Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.

The process of implantation is accomplished by the proliferation, differentiation, migration and invasion of the trophoblasts from blastocyst, together with the regulation of the endometrial decidua. To better understand the detailed process of embryo implantation, the incorporation of the blastocyst or blastocyst surrogates with the in vitro 3D endometrial models are widely studied (Table 2 ), and their applications in association with the endometrial models will be discussed. Table 2 Examples for various implantation models in use Schematic illustration Advantages Physiological process modelling Disadvantages Reference Human/mouse blastocyst - Best recapitulate the morphological features of the blastocyst in vivo - Embryo implantation - Ethical issues - Shortage of human blastocysts - Mouse blastocysts have minor difference compared to human Lalitkumar et al . 2007, Qi, et al . 2014, Ruane, et al . 2020 [ 73 , 102 , 103 ] Trophoblast cell line - Easy to acquire - Relatively easy to set up - Allow the 3D modelling of endoglandular invasion by single extravillous trophoblast cells - Endoglandular invasion by trophoblast cells - Immortalised cell line may contain genetic aberration Buck, et al . 2015 [ 77 ] Trophoblast spheroids - 3D structure better mimic the physiological implantation process - Can be incorporated with stromal cells and ECM (Matrigel /Fibrin) for 3D invasion studies - Trophoblast invasion and early placentation - Mainly derived from cell lines with potential genetic aberrations White, et al . 1988, You, et al . 2019, Akbar, et al . 2020 [ 8 , 104 , 105 ] BAP-EB - 3D structure highly recapitulate human blastocyst - Able to attach to endometrial epithelial cells for implantation studies - Embryo implantation in terms of early adhesion, and attachment to endometrial epithelial cells - High expertise required - Higher cost due to the need of specific differentiation medium Lee, et al . 2015, Yue, et al . 2020 [ 106 , 107 ] Blastoids - Able to differentiate into both embryonic and extra-embryonic lineages - Post-implantation development (especially in mouse) - Extremely high expertise required - Highly complicated experimental set-up - Majority of the studies are in mouse but not in human - May associate with ethical issues Rivron, et al . 2018, Li, et al . 2019, Sozen, et al . 2019, Yu, et al . 2021 [ 108 – 111 ] Examples for various implantation models in use - Ethical issues - Shortage of human blastocysts - Mouse blastocysts have minor difference compared to human - Easy to acquire - Relatively easy to set up - Allow the 3D modelling of endoglandular invasion by single extravillous trophoblast cells - 3D structure better mimic the physiological implantation process - Can be incorporated with stromal cells and ECM (Matrigel /Fibrin) for 3D invasion studies - 3D structure highly recapitulate human blastocyst - Able to attach to endometrial epithelial cells for implantation studies - High expertise required - Higher cost due to the need of specific differentiation medium - Extremely high expertise required - Highly complicated experimental set-up - Majority of the studies are in mouse but not in human - May associate with ethical issues The most straightforward method to study implantation is the culture of blastocysts, either from human or mouse, on endometrial epithelial cells or 3D endometrial models to mimic early implantation [ 102 , 103 , 112 , 113 ]. For example, human blastocysts were cultured on a confluent layer of endometrial epithelial cells to study the impact of mifepristone and levonorgestrel on embryo attachment [ 73 ]. In most cases, however, the use of human blastocysts is restrictive due to their limited supply and ethical considerations. Thus, blastocyst surrogates are largely used in implantation studies. Trophoblast spheroids, which can be formed either from primary trophoblasts or choriocarcinoma cell lines, were commonly used as blastocyst surrogates for implantation studies. The multicellular spheroids can be generated by culture of trophoblast suspension in a rotation platform or an ultra-low attachment culture plate [ 8 , 104 , 114 ]. These trophoblast spheroids can attach onto the surface of endometrial epithelial cells monolayer in vitro. The attachment rate can be quantified to study the impact of molecular or chemical factors on implantation [ 105 , 114 ]. To take one step forward, Wang et al. integrated the trophoblast spheroids derived from a choriocarcinoma cell line with their 3D endometrial culture system (Fig.  3 C) [ 72 ] and demonstrated a significantly higher attachment rate compared to the attachment onto the monolayer of endometrial epithelial cells. Another study established a model investigating the impact of endometrial stromal cells culture on invasion of the trophoblast spheroid through the Matrigel [ 8 ]. In the study, endometrial stromal cells at the bottom of the culture plate were covered with Matrigel diluted with the culture medium at the ratio of 1:1 before addition of the trophoblast spheroids. The trophoblast spheroids were able to penetrate and invade into the Matrigel and interact with the underlying endometrial stromal cells. Although the use of carcinoma-derived cell lines for establishment of the trophoblast spheroids overcomes the problem of availability and ethical considerations of human blastocysts, it is questionable whether it could physiologically resemble the human blastocyst and the dynamic implantation process. To overcome the drawbacks of trophoblastic choriocarcinoma spheroids, human embryonic stem cell-derived trophoblastic spheroids were developed as a novel trophoblast surrogate model in 2015 by Lee et al. [ 106 , 107 ]. Human embryonic stem cell line VAL3 could be differentiated into the trophoblastic cells in specific differentiation medium BAP containing bone morphogenetic protein (BMP) 4, ALK4/5/7 inhibitor and FGF2-signaling inhibitor in mouse embryonic fibroblast-conditioned medium. During the course of BAP-induced differentiation, the expression of the pluripotency marker octamer-binding transcription factor 4 (OCT4) was reduced while that of the trophoblastic markers (Human Leukocyte Antigen-G (HLA-G), β-hCG, Cytokeratin 7 (CK7) and H19) was increased. Moreover, the BAP-treated VAL3 cells showed enhanced capacity of invasion and migration in vitro, consistent with their trophoblastic properties. Embryoid bodies (EB) could be formed by self-aggregation of single-cell culture of VAL3 cells in the AggreWell® plate. BAP treatment converts the EB into a blastocyst-like structure with a cystic structure, termed as BAP-EB. The mRNA profile of BAP-EB resembles that of the trophectoderm of blastocyst before implantation [ 107 ]. BAP-EBs attached specifically to the primary human endometrial epithelial cells at the receptive phase but not at the pre-receptive phase. When co-cultured with endometrial stromal cells, BAP-EBs invaded through the stromal cells resembling the human blastocysts during early implantation. Compared to the classic trophoblast spheroids, BAP-EBs are derived from embryonic stem cells, which allow for long-term culture with an unlimited supply. Moreover, the BAP-EBs selectively attach to the receptive endometrial cells instead of the non-selective attachment ability of the trophoblast spheroids. Morphologically, unlike the multicellular trophoblast spheroids, BAP-EBs possess the blastocoeal cavity-like structure, which resembles the human blastocyst at a more physiological extent. Together, the BAP-EB model may serve as another useful tool for the investigation of early implantation in humans. Due to the unique developmental plasticity of the naive embryonic stem cells, their capacity to be modelled in vitro has been intensively studied. By exposing the naive human embryonic stem cells to designated differentiation media, they can differentiate into the embryonic and the extraembryonic cell lineages, including the SOX2 positive epiblast-like cells, GATA6 positive hypoblast-like cells and GATA3 positive trophoblast-like cells [ 108 – 110 , 115 , 116 ]. The first study on establishment of blastocyst-like structures from embryonic stem cells termed ‘blastoid’ was published in 2018 [ 108 ]. By culturing in non-adherent microwells, the embryonic stem cells self-aggregated to form spheroids, which were then overlaid with trophoblast stem cells. The resulting human blastocyst-like structure possessed a cyst-like structure surrounded by TE-like cells and a cell aggregate inside the cyst resembling the ICM. Molecularly, the blastoids expressed pluripotency markers (OCT4 and NANOG) and a considerable level of trophoblast marker (Caudal-type Homeobox 2 (CDX2)) in the presence of culture supplement (IL-11, 8Br-cAMP, FGF4 and Transforming Growth Factor β1 (TGFβ1)). Moreover, transfer of the blastoids into pseudo-pregnant female mice induced decidualization of the endometrium of the recipient. Human blastoids could be derived solely from human embryonic stem cells [ 110 ]. The resulting blastoids expressed the marker genes present in human blastocyst including SOX2, SOX17 and GATA3. Single-cell RNA sequencing analysis revealed that the blastoids contained epiblast-like cells, hypoblast-like cells and trophoblast-like cells comparable to the cell clusters reported in human blastocysts. Several studies have established the blastoid models using either human or mouse embryonic stem cells, which all showed functional, molecular and morphological similarities to the blastocysts in vivo [ 109 , 111 , 117 ]. The blastoid model possesses the most physiological structure and morphology resembling the blastocyst in vivo, which is unique for studies on preimplantation embryo development and early pregnancy in vitro. However, the specific culture condition and the high expertise required for the blastoid culture are obstacles for their large-scale application. Another concern is public acceptability of research on blastoids, which could potentially give rise to live birth. That possesses an ethical issue that researchers need to consider. The migration and invasion of the EVTs play important roles for successful placentation. After implantation, the TE develops into the cytotrophoblastic shell. CTBs within the shell is disrupted and differentiate into multiple clusters of proliferating EVTs, which invade the decidua via the interstitial or the endovascular routes [ 30 ]. In the decidua, the EVTs remodel the spiral arteries to low-resistance high-flow vessels to provide sufficient feto-maternal exchange. To better understand the molecular drive of this procedure, several EVT migration and invasion models have been established. For the in vitro studies of EVT migration and invasion, the sources of the EVTs are the choriocarcinoma-derived cell lines (e.g. JEG-3, JAR and HTR-8/SVneo), primary trophoblasts isolated from first-trimester placenta, or villous explants from placenta [ 118 – 121 ]. Several EVT migration and invasion assay models have been established in 2D and 3D formats (Fig.  5 ). The most common assay of EVT migration is the scratch/wound healing assay (Fig.  5 A), which simply studies the migration ability of the cells in a 2D condition [ 122 ]. The transwell assay (Fig.  5 B) examines the migration capacity of the cells in a 3D setting [ 123 ]. In the assay, the cells in a culture insert migrated through a semi-permeable membrane under the drive of the chemoattractant of interest below the insert. The number of migrating cells under different experimental conditions could be easily quantified by histological staining of the cells [ 124 ]. To mimic the ECM or endometrial stroma during early pregnancy, the semi-permeable membrane could be coated with Matrigel, which is known as the Matrigel invasion assay [ 125 , 126 ]. Even though majority of the current trophoblast invasion models do not include the maternal endometrial cells, some of the studies have incorporated the conditioned medium secreted by the pre-cultured endometrial cells to study their impact on trophoblast invasion [ 127 – 130 ]. For example, Godbole et al. established the Matrigel invasion assay by adding the conditioned medium produced from decidualized endometrial stromal cells at the lower chamber of the culture insert, and identified a significantly higher number of JEG-3 cells invading through the Matrigel in comparison to the ones cultured with conditioned medium collected from non-decidualized stromal cells [ 127 ]. Alternatively, EVT invasion could be studied by seeding the trophoblast spheroids onto the fibrin gel and quantifying the mean number and length of the invasive protrusions (Fig.  5 C) [ 126 , 131 ]. Fig. 5 In vitro models/assays for trophoblast invasion studies. A Scratch/wound healing assay; B Transwell study; C Trophoblast invasion/protrusion assay; D Villous explant co-cultured with the decidua tissue; E Interstitial invasion model of EVT into spiral artery-embedded gel; F Endovascular invasion model In vitro models/assays for trophoblast invasion studies. A Scratch/wound healing assay; B Transwell study; C Trophoblast invasion/protrusion assay; D Villous explant co-cultured with the decidua tissue; E Interstitial invasion model of EVT into spiral artery-embedded gel; F Endovascular invasion model Both wound healing assay and the transwell assay are easy to experiment thus, widely utilized for the EVT migration studies. However, they lack the physiological environment as only the trophoblasts were involved in the culture system. They cannot fully recapitulate the in vivo condition during placentation. Therefore, a 3D model with maternal tissue may be a further goal to be explored. The in vitro model depicted in Fig.  5 D incorporated the decidua tissue with the co-cultured villous explant to better study trophoblast-decidual invasion in a 3D condition [ 132 ]. In the model, the villous explant tissue from first-trimester placenta was placed on top of the decidua tissue from the same donor. EVT columns showing EVT markers HLA-G could be formed at the bottom of the villous tissue penetrating and invading the decidua tissue in vitro. The model allows study of the regulatory factors of trophoblast invasion and migration, such as the stimulatory action of decidua cell-derived growth factor EGF via regulation of the gap junction protein connexin 40 [ 133 ]. The remodelling processes of the maternal spiral arteries by the invading EVTs can be studied in vitro by measuring the trophoblast invasion into the endothelial cells [ 134 , 135 ]. For study of interstitial invasion, spiral arteries were isolated from the myometrium biopsies and embedded in fibrin gel, and fluorescence-tagged trophoblasts were seeded on top of the embedded spiral artery (Fig.  5 E). Invasion of the trophoblasts into the fibrin gel occurred after 5 days of culture. For investigation of endovascular invasion, the isolated spiral arteries were perfused with fluorescence-labelled trophoblasts followed by closure of the ends of the spiral arteries (Fig.  5 F). The trophoblast-perfused spiral arteries were then immobilized in the fibrin gel and cultured for 3 days. Both models allow the study of the remodelling of the spiral arteries by the invasive EVTs, which serve as suitable models for the pregnancy-associated complications with aberrant spiral artery remodelling, like PE. Although these models are promising and physiological, they are not widely used due to their disadvantages. First, the availability of high-quality primary tissue sample is limited, and inter-patient variation may lead to inconsistent results. Second, the culture of the primary tissue for a long time is challenging due to tissue necrosis in vitro [ 126 ]. The primary CTB cells harvested from first trimester placentae usually show diminished proliferative capacity in vitro. Better EVT migration and invasion model is urgently required, which may allow the participation of the in vitro 3D culture models of endometrium to demonstrate the process more physiologically. Trophoblast organoids have been established as a 3D model for the trophoblast differentiation, invasion and migration studies [ 136 , 137 ]. Placental villi isolated from the first trimester (6 ~ 9 weeks of gestation) placentae are the primary source of the trophoblast organoids, which can self-organize into 3D organoid structures in 2 ~ 3 weeks after embedding in Matrigel and stimulation/inhibition of specific signaling pathways (Fig.  6 ). Physiologically, the placental villi developed from the TE of blastocysts are composed of CTBs, which eventually differentiate into the hormone-secreting STBs and the decidua/spiral artery-invading EVTs. The trophoblast organoids expressed several trophoblast markers, including GATA3, KRT7, EGFR, TFAP2A and TFAP2C [ 136 , 137 ]. Compared to the in vivo condition, the villous cytotrophoblast (VCT) markers Ki67 and TP63, the SCT markers CD46 and CD71 were all expressed in the trophoblast organoids [ 137 ]. Fig. 6 Establishment of trophoblast organoids. Villous tissue collected from first trimester placenta is digested and the trophoblast cells will then be seeded in Matrigel for the formation of trophoblast organoids Establishment of trophoblast organoids. Villous tissue collected from first trimester placenta is digested and the trophoblast cells will then be seeded in Matrigel for the formation of trophoblast organoids The trophoblast organoids are genetically stable and show structural, morphological, functional and metabolic similarities to the villi in vivo. When cultured in a specific EVT differentiation medium, the trophoblast organoids were able to differentiate into EVTs that were positive for EVT marker HLA-G. The HLA-G + EVT cells from the organoids exhibited migratory and invasive activities in Matrigel. The trophoblast organoids harbor all functional trophoblast cell types in the human villi, and they also pocess secretory functions of SCT and several placental hormones and peptides, such as hCG, Growth/differentiation Factor 15 (GDF15) and Pappalysin 1 (PAPPA) of SCT under the stimulation of specific ST differentiation medium [ 137 – 139 ]. The co-culture of the trophoblast organoids with the in vitro 3D culture model of endometrium will potentially allow the recapitulation of trophoblast-endometrium interaction during early placentation under a more physiological environment. Yet the further optimization of this model, for example, to reverse the polarity of trophoblast organoids (from basal-out/apical-in to apical-in/basal out), is required in the near future for the better implications into epithelial biology and trophoblast-endometrium interactions studies.

Although a great number of culture models have been established, majority of them are still in their initial stage of application. Nevertheless, they have the potential to mimic both the embryo implantation and the early pregnancy, which would facilitate the investigation of implantation failure or recurrent pregnancy loss and pregnancy complications such as PE. Combining the endometrial glandular organoids/trophoblast organoids/blastoid models with the currently available endometrial models may better recapitulate the physiological events during trophoblast-endometrium interaction, which has rarely been done now. A comprehensive endometrial model, for example the one established by Wang et al. [ 72 ] which incoporated endometrial stromal cells and epithelial cells with the ECM. By adding the endometrial glandular organoids into the system, their unique properties, such as the preservation of hormone responsiveness and secretory function, would resemble the physiological condition of the human endometrium for a better recapitulation of the in vivo environment. According to the reviewed studies, we proposed a potentially achievable model for the study of trophoblast-endometrium interaction as depicted in Fig.  7 . The model incorporates the endometrial epithelial cells in collagen/Matrigel laying on top of endometrial stromal cells and endometrial glandular organoids-packed in fibrin-agarose gel as the endometrium surrogate, while the blastoids or trophoblast organoids are employed as the blastocyst and early placental villi surrogate, respectively. To further recapitulate the physiological microenvironment, the immune cells and the endothelial cells can be assembled into the system. Fig. 7 Illustration of a 3D model according to the reviewed studies for trophoblast-endometrium interaction investigation. Combining the currently available 3D endometrial models, a novel 3D model is proposed which integrates endometrial cells including endometrial epithelial cells and stromal cells, endometrial glandular organoids, blastoids and trophoblast organoids, for the purpose of better recapitulation of embryo implantation and placentation Illustration of a 3D model according to the reviewed studies for trophoblast-endometrium interaction investigation. Combining the currently available 3D endometrial models, a novel 3D model is proposed which integrates endometrial cells including endometrial epithelial cells and stromal cells, endometrial glandular organoids, blastoids and trophoblast organoids, for the purpose of better recapitulation of embryo implantation and placentation By incorporating the cells and organoids derived from patients of interest, the proposed model may facilitate the demonstration and modelling of different diseases that have been known to contribute to infertility and pregnancy failure. For example, normal blastoids may be added to the 3D culture models of endometrium established from the endometrial cells of patients with repeated implantation failure, which hopefully could demonstrate the defective interaction of the trophectoderm onto the endometrial epithelial cells. In terms of recapitulation of early pregnancy and studies of recurrent pregnancy loss, endometrial biopsies could be collected from respective women to establish the 3D culture models with the endometrial glandular cells and stromal cells from the same patients. Trophoblast organoids can be incorporated into the culture system to mimic the placentation process during early pregnancy, which may allow the demonstration of defective interaction during trophoblast-endometrium interaction at cellular and molecular levels. For the study of diseases associated with trophoblast function, such as PE, trophoblast and endometrial glandular organoids may be developed from PE patients and incorporated into the 3D model to study their functional abnormalities. To establish such a novel endometrial model, however, several hurdles are still present. The selection of the culture medium supporting the co-existence of multiple cell types is the major obstacle for the establishment of in vitro 3D culture models of the endometrium. As the culture condition for the organoid system is comparably complicated due to the requirement of specific growth factors, the optimal culture medium of the culture system would be challenging. Another challenge is the biological variation of clinical samples. Even though the endometrial glandular organoids have been shown with genetical stability for more than six months of culture, their stability and viability when incorporated into the 3D culture model would be unpredicted. Thus, the functional durability of the culture models will still be unknown. In the next step, the proposed culture model could be further improved by assembling of dynamic microfluidics which allows the accomplishment of organ-on-chips systems [ 140 ]. Microfluidics system allows the manipulation of fluids flew into the culture system with multiple cell/tissue compartments incorporated to mimic the blood perfusion in vivo. The incorporation of this microfluidics system into the currently available 3D culture model may further promote the paracrine communications of the cells, enhance the comprehensive functionality, and allow the long-term maintenance of the culture system. By controlling the fluid parameters such as shear force and concentration gradient, the whole culture system could resemble a vascularized tissue or organ, thus bring into the function of endothelial cells which are usually absent in previous 3D models. Meanwhile, the specific shear force that can be manipulated would allow the study of specific cell types and the inter-cellular communications within the system, which may help to model pathophysiological conditions [ 140 – 143 ]. This technique would be especially beneficial for the studies in reproductive tissue, whereby the supplementation of sex hormones into the system may allow to mimic the dynamic hormonal change during the human menstrual cycle in a more physiological setting. It has been shown that under this kind of dynamic environment, the endometrial stromal cells showed a greater extent of decidualization in response to the secretory products from the co-cultured endothelial cells upon the stimulation of designated haemodynamic force [ 141 ], which highlighted the potential of this technique for modelling of human endometrium in terms of paracrine inter-cellular communications. Another study that incorporated multiple female reproductive organs/tissue into the microfluidic culture system has simulated the menstrual cycle for 28 days with sustained circulation [ 143 ]. In their study, successful follicle development in the ovary tissue has been identified, together with a prominently higher secretion of sex hormones in response to the dynamic flow. That result did not only reveal the possibility for this organ-on-chip culture system to be maintained for a longer period of time, but also showed that the cells tend to function more physiologically under the in vivo condition. In sum, the combination of these techniques and culture models would allow the modelling of both physiological and pathological conditions during the course of human reproduction, such as implantation, early pregnancy, pregnancy failure and pregnancy-associated disorders, as majority of those conditions possess the features of relatively long duration and the presence of hormone cues, which are yet difficult to be mimicked according to currently available cell culture techniques. That would definitely be a huge breakthrough if such comprehensive, physiological and manipulatable models could be invented for reproductive studies in the future.

In humans, pregnancy usually takes 39 weeks from embryo implantation to parturition. To establish a successful pregnancy, embryo attachment is the very first step and a synchrony between the receptive endometrium and the competent embryo is a must for the process. As a key event that determines the outcome of pregnancy, embryo implantation is tightly regulated by a plethora of factors from both the maternal side and the embryo side [ 1 ]. Owing to ethical considerations, the collection of human clinical samples, especially at the implantation site, is challenging. However, few studies on farm and laboratory animals have shed some light in this regard, even though the outcomes of the animal studies could not be fully adapted to human due to the physiological and anatomical specificity of human endometrium [ 2 ]. Therefore, it is a daunting task to study the interaction between the embryo and the endometrium in humans during pregnancy. Defects in embryo implantation, placentation, and fetomaternal tolerance may result in pregnancy failures. Several disorders, including recurrent implantation failure, recurrent pregnancy loss, or pre-eclampsia (PE), are known to contribute to the pregnancy failure [ 3 – 5 ]. For majority of these diseases, the exact aetiology remains unknown due to the difficulties in mimicking the pathology in vitro, which makes it challenging for their diagnosis, treatment and prevention. Several in vitro human culture models of endometrium have been developed to facilitate the study of the interaction between endometrium and embryo trophoblasts during implantation and post-implantation development. These models can be categorized into two-dimensional (2D) or three-dimensional (3D) cell/tissue culture models, and the 3D organoid models [ 6 ]. There is an increasing interest in the use of in vitro 3D models to simulate the physiological conditions, especially in corporation with the in vitro blastocyst and trophoblast surrogates. There are many excellent reviews on the evolution and development of in vitro models of the endometrium [ 6 – 8 ]. In this review, the characteristics of the currently established 3D culture models of endometrium will be reviewed and evaluated. Their applications for the study of trophoblast-endometrium interactions upon the association with currently established embryo/trophoblast models under the in vitro conditions will be discussed, together with their potential implications for modelling of defective implantation and placentation. In comparison with other review articles [ 6 – 8 ] which mainly focus on either endometrial cell or trophoblast, our article is distinguished by its emphasis on the integrated 2D/3D system with both cell types for studying the maternal–fetal interaction.

In vitro 2D to 3D models of endometrium are good tools for understanding the molecular mechanism behind embryo implantation and early pregnancy in humans. By introducing the newly established organoid concept, including the endometrial glandular organoids, endometrial assembloids, trophoblast organoids and blastoid model, the in vitro 3D culture models can better recapitulate the trophoblast-endometrium interaction for investigation of the pathophysiology of implantation failure or pregnancy complications such as recurrent pregnancy loss and PE. The outcome of the investigations on patient-derived endometrial organoids/assembloids would enable detection of potential biomarkers and causative factors for early diagnosis and development of novel treatment strategy. Although the integration of these models needs to be optimized, they set the basis for ideal modelling of the endometrium, which would eventually benefit fertility treatment.

In a human menstrual cycle, pregnancy can only be established if implantation of an embryo happens within the window of implantation (WOI) [ 9 – 12 ]. Normally, the WOI occurs at the mid-secretory phase of the menstrual cycle, which generally happens 6 ~ 10 days after ovulation [ 13 ]. Several morphological and molecular signatures have been found to define the WOI, including decidualization of endometrial stromal cells, appearance of pinopodes, optimal development of endometrial glands and local production of cytokines and growth factors [ 12 ]. Together, the uterus is prepared for the implantation of embryo and establishment of pregnancy [ 14 , 15 ]. In humans, implantation failure is a significant cause of infertility. The first three weeks of human pregnancy are generally considered as the implantation period, while placentation occurs from the third to twelfth week of gestation [ 16 ]. Embryo implantation marks the first direct cell-to-cell interaction between the mother and the developing embryo. It consists of a series of complicated and coordinated events. Implantation of the human embryo requires three sequential steps: apposition, adhesion and invasion (Fig.  1 ). During the secretory phase of the menstrual cycle, the endometrial stromal cells start to undergo decidualization under the drive of progesterone, converting the endometrial fibroblasts into the decidualized stromal cells, which facilitate invasion of trophoblasts of the blastocyst [ 17 , 18 ]. In humans, blastocyst is formed from the fertilized zygote with the formation of inner cell mass (ICM) and trophectoderm (TE), which later migrates to the endometrium for the implantation after sequential cleavage and mitosis [ 19 ]. The orientated apposition allows interaction of the TE with the pinopodes (tiny microvilli protrusion) on the apical surface of endometrial luminal epithelium [ 1 ]. The blastocyst then adheres tightly to the endometrium, followed by invasion and penetration into the deeper stroma anchoring the embryo tightly in the endometrium [ 13 , 20 ]. During this process, several molecular factors are produced from the maternal and the fetal sides working together to facilitate a successful implantation. These factors include steroid hormones, vasoactive factors, cytokines, growth factors, as well as the inter-cellular interaction [ 11 , 21 – 25 ]. Fig. 1 The process during the embryo implantation. The blastocyst implants to the maternal endometrium following sequential stages of apposition, adhesion and invasion. Each stage is associated with the production of molecular factors by both the blastocyst and the endometrium. ICM: inner cell mass, TE: trophectoderm, LE: luminal epithelium, GE: glandular epithelialium, STB: syncytiotrophoblast The process during the embryo implantation. The blastocyst implants to the maternal endometrium following sequential stages of apposition, adhesion and invasion. Each stage is associated with the production of molecular factors by both the blastocyst and the endometrium. ICM: inner cell mass, TE: trophectoderm, LE: luminal epithelium, GE: glandular epithelialium, STB: syncytiotrophoblast After implantation occurs, extensive crosstalk takes place at the maternal–fetal interface, which is composed of the maternal decidua and the trophoblasts of the conceptus. A comprehensive schematic illustration of the constitutions of the maternal–fetal interface during early pregnancy is shown in Fig.  2 . Throughout the course of pregnancy, the interface serves as the primary site for physical and functional interaction between the mother and the fetus, and is the site for placentation [ 26 – 30 ]. After embryo implantation, the placenta starts to develop from the third week of gestation, and the nutritional support of the embryo or fetus before the full establishment of the placenta tends to be histotrophic, i.e. originated from the secretion of decidual glands at the maternal–fetal interface. After the placenta is completely formed at the end of the first trimester, the fetus starts to receive full maternal blood supply [ 31 , 32 ]. Fig. 2 The maternal–fetal interface in early pregnancy. This is a simplistic drawing for the maternal–fetal interface. During early pregnancy, the anchored placental villi filled with trophoblast cell column interact with the maternal endometrium, forming the maternal–fetal interface. The inner cytotrophoblasts (CTB) fuse to form the outer syncytiotrophoblasts (STB), and the extravillous trophoblasts (EVT) invade the endometrium, uterine glands and the uterine spiral arteries. Maternal immune cells also participate in this process for maintenance of immune balance The maternal–fetal interface in early pregnancy. This is a simplistic drawing for the maternal–fetal interface. During early pregnancy, the anchored placental villi filled with trophoblast cell column interact with the maternal endometrium, forming the maternal–fetal interface. The inner cytotrophoblasts (CTB) fuse to form the outer syncytiotrophoblasts (STB), and the extravillous trophoblasts (EVT) invade the endometrium, uterine glands and the uterine spiral arteries. Maternal immune cells also participate in this process for maintenance of immune balance On the maternal side, the endometrial glands and the decidual stromal cells are capable to regulate the implantation activity and early embryo development via their secretory products [ 33 – 37 ]. Cytokines and growth factors, for example leukaemia inhibitory factors (LIF), are produced by the endometrial glands in the WOI, which can stimulate decidualization of stromal cells and prime implantation of blastocyst for attachment and adhesion [ 38 – 42 ]. In mice, the endometrial glands are capable to activate the blastocysts for initiation of implantation and acquisition of adhesion competence via the secreted glucose, amino acids (leucine and arginine) and proteins (osteopontin) [ 36 , 43 , 44 ]. The decidualized stromal cells promote the secretory function of the endometrial glands, mainly through the secretion of prolactin [ 45 , 46 ]. Consistently, elevated expression of prolactin receptor has been identified in the endometrial glands both during the secretory phase of the menstrual cycle and during early pregnancy [ 45 , 47 , 48 ]. On the fetal side, the decidualized stromal cells regulate the invasion of trophoblasts [ 49 ]. The cells produce both pro-invasive factors (Interleukin-1β (IL-1β), IL-6, IL-11) and anti-invasive factors (IL-10, Vascular Endothelial Growth Factor (VEGF)), which together maintain the invasion of trophoblasts at a balanced state [ 50 – 53 ]. In addition, the maternal immune cells (e.g. decidual natural killer (dNK) cells, macrophages and leukocytes) with immune-modulatory functions to prevent immune rejection of the fetus also contribute to the organization of the maternal–fetal interface in early pregnancy [ 17 , 54 , 55 ]. The involvement of decidual immune cells during early pregnancy has been extensively studied [ 55 – 57 ]. On the fetal side, the TE develops into cytotrophoblastic shell [ 30 ]. Cytotrophoblasts (CTB) originate from the shell differentiated into the villous CTBs and the extravillous cytotrophoblasts (EVTs). The villous CTBs fuse to form the multinucleated syncytiotrophoblasts (STBs) with the hormone-secreting function, while the EVTs invade the decidua to the spiral arteries during the first trimester of pregnancy. When the EVTs reach the spiral arteries, they induce apoptosis of vascular smooth muscle and endothelial cells and replace the endothelium by fibrinoid material. The remodeling process transforms the spiral arteries to low-resistance, high-flow vessels ensuring sufficient fetal-maternal exchange [ 31 , 58 , 59 ]. Some studies have also reported the invasion of EVTs into the endometrial glands during early-first trimester. By migrating towards the endometrial glands and replacing the glandular epithelial cells, these EVTs may facilitate the endometrial gland secretion and the placental development [ 30 , 60 – 63 ]. In sum, all these compartments exert important functions on the other compartments during the process of implantation and early pregnancy.