organoid, or the embryo model is prepared from human pluripotent stem cells (hPSCs), trophoblast stem cells, or induced / transdifferentiated trophoblast stem cells.

[0384] Embodiment 7: The system of Embodiment 6. wherein the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

[0385] Embodiment 8: The system of any one of Embodiments 1-7, wherein the blastoid, the trophoblast organoid, or the embryo model, is prepared from human cells, and the blastoid or the embryo model is incapable of developing into a human organism.

[0386] Embodiment 9: The system of any one of Embodiments 1-8, further comprises one or more culture media for suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model.

[0387] Embodiment 10: The system of any one of Embodiments 1-9, w herein the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model are brought in contact with each other to allow apposition, attachment, and / or invasion of the endometrial assembloid.

[0388] Embodiment 11 : A method of studying or evaluating embryo implantation and subsequent post-implantation development, wherein the method comprises: placing an endometrial assembloid and a blastoid mimicking a human blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, or an embryo model embryo model capable of interacting with a maternal tissue or the endometrial assembloid near each other such that the blastoid, the trophoblast organoid, and / or the embryo model is able to appose, attach to. and / or invade the endometrial assembloid, w herein the endometrial assembloid comprises a plurality of endometrial stromal cells

[0389] 27

[0390] 56504398 2 Atorney Docket No. 047162-7518WOl(02693) and optionally endothelial cells, enclosed in a layer of endometrial epithelial cells, and wherein the blastoid, the trophoblast organoid, and / or the embryo model mimics a blastocyst or a pre- or post-implantation embryo, optionally wherein the embryo model comprises a human extra-embryoid (hEE) representing a post-implantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a periimplantation embryo model.

[0391] Embodiment 12: The method of Embodiment 11, wherein, in the endometrial assembloid, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

[0392] Embodiment 13: The method of any one of Embodiments 11-12, wherein the endometrial assembloid is prepared from human endometrial and vascular cells.

[0393] Embodiment 14: The method of Embodiment 13, wherein at least one of the following applies:

[0394] (a) the endometrial assembloid is prepared using cells from an endometrial sample of a human subject;

[0395] (b) the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject;

[0396] (c) the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject; or

[0397] (d) the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

[0398] Embodiment 15: The method of Embodiment 14, wherein

[0399] (a) the human subject is suffering from endometriosis;

[0400] (b) the human subject does not suffer from endometriosis;

[0401] (c) it is unknown whether the human subject is suffering from endometriosis;

[0402] (d) the human subject is suffering from infertility; or

[0403] (e) it is uncertain if the human subject is suffering from infertility.

[0404] Embodiment 16: The method of any one of Embodiments 1 1-15, wherein the blastoid, the trophoblast organoid, or the embry o model capable of implanting, is prepared from human pluripotent stem cells (hPSCs).

[0405] Embodiment 17: The method of Embodiment 16, wherein the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

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[0408] Embodiment 18: The method of any one of Embodiments 11-17, wherein the blastoid is prepared from human cells, and the blastoid, the trophoblast organoid, or the embryo model is incapable of developing into a human organism.

[0409] Embodiment 19: The method of any one of Embodiments 11-18, further comprising suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model is suspended in one or more culture media.

[0410] Embodiment 20: The method of any one of Embodiments 11-19, which studies or evaluates the apposition, attachment, and / or invasion of the blastoid, the trophoblast organoid, or the embry o model to the endometrial assembloid.

[0411] Embodiment 21 : An endometrial assembloid, comprising: a plurality of endometrial stromal cells; and a layer of endometrial epithelial cells enclosing the plurality of endometrial stromal cells, wherein the endometrial stromal cells, and the endometrial epithelial cells are from an eutopic or ectopic endometrial sample of a human subject. optionally wherein the endometrial assembloid further comprises a plurality of endothelial cells, and forms a vascular structure, and optionally wherein the endothelial cells are from an eutopic or ectopic endometrial sample of a human subject.

[0412] Embodiment 22: The endometrial assembloid of Embodiment 21. wherein a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid,

[0413] Embodiment 23 : The endometrial assembloid of any one of Embodiments 21-22, wherein the plurality of endometrial stromal cells and layer of endometrial epithelial cells, together with the plurality of endothelial cells, forming vascular structures in an inner region of the endometrial assembloid.

[0414] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

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[0416] 56504398 2

Claims

Attorney Docket No. 047162-7518WOl(02693)CLAIMSWhat is claimed is:

1. A system, comprising: an endometrial assembloid, comprising a plurality of endometrial stromal cells enclosed in a layer of endometrial epithelial cells; and a blastoid mimicking a blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, and / or an embryo model capable of interacting with a maternal tissue or the endometrial assembloid, optionally wherein the endometrial assembloid further comprises endothelial cells and forms a vascular structure, optionally wherein the embiyo model capable of interacting with a maternal tissue or the endometrial assembloid comprises a human extra-embryoid (hEE) representing a postimplantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a peri-implantation embryo model.

2. The system of claim 1, wherein, in the endometrial assembloid, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

3. The system of any one of claims 1-2, wherein the endometrial assembloid is prepared from human endometrial cells.

4. The system of claim 3, wherein at least one of the following applies:(a) the endometrial assembloid is prepared using cells from an endometrial sample of a human subject;(b) the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject;(c) the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject, or(d) the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.3056504398 2Attorney Docket No. 047162-7518WOl(02693)5. The system of claim 4, wherein:(a) the human subject is suffering from endometriosis;(b) the human subject does not suffer from endometriosis;(c) it is unknown whether the human subject is suffering from endometriosis;(d) the human subject is suffering from infertility; or(e) it is uncertain if the human subject is suffering from infertility.

6. The system of any one of claims 1-5, wherein the blastoid, the trophoblast organoid, or the embryo model is prepared from human pluripotent stem cells (hPSCs), trophoblast stem cells, or induced / transdifferentiated trophoblast stem cells.

7. The system of claim 6, wherein the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

8. The system of any one of claims 1-7, wherein the blastoid, the trophoblast organoid, or the embryo model, is prepared from human cells, and the blastoid or the embryo model is incapable of developing into a human organism.

9. The system of any one of claims 1-8, further comprises one or more culture media for suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model.

10. The system of any one of claims 1-9, wherein the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model are brought in contact with each other to allow apposition, attachment, and / or invasion of the endometrial assembloid.

11. A method of studying or evaluating embryo implantation and subsequent postimplantation development, wherein the method comprises: placing an endometrial assembloid and a blastoid mimicking a human blastocyst, a trophoblast organoid mimicking a lineage of the human blastocyst, or an embryo model embry o model capable of interacting with a maternal tissue or the endometrial assembloid near each other such that the blastoid, the trophoblast organoid, and / or the embryo model is able to appose, attach to, and / or invade the endometrial assembloid,3156504398 2Attorney Docket No. 047162-7518WOl(02693) wherein the endometrial assembloid comprises a plurality of endometrial stromal cells and optionally endothelial cells, enclosed in a layer of endometrial epithelial cells, and wherein the blastoid, the trophoblast organoid, and / or the embryo model mimics a blastocyst or a pre- or post-implantation embryo, optionally wherein the embryo model comprises a human extra-embry oid (hEE) representing a post-implantation stage of an embryo, a mouse blastocyst for cross-species assessments, or a human suspension-based epiblast-like spheroid (hSES) representing a periimplantation embryo model.

12. The method of claim 11, wherein, in the endometrial assembloid, a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid.

13. The method of any one of claims 11-12, wherein the endometrial assembloid is prepared from human endometrial and vascular cells.

14. The method of claim 13, wherein at least one of the following applies:(a) the endometrial assembloid is prepared using cells from an endometrial sample of a human subject;(b) the endometrial assembloid is prepared using cells from an eutopic endometrial sample of a human subject;(c) the endometrial assembloid is prepared using cells from an ectopic endometriosis lesion sample of a human subject; or(d) the endometrial assembloid is prepared using endometrial cells from immortalized cell lines.

15. The method of claim 14, wherein(a) the human subject is suffering from endometriosis;(b) the human subject does not suffer from endometriosis;(c) it is unknown whether the human subject is suffering from endometriosis;(d) the human subject is suffering from infertility; or(e) it is uncertain if the human subject is suffering from infertility.

16. The method of any one of claims 11-15, wherein the blastoid, the trophoblast3256504398 2Attorney Docket No. 047162-7518WOl(02693) organoid, or the embryo model capable of implanting, is prepared from human pluripotent stem cells (hPSCs).

17. The method of claim 16, wherein the hPSCs comprises induced human pluripotent stem cells (hiPSCs).

18. The method of any one of claims 11-17, wherein the blastoid is prepared from human cells, and the blastoid, the trophoblast organoid, or the embryo model is incapable of developing into a human organism.

19. The method of any one of claims 11-18, further comprising suspending the endometrial assembloid and the blastoid, the trophoblast organoid, or the embryo model is suspended in one or more culture media.

20. The method of any one of claims 11-19, which studies or evaluates the apposition, attachment, and / or invasion of the blastoid, the trophoblast organoid, or the embryo model to the endometrial assembloid.

21. An endometrial assembloid, comprising: a plurality of endometrial stromal cells; and a layer of endometrial epithelial cells enclosing the plurality7of endometrial stromal cells, wherein the endometrial stromal cells, and the endometrial epithelial cells are from an eutopic or ectopic endometrial sample of a human subject. optionally wherein the endometrial assembloid further comprises a plurality of endothelial cells, and forms a vascular structure, and optionally wherein the endothelial cells are from an eutopic or ectopic endometrial sample of a human subject.

22. The endometrial assembloid of claim 21, wherein a basal side of the endometrial epithelial cells faces the endometrial stromal cells, and an apical side of the endometrial epithelial cells faces the outside of the endometrial assembloid,3356504398 2Attorney Docket No. 047162-7518WOl(02693)23. The endometrial assembloid of any one of claims 21-22, wherein the plurality of endometrial stromal cells and layer of endometrial epithelial cells, together with the plurality of endothelial cells, forming vascular structures in an inner region of the endometrial assembloid.3456504398 2

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