Current strategies using 3D organoids to establish in vitro maternal-embryonic interaction.

Early mammalian development is a complex process that involves the interaction between maternal and embryonic tissue, trophoblast invasion, and placental development. Our understanding of these concepts, in addition to successful implantation, patterning, nutrition, and human pregnancy (including decidualization of the endometrium and formation of the placenta), remains limited and challenging owing to the lack of good in vivo and animal models. Embryo implantation is a complex and time-sensitive coordinated process that plays an important role in achieving successful pregnancy outcomes [ 1 ]. In vitro culture systems have provided insights into the fundamental nature of mammalian development in vivo . However, two-dimensional (2D) do not exhibit the characteristic feature of self-organization during development [ 2 ]. Moreover, 2D cultures of primary endometrial cells are associated with a reduction in their biological activity and may possess potential genetic aberrations associated with prolonged culture after several passages, which is a major disadvantage [ 3 ]. In addition, 2D cell culturing cannot be used to investigate complex environments related to three-dimensional (3D) architecture owing to the absence of cell-to-matrix interactions. The endometrial cell-extracellular matrix (ECM) cannot be used to evaluate the in vivo crosstalk between endometrial stromal and epithelial cells [ 4 ]. These drawbacks of 2D culture and other critical issues have led to the use of 3D culture systems and organoids, which are more effective models, for the investigation of in vivo development and ECM biology [ 5 ]. Organoids represent 3D in vitro cultures that spatially organize into structures mirroring the functional characteristics of organs in vivo , modeling human organs and diseases within a dish, particularly the reproductive diseases, and offering significant potential for translational applications, including regenerative medicine, drug discovery, and precision medicine [ 6 ]. Organoid cultures are derived from tissue samples of human female reproductive tract (FRT), including the endometrium, fallopian tube, and cervix, in addition to trophoblast cells of the placenta from normal and pathological samples [ 7 ]. Different regions of the FRT have been modeled using 3D organoid technology models to determine the normal biology and pathology of FRT [ 8 ]. This review summarizes state-of-the-art organoid technology to provide insights into embryo and maternal environment communication and pregnancy outcomes, as well as the effect of using different cell sources for organoid engineering. Furthermore, this review emphasizes the importance of understanding the integration of organoid systems with advanced technology.

The review presented a comprehensive overview of the current strategies utilizing 3D organoids to establish in vitro maternal-embryonic interaction. Key findings related to the establishment of maternal-embryonic interaction, including 3D organoid culture system, endometrial organoids, menstrual blood flow organoids, placental trophoblast organoids, and in vitro generated embryo modes were discussed in detail. The review identified gaps in knowledge, and proposed future directions for research in this field. Considerations for optimizing 3D organoid models and introduction of microfluidics to better recapitulate in vivo maternal-embryonic interactions and their potential applications in reproductive medicine were highlighted. Pre-, peri-, and post-implantation human organoids have made it possible to research biological processes in vitro [ 9 ]. 3D organoids are self-organizing structures derived from primary cells or induced pluripotent stem cells (iPSCs) that recapitulate the architecture and functionality of native tissues. The unique cellular composition, 3D structure, and functional properties of organoids facilitate better recreation of the complex maternal-embryonic environment, thereby facilitating the study of various aspects of implantation and early placental development. Organoids have been generated using various approaches to establish maternal-embryonic interaction models [ 10 ]. 3D organoids offer several advantages over traditional 2D cell cultures. For instance, 3D organoids facilitate the creation of a more physiologically relevant environment for studying organ development and drug testing [ 11 ]. The ability to generate placenta-like organoids has enhanced our understanding of early placental development by enabling the investigation of key processes, such as trophoblast differentiation, syncytiotrophoblast (SCT) formation, and placental hormone production. The development of placenta-like organoids has also facilitated the study of maternal-fetal nutrient exchange, immune interactions, and the impact of maternal factors on placental function [ 12 ]. The endometrium, the inner lining of the uterus, is a remarkably dynamic mucosal tissue with regenerative capabilities. Functioning as a crucial component in achieving successful pregnancy outcomes, the uterine endometrium creates an optimal environment for the embryo to achieve key developmental milestones, including morphological changes and tissue differentiation. It offers nutritional support before placental development and provides essential biophysical support for successful implantation [ 13 ]. Organoids have been employed to explore the cellular and molecular mechanisms within the non-pregnant endometrium, as well as during early pregnancy stages [ 14 ]. Recent research has established organoid models derived from both mice and humans. These organoid structures, created by embedding dissociated endometrial tissue in Matrigel under WNT-activating conditions, demonstrate a remarkable ability for long-term expansion. Additionally, these structures can replicate the molecular and histological characteristics of the tissue epithelium [ 15 ]. The utilization of synthetic ECM has proven supportive for human postnatal organoid cultures sourced from diverse donors, encompassing organs such as the intestine and endometrium. This suggests its broad applicability for various epithelial organoid cultures [ 16 ]. Notably, endometrial epithelial organoid cultures, derived from human endometrial biopsies and subjected to estrogen (E2) treatment for 2 days or a combination of E2 and medroxyprogesterone acetate (MPA) for 6 days, effectively capture the effects of hormone treatment on gene expression. These cultures faithfully replicate changes observed in both proliferative and secretory phase endometrium [ 17 ]. A receptive endometrial organoid that mimicked in vivo implantation-window endometrial features was developed in another study. The endometrial organoids showcased notable features, including responsiveness to hormones, secretory functions, and specific characteristics associated with the implantation window. These characteristics encompass decidualization, ECM remodeling, proposal formation, cilia generation, and metabolic activities [ 18 ]. A GdA glycosylation pattern significantly different from that of healthy organoids has been observed in organoids derived from the eutopic endometrium of women with endometriosis, which supports the hypothesis that organoids may closely recapitulate the molecular and functional characteristics of the cells/tissue from which they origin, as well as endometrium-embryo crosstalk [ 19 ]. Bovine and human endometrium-derived hydrogels have been used to support organoid cultures from healthy and cancerous tissues. This has led to the growth of mouse and human endometrial organoids with properties comparable with those of Matrigel and the use of organoids to provide insights into endometrial biology and associated pathologies [ 20 ]. A new laboratory model of the human endometrium was developed in a previous study by assembling two main cell types present in the tissue into a 3D structure. These assemblies successfully mimicked the activity of genes in endometrial cells during implantation when treated with hormones [ 21 ]. Endometrial organoids have been created using biopsies from women experiencing primary infertility and those with normal fertility. Both fertile and infertile organoids underwent hormone treatment to simulate the receptive phase of the endometrial epithelium. Subsequently, intra-organoid fluid (IOF) was collected to compare apical protein secretion profiles and trophoblast cell adhesion function. Results revealed that infertile organoids displayed a dysregulated response to estrogen and progesterone treatments. Proteomic analysis identified 150 dysregulated proteins in the fertile and infertile groups (> 1.5-fold change). Trophoblast progenitor spheroids (blastocyst surrogates) treated with apical secretions from infertile organoids exhibited significantly compromised adhesion to organoid epithelial cell monolayers ( p < 0.0001), suggesting that factors released from the apical region of endometrial epithelium regulate the initial attachment and implantation of the blastocyst [ 22 ]. Decellularized pig endometrium, which retained a tissue-specific extracellular matrix, was utilized to create a hydrogel named EndoECM. Subsequently, three lines of human endometrial organoids were derived using this EndoECM. The organoids were cultured under various conditions, including optimal and suboptimal culture expansion media, with or without EndoECM (0.01 mg/mL) as a soluble additive. The study’s findings underscored the significant role of the bioactive environment in sustaining and promoting the proliferation of human endometrial organoids, thereby enhancing the overall models for human endometrial research [ 23 ]. Endometrial organoids have proven valuable in researching adenomyosis, a condition associated with infertility and miscarriages. These organoids were developed from the endometria of patients with adenomyosis and underwent differentiation into secretory and gestational phases, faithfully reproducing native endometrial-tissue-specific features and disease-specific traits. The adenomyosis-derived organoids emerged as promising in vitro preclinical models for investigating impaired implantation and pregnancy disorders [ 24 ]. Notably, unlike mifepristone, which is known to inhibit blastocyst attachment, levonorgestrel did not hinder the attachment of human embryos to the in vitro endometrial construct. This 3D model serves as a valuable tool for comprehending endometrial receptivity and the intricate communication between the embryo and the endometrium [ 25 ]. Recently, Saadeldin et al. [ 26 ] reported a novel model to co-culture embryos with endometrial organoids. They showed that the number of cells in embryos co-cultured and the trophoblast outgrowths' proportion was significantly increased in comparison to control embryos. Embryos co-cultured with endometrial organoids were able to show embryonic elongation for the first time. The use of the 3D organoid model derived from the endometrium has revealed several important aspects of the native endometrial microenvironment, such as secretions and autocrine/paracrine interactions, which play important roles in facilitating embryo development, implantation and achieving successful pregnancy outcomes. The endometrium and FRT samples used as sources for organoid engineering are obtained from biopsy samples of healthy women and women with pathological conditions. Thus, it is considered an invasive method. Moreover, a biopsy can only be performed by a trained clinician. An alternative technique that derives organoids from menstrual flow has been proposed to address these limitations. Organoids have been developed using the menstrual flow of patients undergoing in vitro fertilization (IVF). These organoids, derived from menstrual flows, have demonstrated comparable responses to sex steroids and early-pregnancy hormones. Furthermore, they exhibit alterations in morphology, receptor expression, and the production of ‘uterine milk’ proteins that closely resemble those observed during the late-secretory phase and early pregnancy. These innovative approaches hold promise for addressing common gynecological conditions like endometriosis, as well as reproductive disorders such as failed implantation after IVF and recurrent miscarriages [ 27 28 ]. The placenta is a vital organ that supports fetal development during pregnancy. Placental trophoblasts, the primary cell type present in the placenta, are characterized by the presence of remarkable heterogeneity and complex interactions with the maternal environment [ 29 ]. Placental trophoblast organoids derived from primary trophoblast cells provide a 3D culture system that closely resembles the complex architecture and cellular composition of the placenta [ 30 ]. Placental trophoblast organoids possess the ability to maintain the heterogeneity and self-renewal capacity of trophoblast subtypes, such as villous cytotrophoblasts (VCTs) and SCTs which is a major advantage [ 31 ]. This has facilitated the study of specific trophoblast populations and their interactions with maternal tissues, thereby providing insights into normal placental development and pregnancy-related complications [ 32 ]. Placental trophoblast organoids have also been used to investigate the molecular and cellular mechanisms underlying trophoblast differentiation, specialization, signaling pathways and development [ 33 ]. Current understanding of human placental development and the impact of environmental chemicals on these processes has been limited due to restricted access to developing tissues and the absence of representative experimental models. To address these challenges, several studies have turned to human trophoblast organoids. These organoids exhibit a cellular composition and behavior closely resembling the immature human placenta, offering a valuable tool [ 34 ]. Trophoblast organoids exhibit genetic stability and mirror the structural, morphological, functional, and metabolic characteristics observed in in vivo villi. Under specific extravillous trophoblasts (EVTs) differentiation conditions, these organoids can undergo differentiation, giving rise to EVTs that express the EVT marker HLA-G. The resultant HLA-G+ EVT cells demonstrate both migratory and invasive capabilities when assessed in Matrigel. Crucially, trophoblast organoids encompass all functional trophoblast cell types found in human villi. Moreover, they display secretory functions akin to SCT, releasing various placental hormones and peptides—such as human chorionic gonadotropin, growth differentiation factor 15 (GDF15), and pappalysin 1 (PAPPA)—under the influence of specific SCT differentiation medium stimulation [ 35 36 ]. Co-culturing trophoblast organoids with an in vitro 3D culture model of the endometrium offers the potential to recreate trophoblast-endometrial interactions during early placentation within a more physiological environment. However, additional optimization is required for a thorough understanding of epithelial biology and studies on trophoblast-endometrium interactions. This may include refining aspects such as reversing the polarity of trophoblast organoids—from basal-out/apical-in to apical-in/basal-out—to enhance the fidelity and relevance of the model [ 36 37 ]. These organoids exhibit a villous morphology and HLA-G+ cells during proliferation. Induction of epithelial-mesenchymal transition (EMT) in organoids, along with the switch of differentiation medium, leads to the generation of HLA-G+ EVT that are capable of invading Matrigel in 3D. This approach allows the study of signals inducing VCT differentiation into EVT. Given that various pregnancy disorders exhibit characteristics of impaired EVT differentiation and invasion into the maternal decidua, there is now an opportunity to explore the interactions of EVT with components of the decidual microenvironment. This includes investigations into the role of glandular products that mediate histotrophic nutrition and the involvement of resident uterine natural killer (NK) cells. Such studies can provide insights into the complex dynamics within the uterine environment during pregnancy and shed light on potential factors contributing to pregnancy disorders [ 38 ]. The phenomenon of interest involves the expression of polymorphic HLA-C molecules by EVT both in vivo and those generated from trophoblast organoids. These HLA-C molecules are recognized by killer immunoglobulin receptors (KIRs) on maternal decidual NK cells. Specific combinations of maternal KIR and fetal HLA-C genotypes have been identified as influential factors affecting reproductive success and the risk of pregnancy disorders, such as preeclampsia [ 39 ]. The establishment of co-culture systems and the creation of a biobank featuring patient-derived trophoblast organoids will provide valuable resources for further investigating the intricate interactions between maternal and fetal cells, particularly those impacting EVT during pregnancy. Recent studies have indicated the potential of placental trophoblast organoids as a platform for modeling and studying pregnancy-related disorders, such as pre-eclampsia and intrauterine growth restriction [ 32 ]. Researchers can investigate the dynamics of immune cell infiltration, cytokine production, and immune tolerance at the maternal-fetal interface via the co-culture of trophoblast organoids with immune cells. Zika virus (ZIKV) has been shown to target human trophoblast stem (TS) cells and prevent syncytialization in placental trophoblast organoids [ 40 ]. Placental trophoblast organoids have several advantages: 1) Recapitulation of in vivo -like structure and functions: Placental trophoblast organoids possess a striking resemblance to the native placenta, and exhibit well-defined layers of trophoblasts, syncytialization, and distinct trophoblast subtypes. Furthermore, they also exhibit key functions, such as nutrient and gas exchange, hormone production, and immune modulation. 2) Investigation of early placental development: Placental trophoblast organoids derived from early embryonic tissue have enabled the study of early placental development and morphogenesis, which is a challenging task to perform in vivo . 3) Modeling pregnancy complications: Placental trophoblast organoids have been used to model pregnancy-related disorders, such as pre-eclampsia, intrauterine growth restriction, and gestational diabetes. The molecular and cellular mechanisms underlying these processes can be studied by exposing organoids to specific stimuli. 4) Drug screening and personalized medicine: Placental trophoblast organoids have been used for drug screening and the development of personalized medicines for pregnancy-related disorders. They have facilitated the assessment of drug toxicity, penetration across the placental barrier, and the identification of potential therapeutic targets. Nevertheless, placental trophoblast organoids also have some limitations, such as the requirement for the optimization of culture conditions, standardization of protocols, and incorporation of additional maternal and fetal cell types into organoids [ 12 ]. More sophisticated culture systems would aim to improve the physiological relevance of placental trophoblast organoids by including components of the maternal immune system, endothelial cells, and other supporting cell types to mimic the dynamic and complex intrauterine environment [ 41 ]. The fields of developmental biology and reproductive medicine have undergone a revolutionary shift with the emergence of stem cell-derived blastoids and in vitro embryo models in the last few years [ 42 ]. Early embryonic development is an intricate process involving highly coordinated cellular events and complex molecular interactions. Dealing with human embryos is unlike the mouse embryos, it is associated with limited accessibility and ethical concerns. Thus, the development of alternative models, such as stem cell-derived blastoids and in vitro -generated embryo models has been proposed [ 43 ]. Stem cell-derived blastoids, also known as synthetic embryos are self-organized structures generated from pluripotent stem cells (PSCs) that mimic early embryonic development and display similar spatial organization and cell-type distributions. Blastoids resembling specific stages of embryonic development can be generated by manipulating key signaling pathways and culture conditions [ 42 ]. Investigating the mechanisms underlying lineage specification and tissue morphogenesis throughout early embryogenesis is made possible by this unique platform. In vitro embryo models have become extremely useful tools for revealing the early stages of embryonic development. These models are generated via the assembly of embryonic cells derived from different lineages, thereby providing a more accurate representation of the in vivo environment. Complex structures resembling various organ systems have been created by combining cells from distinct embryonic germ layers [ 44 ]. Stem cell-derived blastoids and in vitro -generated embryo models can be used for disease modeling, which is a major advantage. These models can recapitulate disease phenotypes and offer insights into disease mechanisms by introducing genetic mutations or using patient-specific cells [ 42 ]. Furthermore, their amenability to high-throughput screening has facilitated drug discovery and evaluation, thereby propelling the development of novel therapeutics [ 45 ]. Although in vitro -generated embryo models and blastoids formed from stem cells have numerous advantages, the usage of embryo-like structures in the laboratory is still fraught with ethical issues. Finding a balance between ethical norms and scientific advancements is still an obstacle to overcome [ 46 ]. Future studies must aim to address these concerns while exploring the potential applications of these models in regenerative medicine, understanding birth defects, and unraveling the mystery of early human development [ 47 ]. The initial progress in this field was made possible by the generation of structures resembling blastocysts through the correct proportions of murine embryonic stem cells (mESCs) and murine trophoblast stem cells (mTSCs) in microwell plates ( Fig. 1 ) [ 48 ]. These stem cells have morphogenesis strikingly comparable to that of normal embryos and self-aggregate to build structures. Analogues of epiblast (EPI), trophoblast, and primitive endoderm (PE) are produced by blastoids, which are cells that resemble the latter. In the absence of extraembryonic stem cells, blastoid-like structures can be generated using the more contentious “extended potential” mESCs or 2-cell-like stem cells [ 49 ]. The extent to which these blastoids resemble in vivo blastocysts and their divergence from blastoids created with lineage-true stem cells remains uncertain. However, a recent evaluation of these experiments revealed that the outer cells of blastoids generated from extended potential mESCs do not replicate the essential characteristics of authentic trophectoderm (TE) [ 50 ]. Therefore, even while these cells arrange themselves in a position that makes sense for them, this does not always imply that cell lineage-specific expression patterns will be found [ 50 ]. By using murine extraembryonic endoderm cells (mXEN), which are stem cell types that resemble the PE, these artificial mouse embryo cultures have advanced towards the formation of embryo-like structures that go through specific critical phases of gastrulation. [ 51 ]. By combining reprogramming techniques, it is possible to create embryos that resemble those in the post-gastrulation stage from a single naïve mESC that was induced to differentiate into trophoblasts employing Cdx2 and a PE via Gata4 [ 52 ]. The self-assembly of PE-like cells induced by Gata4, trophoblast-like cells induced by Cdx2, and naïve mECS has shown results comparable to those of bona fide stem cells. However, using genuine mTSCs for co-aggregation greatly increased the efficiency rate of reaching later developmental stages [ 53 ]. Whether artificial embryos and in vivo embryos are different visibly is still up for debate. The self-organizing capacity of stem and progenitor cells, however, is demonstrated by the ability of artificial embryos to go through organogenesis [ 54 ]. Here, cell polarity, along with migratory and adhesive properties, activates a deterministic program that follows the principles of development. Since human development occurs later than firm lineage commitment, blastoids have also been effectively generated from naïve hESCs by adding MEK, TGFB, and HIPPO signaling inhibitors to promote trophoblast differentiation [ 55 ]. The potential application of stem cells to generate an assembly lacking in one or more cellular components is a potent outcome of current technological advancements. Early blastoid formation examples demonstrated that Klf6 is a crucial response gene and that TE development is guided by a NODAL signal coming from the EPI [ 56 ]. These cell combinations were less capable of forming fluid-filled blastoid structures when mTSCs deficient in Klf6 were combined with wild-type mESCs [ 57 ]. The emergence of CRISPR technology allows for the targeted removal of nearly any gene or genomic feature from stem cells, showcasing the profound conceptual impact of this approach [ 58 ]. This advancement facilitates the expedited exploration of cell-lineage relationships and inductive processes involving the EPI, PE, and trophoblast tissues—complexities challenging to replicate in conventional conditional mouse mutant studies. A major obstacle to in vitro developmental progression is the absence of actual placenta and blood flow. Only up to E8.5 may mice be developed in vitro ; before then, yolk sac-mediated feeding is adequate. At E10.5, the placenta’s functioning becomes crucial. Placenta formation requires three distinct cell lineages: trophoblasts, extraembryonic mesoderm (responsible for the fetal placental vasculature and the mesenchymal villous core in humans), and decidua derived from the maternal side. The close and functional interaction of these lineages is essential for proper placental function. Although the reconstruction of the placenta as a fully functioning organ is currently beyond reach, the study of intricate interactions among these entities has become more feasible with the aid of cellular models [ 59 ]. There are two different pluripotent states for human and mouse PSCs: primed and naïve. Primed PSCs are comparable to peri-gastrulation EPI, while naïve PSCs match pre-implantation EPI [ 60 ]. As our comprehension of the self-organizing features of PSCs has advanced, the procedures for three-dimensional cultures of PSCs to form multicellular structures, mimicking embryo, and organ development, have significantly improved. The development of sophisticated bioengineering tools has allowed dynamic control of chemical and physical conditions in 3D cultures. Drawing on the enhanced understanding of early mammalian development, PSC characteristics, and 3D cultures, both naïve and primed pluripotent PSCs have been employed to create mammalian embryo models that replicate the pre- and post-implantation phases of mammalian development, respectively [ 61 ]. Structures resembling peri-implantation mouse embryos have been successfully developed and continue to progress into the early stages of organogenesis. This achievement is attributed to a combination of assembled-like structures and ex-utero embryo culture systems [ 62 63 ]. In both studies, mPSCs were aggregated with PE-like cells, formed from mPSCs through transient overexpression of Gata4, and TE-like cells derived from mPSCs via transient overexpression of Cdx2. Surprisingly, the mixed cells demonstrated self-organization, forming cylindrical structures resembling eggs. Over 8 days in ex-utero embryo culture systems (also known as rotator-type bottle culture systems), these assemblies developed primitive organs such as the neural tube, primitive gut, beating heart, and bilateral and paired somites. Histological and transcriptome analyses confirmed that these post-gastrulation mouse embryo-like formations closely resembled 8.5-day in vivo mouse embryos. Despite their limited efficiency, these structures exhibiting post-gastrulation characteristics show substantial, if not complete, potential for bone fiber development. Human blastoids can be generated through the induction of self-organization in naïve human pluripotent stem cells (hPSCs) [ 64 65 ]. These human blastoids have been employed in the development of implantation models involving endometrial cell cultures, to mimic post-implantation human development [ 31 ]. Post-implantation developmental models associated with the EPI lineage have been established using primed hPSCs. Using a bioengineered microfluidic device and primed hPSCs, an embryonic-like structure resembling a bipolar-patterned amnion-EPI sac has been developed before gastrulation [ 64 ]. As this amnion-EPI-like sac continues to take shape, clear indications of gastrulation initiation and primordial germ cells (PGCs) specification along their lineage become apparent. Primed hPSCs exposed to exogenous Wnt signals give rise to a distinct human embryo model known as “gastruloid.” Throughout post-gastrulation development and early organogenesis, human gastruloids undergo symmetry-breaking and morphogenetic events mirroring specific developmental characteristics [ 66 ]. The progress made in mouse embryo models suggests the potential development of stem cell-derived human embryo models soon. While a patterned amnion-EPI-like sac constantly develops, it is clear that gastrulation has begun and that PGCs are being specified along their lineage [ 67 ]. It is possible that stem cell-derived human embryo models will soon follow suit, based on the work on mouse embryo models. Efforts to extend the culture and development period of natural monkey embryos or embryo models beyond the gastrulation stage are continually advancing. Techniques for cultivating natural primate embryos are being refined with the specific goal of studying human and non-human primate (NHP) embryos during in vivo post-implantation stages [ 68 69 70 ]. Additionally, the creation of monkey (NHP) blastoids has been explored, offering potential applications in implantation studies to support ongoing development within a surrogate uterus [ 71 ]. Initial cells and techniques utilized to create human blastoids have a significant impact on the model’s accuracy [ 64 72 ]. Precocious differentiation into cells outside of the blastocyst stage can occur from suboptimal conditions or abnormalities during the creation of human blastoids [ 72 ]. It is still challenging to create hypoblast and TE stem cells that are authentically human to use in human embryo models. Furthermore, it is unacceptable to conduct implantation studies using human embryo models with a uterine surrogate. Instead of simulating post-implantation primate growth, monkey blastoids may be a more morally acceptable option that can offer helpful insights for advancing human blastoid techniques. Moreover, models of mammalian embryogenesis offer crucial experimental tools for understanding the cellular and molecular processes that underpin mammalian development. This new area of study will help reduce teratogenesis, birth abnormalities, and pregnancy loss as well as improve assisted human reproduction [ 73 ]. In general, the blastoids’ diameter was similar to that of E3.5 blastocysts found in pregnant women. When blastoids are introduced into the uterus of a female pseudopregnant, they implant, as is the case with many foreign bodies—such as BioRad AffyGel blue beads—and induce a decidual tissue reaction [ 74 ]. This does not show that the TE-like layer produced from TS cells can trigger a physiological implantation response. The proliferation of blastoids is more extensive than that of trophoblast vesicles [ 75 ], demonstrating the interaction of the trophoblast and EPI components in both compartments to mediate proliferation and self-renewal. Since blastoids lack a fundamental endoderm layer from their initial manifestation, they are not able to undergo further development. Nevertheless, by filtering the media conditions, a PE population enclosing a cavitated EPI epithelium was obtained [ 76 ] and it was via this means that the third lineage was introduced into the blastoids. Following experiments, the whole repertoire of three blastocyst lineages in the proper arrangement has been successfully created using mouse expanded potential stem cells. In terms of morphology and cell lineage allocation, extended pluripotent stem (EPS)-derived blastoids are similar to blastocysts. Upon transfer, some EPS-derived blastoids underwent implantation in the uterus and triggered decidualization, resulting in the creation of disorganized tissues in utero. EPS-derived blastoids comprise all three mouse blastocyst cell lineages, and they exhibit transcriptional similarities with blastocysts recovered from pregnant females, according to single-cell and bulk RNA sequencing data. However, no blastoid investigation has thoroughly examined the characteristics of cells that represent the PE/hypoblast lineage. Furthermore, it is not evident if PE cells polarize and develop an epithelium on the basement membrane or inner cell mass surface. In blastoids, one is located between the PE and the EPI, and the other one divides the polar TE from the EPI. For proper embryonic development, these basement membranes are necessary. At these stages, mouse mutants deficient in the constituents of these basement membranes are fatal to the embryo. Therefore, a thorough investigation into peri-implantation mouse embryos will assist in exposing the flaws in artificial systems [ 77 ]. EVs are involved in extensive communication between various organs and tissues, as well as between various cells within tissues alongside interactions, during oocyte maturation, fertilization, and embryo-mother crosstalk. In the organoids model, EVs have a crucial role in maintaining intercellular communication [ 78 ]. EVs are small nano-size lipid bilayer membrane-bound vesicles that contain genetic material, proteins, and lipids. EVs, which can be released by various cell types in organoid models [ 79 ], facilitate the transfer of cargo, including RNA, DNA, and proteins, between donor and recipient cells in an organoid model [ 80 ]. Within the organoids model, EV-mediated transfers play a role in controlling cellular processes such as proliferation, differentiation, and apoptosis [ 81 ]. EVs derived from various cell types in the organoid model can possess distinct cargo compositions, which highlight their potential role in specific cellular functions and tissue development [ 82 ]. The interplay between EVs and organoid models provides valuable insights into the intricate processes underlying cellular communication and tissue development that will transform basic research and clinical applications [ 79 83 ]. Saadeldin et al. [ 83 ] reported that embryos produced in vitro could secrete EVs as a possible tool to communicate within their microenvironment. It is possible to examine EVs from particular cell and/or tissue types more successfully and sustainably. Research has been done on the biogenesis of organoid-derived EV interactions and their possible therapeutic applications [ 84 ]. Moreover, biochemical variations between the secretions from the basal and apical cellular surfaces of endometrial organoids were found [ 85 ]. Patients with adenomyosis have been shown to secrete EVs carrying miRNAs associated with failed embryo implantation, pregnancy complications, and progression of adenomyosis [ 86 ]. Advanced bioengineering tools can be used in direct strategies for creating high-fidelity, next-generation embryoids and organoids to mimic the microenvironments at the tissue and organ level [ 87 ]. Three-dimensional bioprinting techniques have been used to precisely arrange cells and biomaterials spatially, resulting in well-defined tissue architectures that closely resemble native organs [ 88 ]. Biomaterial-based scaffolds have been used to provide mechanical support to the developing organoids and aid in their maturation and vascularization [ 89 ]. The repeatability of organoids can be enhanced using micropatterning to limit the number of cells [ 90 ]. With an emphasis on structure and function, the integration of bioprinting with organoids can open the door for the generation of actual organs [ 91 ]. Organoid systems have successfully integrated microfluidic technology, allowing for improved tissue functionality and fine-grained control of the cellular microenvironment. Organoids can be perfused with nutrition, oxygen, and growth stimulants via microfluidic technology, which mimics an in vivo natural vascular network [ 92 ]. Moreover, they also enable the dynamic monitoring of cellular behavior, leading to a better understanding of organoid physiology and responses to external stimuli [ 93 ]. Thus, microfluidic models are valuable experimental systems that can be used to expand our understanding of reproduction, embryonic development, and related fertility diseases. This model could help with high-throughput medication and toxicity screens to avoid miscarriage and birth defects as well as the logical design of hPSC differentiation processes for disease models and cell treatment [ 66 94 ].

A systematic search of electronic databases including PubMed, Web of Science, and Google Scholar was conducted to identify relevant studies that were recently published. Keywords including “3D organoids,” “maternal-embryonic interaction,” “ in vitro models,” and related terms were used to ensure a comprehensive search strategy. Studies included in this review met the following criteria: (1) investigated the use of 3D organoids to study maternal-embryonic interaction, (2) utilized in vitro models, (3) published in peer-reviewed journals, and (4) written in English.

3D organoids are an important tool for exploring maternal-embryonic interactions and developing implantation models ( Fig. 2 ). Three-dimensional organoids have been used to mimic the complex interactions that occur between the maternal tissues and the developing embryo during implantation ( Fig. 3 ). A common strategy involves culturing maternal endometrial and embryonic organoids separately and allowing them to mature in a controlled environment. The two types of organoids are subsequently combined to create a co-culture system that mimics the interface between the maternal endometrium and embryo. This coculture system has enabled the study of the biological processes, such as cell adhesion, communication, and tissue remodeling, that occur during implantation. Various factors, such as hormone levels, growth factors, and gene expression, can be manipulated to evaluate their effects on maternal-embryonic interactions and implantation. Furthermore, the use of 3D organoids has aided in overcoming some limitations of traditional cell culture models. The 3D structure of organoids better reflects the complexity of real tissues and provides a more accurate representation of cell-cell interactions and tissue architecture ( Table 1 ) [ 18 19 21 22 24 25 26 27 28 30 95 96 97 98 99 100 ]. 3D, three-dimensional; EV, extracellular vesicle. SCT, syncytiotrophoblast; EVT, extravillous trophoblast; EV, extracellular vesicle. 3D, three-dimensional; WOI, window-of-implantation; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; IVF, in vitro fertilization. It is worth mentioning that research in this field is progressing rapidly, with new strategies emerging continuously. These strategies aim to enhance our understanding of maternal-embryonic interactions and implantation, thereby contributing to advancements in reproductive medicine and maternal health. Nevertheless, several challenges must be addressed when using 3D organoids to establish maternal-embryonic interaction and implantation models, such as the need for the standardization of protocols, improved scalability, and better representation of cellular heterogeneity within organoids. Utilizing organoids along with organ-on-a-chip and microfluidic sensors can yield more dynamic and physiologically relevant models. Overall, much work remains to be done on these in vitro models which will greatly advance our understanding of mammalian development and potentially contribute to the elucidation of the causes of infertility and other pregnancy complications as well as the dialogs between maternal-embryonic interaction.