A successful human pregnancy requires a complex cross-talk between the maternal and foetal cells during the development of the placenta. The aim is to create an efficient system for adequate oxygen and nutrient transport for the growing foetus. Any defect in this series of events can result in severe complications in late stages of pregnancy. This essay shall first give an introductory background on human implantation and placentation, to which, further discussion will then be made on specific factors and pathways involving trophoblast invasion and spiral artery remodelling. The clinical relevance of the placental-decidual interactions will be clarified using the examples of pre-eclampsia and intrauterine growth restriction.

Background

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Despite of the growing population, human reproduction can be considered to be relatively inefficient when compared with other species such as rats of chimpanzees, with the rate natural conception per cycle around 30% (1), of which only 50-60% progress beyond 20 weeks of gestation and 75% of failed pregnancy are due to implantation and placentation defects (2).

Therefore, implantation and placentation, which occur during the first trimester, represent critical roles in ensuring a successful pregnancy, as their defects lead to the manifestation of complications in later gestation stages, such as pre-eclampsia, intra-uterine growth restriction (IUGR) and miscarriage (1).

After fertilisation, the embryo begins to move to the uterine cavity, where it develops into a blastocyst. There, implantation occurs around 6-7 days post-conception, where the blastocyst comes in contact with the maternal endometrium for the first time, through a sequence of apposition, adhesion, attachment and penetration (3). For implantation to succeed, it requires a crosstalk between a competent blastocyst and a receptive uterine endometrium within a specific period of time called the “window of implantation” (3). This is mediated by ovarian hormones (oestrogen and progesterone) and local signalling molecules such as cytokines, growth factors, homeobox transcription factors, lipid mediators and morphogen genes in the basis of autocrine, paracrine and juxtacrine (4).

Simultaneously, decidualisation occurs in which the cells within uterine endometrial layer undergoes extensive proliferation and differentiation into decidual cells, in order to accommodate embryonic growth and invasion. In humans, this process is initiated after ovulation, but becomes more robust in the presence of the blastocyst. Moreover, decidua contributes to the protection of embryo against the maternal immune system (3).

Upon attachment, syncytiotrophoblasts (of the blastocyst) soon begin penetrating through the uterine epithelium until the entire blastocyst becomes embedded in the maternal subepithelial stromal tissues (1). Thereafter, cytotrophoblasts (the inner layer of trophoblasts) move out to invade the entire endometrium, inner third of the myometrium and maternal endovascular structures. Once endovascular invasion has occurred, placentation proceeds to establish a definitive uteroplacental circulation via the placenta (1).

Weighing up to 500-600 grams when fully matured, a placenta is an organ consisting of branching villi, each containing foetal blood vessels and macrophages (6). They are surrounded by two layers of trophoblasts, syncytiotrophoblasts (outer) and cytotrophoblasts (inner), thus providing preventing the mixing of foetal and maternal blood. Cytotrophoblasts fuse into the overlying syncytiotrophoblasts to form a site for nutrient exchanging and hormonal synthesis, such as steroidogenesis. In addition, cytotrophoblasts also differentiate into extravillous trophoblasts which invade into the decidua and remodel decidual spiral arteries (5). This is also the basis for many placental defects which will be further discussed in later sections.

Trophoblast differentiation and invasion

Derived from the trophectoderm of the developing blastocyst, trophoblasts are vital in establishing and maintaining a successful pregnancy (7). Once implanted, trophoblasts differentiate into two major cell lineages, the syncytiotrophoblasts and cytotrophoblasts stem cells. The syncytiotrophoblasts, forming chorionic villi, are responsible for placental nutrient and gas exchange, placental hormone and growth factor production. Some of the cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which migrate from the villous tips and invade deep into the maternal decidua and inner third of the myometrium, as well as shelling around it. When endovascular trophoblasts, a subtype of EVTs, penetrate uterine vasculature and replace maternal endothelial cells, this process induces transformations of uterine spiral arteries to result in dilated, non-vasoactive vessels by the mid-second trimester, thus allowing a greater supply of maternal blood into the intervillous space (7).

There are several regulator factors involved in these mechanisms, one of which has to be oxygen tension. There is already a hypoxia in the initial phase of placentation, with oxygen concentration reported to be less than 20 mmHg (1-2% O2) during the first 10 weeks and 60 mmHg (8% O2) at the second trimester (8). Once haemochorial placentation is completed, the initial burst of blood into the intervillous space raises oxygen tension which creates oxidative stress, thus promoting trophoblast proliferation into an invasive phenotype (8). Hypoxia-inducible factor (HIF), a heterodimeric transcription factor consisting of ? and ? subunits, has been implicated in this process, due to its upregulation in hypoxic conditions. HIF-1 ? and HIF-2? proteins are found to be overexpressed in pre-eclampsia, thus making HIF as the marker of cellular oxygen deprivation (9).

Most cell adhesion molecules (CAMs) can be categorised into four families – the immunoglobulin superfamily, the integrins, the selectins and the cadherins (10). Cadherins include E-, N-, P-, R-, B- and VE-cadherins. Integrins are molecules that bind to ECM proteins and members of immunoglobulin superfamily, including intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3 and vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial CAM (PECAM-1). ECM proteins encompass fibronectin, laminin and collagen. E-, P and L-selectin are CAMs involved in leukocyte extravasation (10).

CAMs are expressed on the surface of invasive trophoblasts, and their ligands control adhesion and invasion of trophoblasts. To do so, it requires a ‘switching’ of adhesion molecule expression. Indeed, examination of an in vitro model of cytotrophoblast invasion shows a switching in their adhesion molecule repertoire to resemble an endothelial phenotype (10,11). For instance, the upregulations of VCAM-1, PECAM-1, VE-cadherin and E-selectin have been reported in endovascular EVTs, despite not being expressed previously on cytotrophoblast stem cells (10).

Apart from adhesion, trophoblasts also require gene expression of several proteases, such as serine protease, cathepsins and matrix metalloproteinases (MMPs), in order to digest ECM (11,12). Their importance cannot be underestimated as human trophoblast invasion is required to highly aggressive but tightly regulated (12). Urokinase plasminogen activator (PLAU) is responsible for EVT migration through matrix degradation and activation of EVT-produced MMPs (13). MMPs, namely MMP-1, MMP-2, MMP-3, MMP-9, MMP-11 and MMP-14, are secreted from the blastocyst stage, to break down collagen, gelatin and collagen stromelysins. In addition, tissue inhibitor of matrix metalloproteinases (TIMPs), of which TIMP-1, TIMP-2 and TIMP-3 being the most well-known, are involved in regulating this process by inhibiting MMPs (11). A normal invasion depends on the balance between MMPs and TIMPs – both produced by trophoblasts and decidual cells. Other regulatory factors include IL-1?, LIF and corticosteroids (10). Excessive invasion can result in choriocarcinoma and restricted invasion is found in early pregnancy failure, pre-eclampsia and IUGR (11).

During early stages of gestation, placental development occurs without blood supply from the mother. This means placentation also relies on factors produced from endometrial glands such as endothelial growth factor (EGF), vascular EGF (VEGF), placental growth factor (PlGF), CSF-1, insulin-like growth factor-1 (IGF-1) and IGF-2 (25). At the maternal-foetal interface, these factors have been shown to promote proliferation, adhesion and invasion. In contrast, several inhibitory proteins, produced by decidual cells and cytotrophoblasts, work to regulate these processes, including transforming growth factor (TGF-?) family members, interferon-?, endostatin, kisspeptin-10 and TNF?.

Immune tolerance by the maternal tissue plays a key role in the survival of the conceptus, trophoblast invasion and vascular growth, and is mainly mediated by decidual natural killer cells (dNK) (11,14). Other decidual immune cells include macrophages, T cells and T cells (14). The potential roles of dNK cells are summarised in figure 1.

the dNK cells, recruited by decidualisation, constitute up to 70% of maternal immune cells in the decidua basalis (14). In contrast to peripheral blood NK cells, dNK cells are more involved in cytokine synthesis rather than cytotoxicity (15). Transcripts encoding several anti- and pro-angiogenic and endothelial mitogenic stimulants such as angiopoietin-1 (ANGPT1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and NKG5 (mitogenicity stimulant) have been identified in dNK cell profiles (15). Moreover, cytokines and chemokines such as IL-1, IL-6, IL-8, IL-10, granulocyte macrophage colony stimulating factor (CSF1, CSF2), TNF, also produced by dNK cells, have been implicated in trophoblast invasion as well (15).

The targeting mechanism of dNK cells is achieved by the expression of inhibitory and activating receptors, which are the killer immunoglobulin-like receptors (KIR), C-type leptin heterodimer family (CD94/NKGs), the natural killer cytotoxicity receptors (NCR) and the immunoglobulin-like transcripts (ILT) (16). They interact with major histocompatibility complex (MHC) antigens, which are mostly HLA class I molecules expressed on trophoblasts. The inhibitory KIR-HLA interaction and the general downregulation of the majority of polymorphic MHC molecules on trophoblasts together indicate immune tolerance at maternal-foetal interface (16). 

 

Spiral artery remodelling

Spiral artery development begins in the luteal phase of the menstrual cycle, under the priming of progesterone, modifying into a high-flow, low resistance vasculature capable of meeting the demands of growing foetus (17). They arise from radial arteries which possess well developed muscular walls and elastin lamina. In the absence of an implanting blastocyst, spiral arteries will eventually regress due to the decrease in progesterone level and subsequently result in menstrual shedding (17).

The remodelling begins prior to implantation for the first 22 weeks of gestation and can be divided into two stages. The first stage of spiral artery remodelling is trophoblast-independent as decidualisation leads to vascular changes such as endothelial basophilia, vacuolation, increased endothelial activation and vasodilation (18). The second stage requires the presence of trophoblasts and is essential to complete these physiological changes, such as the removal of vascular smooth muscle cells (VSMC) and endothelial cells (EC), VSMC de-differentiation and fibrinoid deposition (17). The balance between pro- and anti-apoptotic stimuli is critical for an effective transformation of spiral arteries, as vascular cell loss is achieved without compromising vessel integrity (19). Tumour necrosis factor-? (TNF?), TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL), expressed by trophoblasts, have been implicated in the regulation of vascular cell apoptosis (17, 19). TNF? binds to TNF-receptor 1 (TNF-R1) and TNF-R2 on EC and VSMC, leading to the recruitment of the intracellular TNF-receptor-associated death domain (TRADD). Together with several proteins such as TNF-receptor-associated factor 2 (TRAF2), it subsequently activates JNK pathway to promote cell survival.

On the other hand, the interaction between TRAF2 and Fas activating death domain (FADD) results in activation of caspase 8 and the subsequent apoptosis (19). FasL, expressed by villous, extravillous and syncytiotrophoblasts, activates Fas receptors (CD95) which are expressed on maternal lymphocytes, to induce their apoptosis, in order to ensure immune privilege at the maternal-foetal environment (22). Apoptotic cells are then phagocytosed by decidual macrophages and trophoblasts to prevent surrounding tissue damage by toxic intracellular contents (20), under the signalling of phosphatidylserine (21). This process can be considered as immunosuppressive as it does not involve inflammatory response, in contrast to necrotic syncytiotrophoblast microparticles seen in pre-eclampsia (23).

In addition, other changes include ECM restructuring, migration and changes in cellular adhesion (14). In a similar manner to trophoblast invasion, degradation of ECM proteins, particularly endothelial elastin, by MMP-1, -2, -9 and -12, TIMPs regulation and PLAU activation are also involved in these processes (14). 

Finally, extravillous trophoblasts secrete and deposit fibroid material made up of fibronectin, collagen type IV and laminin. This deposition serves to maintain the integrity of the newly transformed spiral arteries and also form a basement membrane for re-endothelialisation (18).

 

Clinical relevance

Pre-eclampsia

Affecting 5-8% of the population, pre-eclampsia is a pregnancy disorder characterised by a classical triad of gestational hypertension (>140/90 mmHg), proteinuria (?300 mg in 24h or ?2+ on urine dipstick in absence of renal pathology) and systemic endothelial cell (EC) activation presenting from 20 weeks onward (5, 9). Pre-eclampsia can lead to serious health complications to both the mother and child, such as end-organ failure, seizure (eclampsia) and death (5, 9, 14). 

Figure 2 shows an integrated model of pre-eclampsia pathophysiology. Genetic and environmental factors induce shallow trophoblast invasion and defective spiral artery remodelling, by the first trimester, thus producing uterine ischaemia depending on placental capacity and foetal demand on blood supply (9). Hypoxia instigates several adaptive responses comprising pro-inflammatory cytokine release, oxidative and ER stress, antibodies against type 1 angiotensin II receptor and necrotic trophoblast debris (9, 23). Collectively, these factors shift the balance to an antiangiogenic state, in which leukocyte activation, intravascular inflammation, EC dysfunction and inappropriate thrombin synthesis occur, leading to adverse consequences portrayed on the diagram (9).

IGF has been associated with pre-eclampsia as it’s been found that women suffering from this disorder exhibit higher levels of IGF binding protein-1 (IGFBP-1), which is known for disrupting integrin function during adherence of trophoblasts to endometrial layer (26). Consequently, this leads to shallow invasion which has negative ripple effects on the following spiral artery transformation.

Another factor is the expression of glial cell missing-1 (GCM1) which is found to be low in pre-eclamptic patients (27). As GCM1 is a transcription factor known for regulating syncytiotrophoblast formation or turnover, it has been suggested that GCM1 is degraded by HIF-1 from persistent hypoxia. In addition, oxidative stress can downregulate TGF-?3 expression after 9 weeks, hence resulting in defective differentiation of trophoblast and inadequate spiral artery transformation (9).

Non-transformed spiral arteries have narrow lumens which disturb normal blood flow. This increases the chance of atherosis which involves lipid-laden macrophages, fibrinoid necrosis and mononuclear perivascular infiltration of the vessel walls. Over time, these lesions create oxidative and endoplasmic reticulum (ER) stress and further restrict blood supply to foetal tissues (9). In the state of energy crisis, ER becomes unfolded, hence ceasing trophoblast proliferation and inducing trophoblast cell death (29). The release of micro-particles and nanoparticles into maternal circulation as the response instigates intravascular inflammation. Oxidative stress occurs when reactive oxygen species (ROS) is more abundant than the available antioxidant molecules. As a result, damages to cellular lipids, DNA and proteins are seen (28).

Intra-uterine growth restriction

IUGR, seen in 30% of pre-eclampsia cases, is the failure of the foetus to reach its growth potential, and characterised by inadequate maternal-foetal blood and nutrient supply across the placenta (14). However, IUGR is not secondary to pre-eclampsia as it generally does not present with hypertension nor pro-inflammatory and anti-angiogenic factors seen otherwise (9). For instance, fms-like tyrosine kinase 1 (sFlt-1), an anti-angiogenic factor is seen to be increased in pre-eclampsia but not in normotensive pregnancies (30).

Several mechanisms have been proposed, including reduced uteroplacental circulation, compromised foetal-placental angiogenesis (14). The immune system is a shared feature between pre-eclampsia and IUGR. In both cases, placental biopsies reveal decreases in the number of dNK cells, T lymphocytes and macrophages, thus suggesting the significance of cytokines and growth factors produced by these cells (24).

 

Conclusion

From a plethora of research materials, human implantation and placenta have been shown to be incredibly delicate and its success depends on careful interactions between all individual elements. When the blastocyst aligns and attaches itself to the maternal endothelium, the communication at maternal-foetal interface instigates the trophoblast invasion and differentiation, which induce physical and physiological adaptations of uterine spiral arteries – the main source of blood supply – into a high-flow, low resistance system. This total embedment allows for a proper uteroplacental circulation to be formed so that the increasing demands of a developing foetus will always be met.

For trophoblast invasion and differentiation, several factors have been indicated, such as oxygen tension (HIF) stimulating invasive proliferation, the switching of cell adhesion molecule gene expression on extravillous trophoblasts into an endothelial phenotype, the balance between matrix degradation by proteases (e.g. MMPs) and its inhibition by TIMPs and decidual natural killer cells which produce growth-stimulant cytokines and ensure immunological privilege at the maternal-foetal border. In terms of spiral artery remodelling, it develops on the basis of pre-existing decidualisation of maternal endometrium and the success of trophoblast invasion and differentiation. The balance between pro- and anti-apoptotic factors must be maintained to achieve adequate removal of vascular smooth muscle cells and endothelial cells, without triggering inflammatory responses. In addition, MMP-TIMP interactions during this stage play homage to the occurrence in invasion process as well.

When there is a disturbance in the human implantation and particularly placentation during the 3 months of gestations, this can result in later complications as discussed using pre-eclampsia and intrauterine growth. Impaired uteroplacental circulation has been implied in both cases. However, only pre-eclampsia involves inflammatory response due to endothelial and molecular damages caused by persistent hypoxia, thus leading to organ damage and fatality. 

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