Unraveling the molecular mechanisms driving enhanced invasion capability of extravillous trophoblast cells: a comprehensive review

Abstract

Precise extravillous trophoblast (EVT) invasion is crucial for successful placentation and pregnancy. This review focuses on elucidating the mechanisms that promote heightened EVT invasion. We comprehensively summarize the pivotal roles of hormones, angiogenesis, hypoxia, stress, the extracellular matrix microenvironment, epithelial-to-mesenchymal transition (EMT), immunity, inflammation, programmed cell death, epigenetic modifications, and microbiota in facilitating EVT invasion. The molecular mechanisms underlying enhanced EVT invasion may provide valuable insights into potential pathogenic mechanisms associated with diseases characterized by excessive invasion, such as the placenta accreta spectrum (PAS), thereby offering novel perspectives for managing pregnancy complications related to deficient EVT invasion.

Introduction

Precise trophoblast invasion is essential to successful placentation and pregnancy. The decidua contributes to regulating this intricate process [1]. Extravillous trophoblasts (EVTs) break through the cytotrophoblast shell, destroy the smooth muscle media, and subsequently migrate toward the uterine spiral artery located in the decidua. Consequently, endovascular trophoblast cells (EVCTs) develop a plug near the cytotrophoblast shell and take over the role of the endothelium, resulting in increasing the diameter and blood flow of those vessels, which allows the maternal–fetal circulation to be established. Giant cells (GCs) formed by the fusion of EVTs at the boundary between decidua and myometrium frequently invade the inner third of the myometrium.

Placenta accreta spectrum (PAS) is a pregnancy complication with catastrophic outcomes [2, 3]. According to the depth of invasion of the villous tissue into the myometrium, PAS is classified as accreta, increta, and percreta. The more profound the invasion of EVT, the worse the prognosis of PAS [2]. Conversely, EVT invasion deficiency acts as a pathological characteristic of various pregnancy complications such as preeclampsia (PE) [4–6], recurrent spontaneous abortion (RSA), fetal growth restriction (FGR), preterm labor, and placental abruption [7, 8]. A more comprehensive understanding of the role of over-invasion in placental diseases can improve understanding of the molecular pathology of PAS and potential targets for the diseases attributed to invasion deficiency.

We review the articles in the past 10 years and summarize their highlights. Therefore, this review will delve into the influence of hormones, immunity, inflammation, angiogenesis, the extracellular matrix microenvironment, hypoxia, stress, programmed cell death, epigenetic modifications, and microbiota on the heightened invasion of EVT. This review examines the mechanisms facilitating EVT invasion and explores potential pathogenic mechanisms associated with over-invasion. Subsequently, the pathogenesis and treatment of placental diseases will be hypothesized.

Mechanisms of promoting EVT invasion

Hormones

During pregnancy, steroid hormones such as estrogen (E2) and progesterone synthesized in the corpus luteum act through their receptors and are essential for embryo implantation and maintenance of pregnancy during the first trimester [9]. Previous studies have demonstrated that hormones and their receptors intricately regulate EVT invasion, as depicted in Fig. 1.

Fig. 1.

Mechanisms of hormone promoting EVT invasion. Prostaglandins F2α, PGF2α; PGF2α receptor, PTGFR; mechanistic target of rapamycin, mTOR; focal adhesion kinase, FAK; mitogen activated protein kinases, MAPK; matrix metalloproteinase, MMP; angiopoetin-like 4, ANGPTL4; yes-associated transcriptional regulator, YAP; 17-β-estradiol, E2; G protein-coupled estrogen receptor, GPER or GPR30; phosphoinositide 3-kinase, PI3K; protein kinase B, Akt; Serum- and glucocorticoid-inducible kinase, SGK1; brain and muscle ARNT-like protein 1, BMAL1; specificity protein 1, SP1; DNA 5′-cytosine-methyltransferases 1, DNMT1; DAB2 interaction protein, DAB2IP; Runt-related transcription factor 2, RUNX2; extracellular signal-regulated kinase 1/2, ERK1/2; Neural-cadherin, N-cadherin; gonadotrophin-releasing hormone, GnRH; gonadotrophin-releasing hormone receptor, GnRHR. All figures were drawn with Biorender 

E2 facilitated the expression of SGK1, stimulating the migration and invasion of HTR-8/Svneo cells [10]. GPR30, E2 receptor, promoted EVT cell invasion by the YAP-enhanced ANGPTL4 expression and activation of the PI3K/Akt signaling pathway [11, 12]. Progesterone enhanced the invasion capacity of EVT cells by activating the PI3K/Akt signaling pathway [13]. Simultaneously, progesterone-induced SP1 expression by the upregulation of BMAL1, thereby increasing the expression of DNMT1 and DAB2IP, further enhancing the migration and invasion of HTR-8/SVneo cells mediated by MMP2/9 [14]. PGF2α, a luteolytic hormone, promoted invasive capacity through the FAK and MAPK1/3 signaling pathway, mTOR pathway activation, and upregulated MMP2/9 expression [15, 16].

Besides, protein and peptide hormones such as GnRH, leptin, and ELA facilitated EVT invasion. GnRH induced c-FOS and c-JUN activation, promoting cadherin-11 expression and EVT cell invasion. It also activated Akt and ERK1/2 signaling to increase RUNX2 expression, facilitating MMP2/9-mediated invasion of EVT cells while activating Akt signaling to upregulate TWIST expression and enhance invasion of EVT cells [17–19]. In addition, leptin can induce Notch1 expression to stimulate the PI3K/Akt signaling pathway, upregulate MMP14 expression, and promote EVT cell invasion [20]. Moreover, ELA activated the APJ-associated pathway and enhanced the invasion and proliferation of EVT cells [21].

Angiogenesis-related factors

The invasion of EVT is synchronized with angiogenesis, a process that facilitates maternal–fetal circulation [22]. Various angiogenesis-related factors have been identified to play crucial roles in this invasion process and are shown in Fig. 2.

Fig. 2.

Mechanisms of angiogenesis-related factors in promoting invasion. Bone morphogenetic protein 2, BMP2; epidermal growth factor, EGF; semaphorin 4D, SEMA4D; endocrine gland-derived vascular endothelial growth factor, EG-VEGF; bone morphogenetic protein and activin membrane-bound inhibitor, BAMBI; Angiopoietin-2, Ang-2; wingless/integrated, Wnt; mesenchymal-epithelial transition factor, Met; growth differentiation factor, GDF; epidermal growth factor receptor, EGFR; prokineticin receptors, PROKR; phosphoinositide 3-kinase, PI3K; inhibitor of DNA-binding protein, ID; mammary serine protease inhibitor, Maspin; signal transducer and activator of transcription, STAT; SRY-box transcription factor 4, SOX4; protein kinase B, Akt; snail family transcriptional repressor 1, Snail; insulin-like growth factor binding protein 3, IGFBP3; snail family transcriptional repressor 2-like 1, Slug; IgG-like domain, AMIGO; c-Jun N-terminal kinases, JNK; extracellular signal-regulated kinase, ERK; connective tissue growth factor, CTGF; matrix metalloproteinase, MMP; vascular endothelial growth factor, VEGF; mitogen-activated protein kinases, MAPK; neural-cadherin, N-cadherin; cellular Jun proto-oncogene, c-JUN; Hes-related family BHLH transcription factor with YRPW motif 1, HEY-1; microRNA, miR; zinc finger e-Box binding Homeobox 1, ZEB1; insulin-like growth factor 1, IGF-1; proteasome 26S subunit, non-ATPase 14, PSMD14; Grb2-associated binder-1, GAB-1; follistatin-like 3, FSTL3; kisspeptin-1, KISS1; placenta growth factor, PIGF

EGF family

The members of the EGF family, including amphiregulin, EGF, VEGFC, and EG-VEGF, have been found to influence trophoblast cell invasion significantly through various mechanisms. For example, amphiregulin enhanced invasion by upregulating MMP 9, TIMP-1, and the MMP 9/TIMP-1 ratio through ERK 1/2 and Akt signaling pathways [23]. EGF promoted invasion by activating STAT5, elevating STAT5B, and phosphorylating ERK1/2, STAT1, and STAT3. It is also bound to EGFR, activating the PI3K/Akt pathway and downregulating CTGF expression, ID3 protein, and KISS expression [24–27]. Additionally, VEGFC induced by maspin enhanced the invasion of normoxic EVT cells [28]. EG-VEGF modulated trophoblast cell invasion by activating the ERK 1/2 pathway and increasing MMP 2 and MMP 9 expression [29]. This effect is achieved through either PROKR 2 or primary cilia regulation, further contributing to trophoblast cell invasion [30].

TGF-β superfamily

A member of the TGF-β superfamily BMP2 contributed to the endothelial-like tube formation [31]. BMP2 exerted its functions through autocrine and paracrine mechanisms and interacted with ALK2/3/4 type I receptors and BMPR2 or ACVR2A type II receptors. Promoting trophoblast cell invasion by BMP2 involves several intricate pathways [32]. Specifically, BMP2 promoted trophoblast invasion, and its activity was closely associated with the presence of AMIGO2 [31]. BMP2-induced IGFBP3 occurred through ID1 mediation, stimulating the charge of trophoblast [33]. Furthermore, BMP2 regulated specific genetic elements by upregulating LncRNA NR026833.1 and competitively binding to miR-502-5p to promote the expression of Snail, consequently increasing MMP2 expression in primary extravillous trophoblast cells, ultimately facilitating cell invasion [34]. Additionally, BMP2 activated the canonical WNT/β-catenin signaling pathway via BAMBI, which typically acted as an antagonist of TGF-β signaling by interfering with the regular interaction between TGF-β type I and Type II receptors [35]. This activation of inhibiting βA led to activin A production through SMAD1/5/8-SMAD4 signaling transduction and enhanced the trophoblast invasion [32].

Activins A, B, and AB are members of the TGF-β superfamily and can promote invasion by activating SMAD2/3-SMAD4 [36]. Furthermore, activin A, when binding to two types of transmembrane serine/threonine kinase receptors, activates the SMAD2/3-SMAD4 signaling pathway and subsequently upregulates the expression of Snail-induced MMP2 and integrin β1/β3, thus enhancing the invasion of EVT cells [37–39].

Regarding members of the TGF-β superfamily GDF-8 and GDF-11, they can facilitate trophoblast invasion by affecting ECM remodeling. The expression of FSTL3, MMP2, and N-cadherin induced by GDF8 and ID2-mediated MMP2 expression induced by GDF11 can promote the invasion of EVT and HTR-8 via SMAD2/3 signaling pathways [40–42].

Other related factors

IGF-1 and Ang-2 are angiogenic growth factors. IGF-1 upregulated the expression of ZEB-1 through the ERK/MAPK pathway, which in turn bound to the miR-183 promoter to suppress the expression of miR-153, thus promoting EVT invasion [43]. Ang-2 regulated trophoblast migration and invasion during early pregnancy by activating Tie2 and the JNK/c-JUN signaling pathway [44].

Transcription factors, such as HEY-1-induced PSMD14 transcription, have been identified to promote angiogenesis and EVT invasion [45]. Moreover, acting as a transcription factor, EGR1 downregulates miR-574, consequently weakening the inhibitory impact of miR-574 on GAB-1 and ultimately leading to the upregulation of invasion among EVT cells [46].

Additionally, previous studies have elucidated the relationship between DLL4-Notch signal transduction, SEMA4D, and Wnt5a in angiogenesis. DLL4 has been identified as a pivotal factor in enhancing migration and invasion abilities of HTR-8/SVneo cells by directly binding to Notch receptors and upregulating the expression of EphrinB2 [47]. Additionally, SEMA4D can regulate angiogenesis and attach to Plexin-B1, activating the Met/PI3K/Akt pathway, leading to upregulation of MMP-2 and enhancing trophoblast invasiveness [48, 49]. Wnt5A promoted invasion through the Wnt-induced JNK pathway and Wnt/PKC pathway [50].

Hypoxia and stress-related factors

Hypoxia is a common feature of the microenvironment surrounding EVT cells, which is pivotal in cell proliferation, invasion, and migration during the initial stages of human placentation [51]. Hypoxia causes an imbalance of oxidants and antioxidants, contributing to oxidative stress, which could affect invasion [52]. The detailed mechanisms by which hypoxia and stress affect EVT invasion are shown in Fig. 3.

Fig. 3.

Mechanisms of promoting invasion under hypoxia and stress. Extravillous trophoblast, EVT; alpha-1 antitrypsin, A1AT; high temperature requirement A1, HTRA 1; protein kinase B, Akt; hepatocyte growth factor, HGF; matrix metalloproteinase, MMP; hypoxia-inducible factor 1α, HIF-1α; neurogenic locus notch homolog protein 1, Notch1; extracellular signal-regulated kinase, ERK; endothelin receptor type B, ETBR; gene-thioredoxin-interacting protein, TXNIP; signal transducer and activator of transcription, STAT; microsomal glutathione transferase 1, MGST1; phosphoinositide 3-kinase, PI3K; mechanistic target of rapamycin, mTOR; glycogen synthase kinase-3beta, GSK3β; macrophage inflammatory protein-1β, MIP-1β; adrenomedullin 2, ADM2; urokinase-type plasminogen activator receptor, uPAR; urokinase-type plasminogen activator, uPA; interleukin, IL; endothelin receptor type B, ETBR; endoplasmic reticulum, ER; reactive oxygen species, ROS

Previous studies have suggested that HIF-1α, a crucial protein that safeguards EVT cells from hypoxia, also promotes invasion of EVT cells. HIF-1α may occur by activating the Notch1/STAT3/ETBR signaling and the uPA-uPAR pathways [51, 53]. HIF-1α also triggers upregulated expression of ADM2 and increases the expression of MMP9 and Rac1, resulting in cell invasion [54]. Under a hypoxic environment, HGF facilitates the migration/invasion of HTR-8/SVneo cells. This phenomenon is correlated with augmentation of MMP1/9 and activation of Akt and ERK1/2 pathways. Meanwhile, phosphorylation of Akt and ERK1/2 recruited HIF-1α and synergistically enhanced the migration in HTR-8/SVneo cells [55].

H2O2 promoted the invasion of HTR-8/SVneo by triggering STAT-1 and STAT-3 to induce IL-8 and MIP-1β secretion and increase the MMP-9 without affecting cell proliferation, viability, or apoptosis [56]. TXNIP, an axiomatic ROS regulator, accelerated the migration and invasion of the trophoblast cells through STAT3 and Vimentin-related pathways [57]. MGST1 and Nesfatin-1 attenuated oxidative stress in trophoblast cells. They increased trophoblast migration and invasion via the activation of the Akt signaling pathway [58, 59].

Additionally, the endoplasmic reticulum is also involved in the regulation of EVT invasion. A1AT-induced ER stress upregulated the expression of HTRA1 and resulted in the invasion of EVTs [60].

Extracellular matrix microenvironment

The extracellular matrix (ECM) provides a supportive environment for trophoblast invasion. Various factors have been implicated in regulating ECM remodeling facilitating trophoblast invasion, as described in Fig. 4, through interactions with molecules and activating specific pathways [61].

Fig. 4.

Invasion-promoting mechanisms in the extracellular matrix microenvironment. Wingless/integrated, WNT; C–C motif chemokine ligand, CCL; collagen triple helix repeat containing-1, CTHRC1; galectin-9, Gal-9; hyaluronic acid, HA; chemokine receptor 3, CCR3; a cluster of differentiation 44, CD44; extracellular signal-regulated kinase 1/2, ERK1/2; phosphoinositide 3-kinase, PI3K; protein kinase B, Akt; endothelial nitric oxide synthase, eNOS; mitogen-activated protein kinases, MAPK; extravillous trophoblast, EVT; matrix metalloproteinase, MMP

The metabolism of ECM components, such as fibulin-5, HA, CTHRC1, and VGVAPG, provided the environment for invasion [62]. HA promotes the invasion of EVT by binding to the membrane receptor CD44 [63]. Furthermore, CD44 and MMP9 expressions were increased by WNT3A, WNT5A, and WNT10B, promoting invasion in EVT [64]. CTHRC1 could impact trophoblast function by inhibiting the Wnt/β-catenin pathway, thereby promoting invasion of HTR-8/SVneo cells, and this effect might be associated with the inhibition of collagen formation and the induction of cell migration [65]. Additionally, elastin-derived peptide VGVAPG, released during the ECM components elastin breakdown in the walls of uterine spiral arteries, promotes EVT migration and invasion through the phosphorylation of eNOS and activation of the MAPK pathway. Furthermore, VGVAPG can establish a positive feedback mechanism, leading to excessive elastin degradation and further invasion of EVT cells [66].

Trophoblast-derived CCL24 and Gal-9 were secreted into the extracellular matrix and acted on its receptors as essential mediators in trophoblast self-regulation. CCL24 promotes the expression of Ki67 and MMP9 via ERK1/2 and PI3K signaling pathways [67]. Gal-9 induced the p38 pathway and promoted the invasion [68].

Epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is a morphological change that transforms epithelial cells into mesenchymal phenotype, which contributes to trophoblasts’ invasiveness [69]. Figure 5 shows how various factors regulate EMT to promote EVT invasion.

Fig. 5.

Mechanisms of EMT and immune inflammation-related factors promoting-invasion. Extravillous trophoblast, EVT; epithelial-mesenchymal transition, EMT; interleukin 1β, IL-1β; interleukin 17, IL-17; interleukin 1 receptor, IL-1R; C–C motif chemokine ligand 5, CCL; CXC chemokine receptor 6, CXCR6; galectin-14, Gal-14; forkhead box C2, FOXC2; granulocyte colony-stimulating factor, G-CSF; granulocyte colony-stimulating factor receptor, G-CSFR; abnormal polarization of decidual macrophages, dMϕ; matrix metalloproteinase, MMP; sonic hedgehog, Shh; phosphoinositide 3-kinase, PI3K; protein kinase B, Akt; wingless/integrated, Wnt; zinc finger e-Box binding homeobox 1, ZEB1; glioma-associated oncogene homolog 1, Gli1; peroxisome proliferator-activated receptors, PPARs; retinoid X receptor-α, RXR-α; serum amyloid A, SAA; E2F transcription factor 1, E2F1; a disintegrin and metalloproteinase with thrombospondin type 1 motif 7, ADAMTS-7; focal adhesion kinase, FAK; neural-cadherin, N-cadherin; extracellular signal-regulated kinase1/2, ERK1/2; nuclear factor-kappa B, NF-κB; extravillous trophoblast, EVT; toll-like receptor 4, TLR4; alternatively activated macrophages, M2

It has demonstrated that chemokines CXCL5 bound to CXCR2, and CCL21 combined with CCR7 upregulated the expression of N-cadherin by activating the ERK1/2 pathway and promoted migration and invasion in HTR-8/Svneo cells via inducing the EMT process [70, 71].

CD97 and FOXC2 are put forward to regulate EMT in tumor invasion. CD97 upregulated FOXC2 expression, which increased invasion in trophoblasts by the PI3K/Akt and Hedgehog signaling pathways [72, 73].

Gal-14, a galectin family member, has high-level expression in the placenta. Gal-14 promoted EMT induced by N-cadherin and enhanced HTR-8/Svneo cell invasion by upregulating MMP9 via the Akt signaling pathway [74].

E2F1, a member of the E2F family, has been shown to contribute to the activation of EMT of trophoblast cells and facilitate trophoblast proliferation and invasion of HTR-8/Svneo by increasing ZEB1 expression [69].

Immune and inflammatory related-factor

Pregnancy is similar to semi-allograft transplantation for the pregnant woman [75]. Maternal–fetal immune tolerance allows semi-allogenic fetus survival in the uterine. An appropriate immune microenvironment provides conditions for the invasion of EVT [76]. Figure 5 displayed the mechanisms of immune and inflammatory-related factors in EVT invasion.

The immune cells, such as macrophages and T cells, enrich the maternal–fetal interface and contribute to immune tolerance [77]. Macrophages can affect the invasion in the trophoblast by secreting various factors, such as G-CSF and CCL5. M2 polarization was induced by abnormal trophoblasts and increased the secretion of G-CSF [78]. M2 macrophages induced by IL-4/IL-13 stimulated the expression of G-CSFR in HTR-8. The combination of G-CSFR and G-CSF enhanced the invasion and proliferation of trophoblast cells, which may eventually lead to PAS [78]. Additionally, the secretion of CCL5 by M2 macrophages activated the PI3K signaling pathway and promoted the invasive capacity of trophoblast cells [79]. Moreover, the interaction of CXCL16 with CXCR6 expressed by decidual γδ T cells led to the increased invasion of trophoblasts [80].

The overactivation of inflammation at the maternal–fetal interface may impair EVT invasion [81]. The release of proinflammatory factors such as IL-1β and IL-17 mediates the activation of inflammation. IL-1β increased the expression of ADAMTS-7 via the FAK signaling pathway, thereby enhancing the migration and invasion of HTR8/SVneo cells [82]. Another study showed that elevating the expression of IL-17 can mediate the PPAR-γ/RXR-α/Wnt signaling pathway, enhancing the proliferation, migration, and invasion of HTR8/SVneo cells [83]. SAA is an acute-phase protein produced after inflammation acts on the body. It induces the invasion of EVT cells by affecting the expression of TLR4. The above mechanism may alleviate the injury of overactivation of inflammation to EVT invasion [84].

Programmed cell death

EVT invasion is associated with post-apoptotic cell membrane eversion and LC3-mediated apoptosis. Several factors in Fig. 6 modulate trophoblastic cells, inhibiting apoptosis and inducing autophagy. Extensive research has consistently linked these processes to enhanced EVT invasion capabilities.

Fig. 6.

Mechanisms of programmed death-related factors promoting EVT invasion. Chorionic villous mesenchymal stem cell, CV-MSC; human umbilical cord mesenchymal stem cell, hUCMSC; decidual stromal cell, DSC; interleukin, IL; LDL receptor-related protein 6, LRP6; wingless/integrated, Wnt; transcription factor like transcription factor CP2, TFCP2; p57 kinase inhibitory protein(KIP)2, p57KIP2; left–right determination factor, Lefty; claudins3, CLDN3; annexin A4, ANXA4; extravillous trophoblast, EVT; Janus kinase, JAK; signal transducer and activator of transcription 3, STAT3; protein kinase B, Akt; extracellular signal-regulated kinase, ERK; phosphoinositide 3-kinase, PI3K; c-Jun N-terminal kinases, JNK; matrix metalloproteinase, MMP; endothelial nitric oxide synthase, eNOS; Ras-related protein 7, Rab7

A member of the TGF-β superfamily Lefty, a member of the tight junction protein family CLDN3, and a member of the annexin family ANXA4 were suggested to inhibit HTR8/SVneo cell apoptosis. Lefty-induced inhibition of nodal signaling pathways, CLDN3-induced activation of ERK1/2 signaling pathway, and ANXA4-induced activation of the PI3K/Akt/eNOS pathway upregulated the expression of MMP2/9. Furthermore, it facilitated HTR8/SVneo cell proliferation, migration, and invasion [85–87].

Human umbilical cord mesenchymal stem cell (hUCMSC)-derived TFCP2 and decidual stromal cells (DSCs)-derived WNT16 inhibited cell apoptosis. Both of them increased the expression of MMP2 by the β-catenin pathway and resulted in the invasion of trophoblasts [88, 89].

Additionally, p57KIP2, an enzyme inhibitor, promoted migration and invasion in HTR8/Snevo by suppressing the JNK/SAPK signaling pathway and inhibiting apoptosis [90].

IL-6 and LRP6 can promote invasion by promoting autophagy. IL-6 secretion of chorionic villi-derived mesenchymal stromal cells (CV-MSCs) accelerated autophagy of placental explants and HTR-8 cells through the JAK2/STAT3 signaling pathway, further enhanced their proliferation and invasion [91]. Additionally, LRP6 induced autophagy by activating the Wnt/β-catenin pathway and increased migration and invasion in HTR-8/Svneo cells [92].

Epigenetic modifications

Epigenetics, such as histone modification, RNA modification, and non-coding RNA can affect gene expression by affecting chromatin structure, and then affect trophoblast cell invasion [93]. Figure 7 depicts the effects of enzymes that modulate RNA and histone modifications and non-coding RNA on trophoblast cell invasion.

Fig. 7.

Mechanisms of epigenetic modification-related factors promoting invasion in EVT. microRNAs, miRNAs; circular RNAs, circRNAs; long non-coding RNAs, lncRNAs; alkB homolog 5, ALKBH5; histone deacetylase sirtuin 2, SIRT2; circular RNA homeodomain interacting protein kinase 3, circHIPK3; circRNA furin, paired basic aminoacid cleaving enzyme, circ-FURIN; N6-methyladenosine, m6A; transforming growth factor, TGF-β; nuclear factor kappa B, NF-kB; atypical chemokine receptor 2, ACKR2; potassium channel modulatory factor 1, KCMF1; transcription factor AP-2 alpha, TFAP2A; metastasis-associated lung adenocarcinoma transcript-1, MALAT1; F-box and WD repeat domain–containing 7, FBXW7; cryptochrome 2, CRY2; SMAD family members, SMADs; ephrin receptor B4, EPHB4; activin receptor-like kinase 7, ALK7; amine oxidase copper containing 1, AOC1; thrombospondin 2, THBS2; C-X-C motif chemokine ligand 11, CXCL11; nuclear factor kappa B, transcription-κB; soluble fms-like tyrsine kinase-1, sFlt-1; E3 ubiquitin-containing protein ligases, β-TrCP; GATA binding protein 2, GATA2; wingless/integrated, Wnt; cluster of differentiation 97, CD97; vascular endothelial growth factor, VEGF; matrix metalloproteinase, MMP; bromodomain containing 4, BRD4

Studies shed light on the intricate role of RNA epigenetic modifications in trophoblast invasion, highlighting the involvement of various coding and non-coding RNAs. It has shown that miR-101 inhibited the expression of BRD4 and subsequently inhibited NF-κB/CXCL11 axis expression, thereby promoting trophoblast cell invasion [94]. Recent evidence suggests that miR-424-5p inhibited AOC1 expression and facilitated HTR-8/Svneo and TEV-1 invasive capacity via the Wnt/β-catenin signaling pathway [95]. Moreover, multiple studies have provided evidence that miRNAs can facilitate trophoblast invasion. Examples of such miR-18a, miR-135a-5p, miR-139-5p, miR-150-5p, miR-218-5p, miR-221-3p, miR-454, and miR-520c-3p [96–104].

It is shown that RNA demethylase ALKBH5 and histone deacetylase SIRT2 play pivotal roles in increased trophoblast cell invasion. The overexpression of ALKBH5 caused demethylate methylation modification of the N6-methyladenosine(m6A), a common RNA modification, promoting the expression of SMAD1/5. SMAD proteins acted as a transcriptional regulator that mediated the TGF-β signal to increase the expression of MMP9 and ITGA1 and thus promoted HTR-8/SVneo cell invasion [105]. SIRT2 stimulated the deacetylation of the p65 NF-κB subunit. This pathway will increase the miR-146a and produce more ACKR2, promoting EVT invasion [106]. The mechanisms by which these miR RNA regulate EVT invasion are detailed in Fig. 7.

CircRNAs played a role in EVT invasion by sponging miR RNA, such as hsa_circ_0000848 acting as miR-6768-5p sponge [107]. The overexpression of circHIPK3, which functioned as a miR-346 sponge, can inhibit the function of miR-346 and increase KCMF1, thus promoting HTR8/SVneo cell invasion [108]. Besides, circFURIN, a converting enzyme under hypoxic conditions, can enhance trophoblast invasion by inhibiting miR-34a-5p from increasing the target gene TFAP2A expression [109].

For lncRNA, it was found that overexpression of lncRNA-H19 promoted autophagy and enhanced invasion of EVT cells through activation of PI3K/Akt and mTOR pathways [110]. Meanwhile, lncRNA H19-induced miR-675-5p decreased GATA2 expression and upregulated the transcription of MMP14 and MMP13 proteins, which catalyzed trophoblast invasion [111]. Moreover, it has been demonstrated that LncRNA MALAT1 impaired CRY2 protein stability by recruiting FBXW7 and further enhanced HTR-8/SVneo cell migration and invasion[112].

Microbiota

Recently, the human microbiome has been suggested to play a role in the endometrium [113]. Current research indicates that, particularly during pregnancy, specific uterine microbiota, such as Lactobacillus crispatus (L. crispatus) or gut microbiota (GM), such as Fusobacterium nucleatum (F. nucleatum) and Akkermansia muciniphila (Am), affect EVT cells proliferation, invasion, and migration. Those specific bacterial species increase the invasive ability of EVT by promoting the secretion of hormones or specific proteins, as shown in Fig. 8.

Fig. 8.

Mechanisms of microbiota promoting EVT invasion and other related factors promoting invasion. Eukaryotic translation initiation factor 5A1, EIF5A1; extracellular signal-regulated kinase, ERK; nuclear receptor coactivator 6, NCOA6; nuclear factor kappa B, NF-kB; signal transducer and activator of transcription 3, STAT3; CXC chemokine receptor 2, CXCR2; phosphoinositide 3-kinase, PI3K; protein kinase B, AKt; syntaxin2, STX2; EP300 interacting inhibitor of differentiation 1, EID1; receptor tyrosine kinase-like orphan receptor 1, ROR1; mechanistic target of rapamycin, mTOR; MARVEL domain-containing 1, MARVELD1; Lactobacillus crispatus, L. crispatus; Fusobacterium nucleatum, F. nucleatum; Akkermansia muciniphila, Am; epidermal growth factor receptor, EGFR; matrix metalloproteinase, MMP

L. crispatus can release soluble factors that enhance the activity of MMP1 and MMP2, significantly increasing the invasive capability of HTR-8/SVneo cells [114]. F. nucleatum triggers the invasion of HTR8/SVneo cells by upregulating the secretion of soluble mediators, including CXCL1, IL-6, and IL-8, as well as metalloproteinases like MMP-2 and MMP-9 [115]. Another study demonstrates that Akkermansia muciniphila extracellular vesicles (Am-EVs) influence the EGFR/PI3K/Akt signaling pathway, leading to increased proliferation, migration, and invasion of HTR-8/SVneo cells [116].

Others

Previous studies have shown that various factors promoted invasion in HTR8/Svneo cells. Still, they did not act through the above biological processes and may affect trophoblast invasion through other biological processes drawn in Fig. 8.

RTK family member ROR1, CXCR2 protein, and epithelial morphoregulator STX2 promoted trophoblast proliferation, migration, and invasion by triggering the PI3K-Akt pathway [117–119].

Transcription-related factors such as EID1, EIF5A1, and NCOA6 were essential in regulating signaling pathways and influencing trophoblast invasive capacity. EID1 has been found to encourage trophoblast invasion by regulating the Akt/β-catenin signaling pathway [120]. Additionally, EIF5A1 improved trophoblast invasion and migration in vitro and villous explant by activating the integrin/ERK signaling pathway mediated by ARAF, shedding light on the complexities of trophoblast behavior and signaling pathways [121]. Moreover, NCOA6-induced activation of the NF-κB pathway [122].

The nuclear factor MARVELD1, identified as a novel candidate for tumor suppression, was put forward that it may be involved in the occurrence of PA in recent years. The reduction of MARVELD1 and subsequent decrease in integrin β4 contribute to trophoblasts’ heightened invasion and migration [123].

Zinc, an essential micronutrient, has been shown to enhance migration and invasion in trophoblast cells with the upregulation of MMP-2/9 expression mediated by STAT3 [124].

Summary and outlook

Herein, this review describes the mechanism of EVT abnormal invasion from the following aspects: hormones, angiogenesis, hypoxia and stress, extracellular matrix remodeling, EMT, immune and inflammation, programmed cell death, epigenetic modifications, and microbiota.

In our review, several signaling pathways appeared to play crucial roles. The MAPK and PI3K/Akt pathways play a role in most physiological processes summarized here and promote invasion. Additionally, JNK, Wnt/β-catenin, and FAK signaling pathways also play vital roles in promoting EVT cell invasion, providing essential clues for future studies on the mechanisms of trophoblast cell invasion and potential therapeutic targets for diseases.

Multiple studies have indicated that some factors have complex and variable regulatory functions to EVT invasive capacity. Several studies have suggested that proliferation, migration, and invasion may not necessarily be co-directional. Adu-Gyamfi et al. found that DiO2 could decrease trophoblast cell proliferation but can promote the migration and invasion of EVT cells by upregulating the expression of Twist1 and HIF-α. Xia Xu et al. [125] reported that FtMt enhances invasion and angiogenesis capabilities through the HIF-1α/VEGF signaling pathway but inhibits cell proliferation [52].

The interaction between EVT and mesenchymal stem cells (MSCs), decidual stromal cells (DSCs), and macrophages has been a hotspot in research on EVT invasion in recent years. Macrophage polarization produces M1 and M2 phenotypes, and M2 has been demonstrated to improve EVT cell invasion [78]. Liu M. et al. proved that human decidual stromal cells (HDSCs)-sEVs could be uptaken by trophoblast cells and promote charge [126]. DSCs can regulate inflammatory factors such as CXCL5 to promote the EVT invasion and EMT process and secrete more WNT16 to promote the survival and invasion of EVT cells [70, 89]. hUCMSCs, which are easy to obtain noninvasively, can self-renew, have multi-directional differentiation, and have paracrine properties. hUCMSCs can promote the proliferation, migration, and invasion of HTR-8/SVneo trophoblast cells in vitro or co-culture. Thus, it might be a potential source for cell therapy [127]. Chorionic villus-derived mesenchymal stem cells (CV-MSCs) can also mediate EVT autophagy under hypoxia to promote invasion [91].

Presently, researchers are more likely to believe that PAS results from damage to the uterine myometrium [3], such as uterine surgery. Studies have shown that about 25% of PAS patients have not undergone uterine surgery [128]. Besides, some previous studies speculated that some factors that increased the invasion ability of trophoblast cells may be also related to the development of PAS, such as LAMC2, CXCL12, YKL-40, AGGF1, and G-CSF [78, 129–132]. Some studies have shown that the depth of EVT invasion is related to the prognosis of PAS [2]. The above mechanisms may contribute to deepening our understanding of the development of PAS.

The first stage of preeclampsia occurs in the uterine decidua, where the insufficient expression of various factors leads to abnormal spiral artery remodeling and inadequate trophoblast cell invasion. We reviewed the roles of various causative factors in 62 studies categorically based on preeclamptic patients, including the modulation of epigenetic modifications through ALKBH5 and SRIT2; the influence of hormones such as progesterone, E2; the role of hypoxia-related factors including HIF-1α and ADM2; the impact of oxidative stress-mediated by MGST1 and Nesfatin-1; the involvement of angiogenesis via PTPRO, EGR1, and EGF; the contribution of programmed cell death through TFCP2 and CLDN3; and the significance of immune responses involving IL-25, TIPE2, CXCL3, and Apelin-36 [10, 27, 29, 46, 52–54, 58, 62, 71, 87, 88, 105, 106, 133–138]. These findings offer promising insights into the mechanisms underlying the pathogenesis of preeclampsia and suggest various therapeutic targets for intervention. Targeting these pathways and factors may enhance trophoblast function, ultimately preventing preeclampsia and improving the fetal pregnancy environment.

This review includes articles that utilized extravillous trophoblast cell lines as experimental models. The results of most experiments were derived from the HTR-8/SVneo cells, which belong to the EVT cell line. There is evidence that HTR-8/SVneo cells cannot completely represent the properties of extravillous trophoblast cells [11]. Horvath G et al. observed that PACAP had no effect on the invasiveness of HTR-8/SVneo cells but contributed to the proliferation and invasion of HIPEC 65 cells, which also belong to the EVT cell line. The result suggested that its effects on invasion depend on the cell type [139]. We anticipate that further experiments will employ human primary EVT cells and validate through vivo experiments in the future.

Organoid cultures are a significant development in the trophoblast cell field. Compared to the EVT cell line cultures in 2D, the advantage of 3D organ culture is the preservation of the typical villous structure and it can study the morphogenetic changes [140, 141]. Trophoblastic organoid cultures were generated by culturing villi from first-trimester placentas in the organoid medium. The medium was subsequently changed to EVT medium to differentiate the villous cytotrophoblasts (VCTs) in the trophoblast organoids into EVTs [142]. The EVT organoid cultures have gradually emerged as an experimental model to study the invasive capacity of EVTs [143]. It can be used to study physiology during pregnancy and the pathophysiology of placental diseases such as pre-eclampsia and fetal growth restriction [142, 144–147]. In addition, it may allow the study of the interaction of the uterine decidual microenvironment with the invasiveness of EVT cells, such as constructing a model of the interaction between decidual NK cells and EVT [148].

With recent advances in technology, the research on the type and status of single cells has been deepened, which has exacerbated the understanding of the significant obstetric diseases caused by defective arterial transformation, such as preeclampsia, fetal growth restriction, and preterm birth. Single-cell RNA is used to characterize the transcriptomics of EVT subpopulations and divide EVT into two subpopulations, improving our understanding of the heterogeneity of EVT and tissue [149]. Spatial transcriptomics is frequently used with single-cell sequencing, which has the advantage of studying single-cell characteristics and cell distribution and function at the tissue level. By characterizing the trophoblast invasion trajectory thoroughly and analyzing the communication between endothelial cells and EVT cells, the effect of trophoblast invasion defects on arterial transformation of pregnancy is explained. Single-cell proteomics can detect protein modifications and contribute to refining the study of the pathogenesis of pregnancy complications. The difficulty of single-cell proteomics technology is the low abundance of proteins in single cells, thus, it has not been widely used. This technology is mainly used in cancer and rarely in placental diseases [150]. Single-cell ATAC analysis is conducive to identifying upstream regulatory factors of placental diseases and studying gene regulatory networks in trophoblast lineage differentiation and function [151]. The above high-throughput detection technology will contribute to exploring the precise mechanism of genes and their upstream and downstream regulation at single cells and space levels. We anticipate that these advanced techniques will be used to explore the development and function of EVT cells, provide clinical diagnostic markers for obstetric diseases, and contribute to the early intervention of diseases.

Abbreviations

EVT

Extravillous trophoblast

EVCTs

EVT endovascular trophoblast cells

GCs

Giant cells

PAS

Placenta accreta spectrum

PE

Preeclampsia

RSA

Recurrent spontaneous abortion

FGR

Fetal growth restriction

GnRH

Gonadotrophin-releasing hormone

GnRHR

Gonadotrophin-releasing hormone receptor

PGF2α

Prostaglandins F2α

GPER or GPR30

G protein-coupled estrogen receptor

E2

17-β-Estradiol

ELA

ELABELA

RUNX2

Runt-related transcription factor 2

c-FOS/c-JUN

Activator protein-1 components

SGK1

Serum- and glucocorticoid-inducible kinase

ANGPTL4

Angiopoetin-like 4

GPR30

G protein-coupled receptor 30

BMAL1

Brain and muscle ARNT-like protein 1

SP1

Specificity protein 1

DNMT1

DNA 5′-cytosine-methyltransferases 1

DAB2IP

DAB2 interaction protein

APJ

Apelin receptor

ELABELA

ELA, also known as APELA

PTGFR

PGF2α receptor

FAK

Focal adhesion kinase

APJ

Apelin receptor

E2

Estrogen

YAP

Yes-associated protein

MMP

Matrix metalloproteinase

BMP2

Bone morphogenetic protein 2

BMPR

Bone morphogenetic protein receptors

ACVR

Receptor activin receptor

EGF

Epidermal growth factor

SEMA4D

Semaphorin 4D

ALK

Activin receptor-like kinase

TIMP-1

Tissue inhibitors of matrix metalloproteinase-1

TGF-β

Transforming growth factor-beta

ECM

Extracellular matrix

HTR-8/SVneo

Human trophoblast research-8/Swan-71 neo

ZEB-1

Zinc finger E-box binding homeobox 1

EGR1

Early growth response protein 1

DLL4

Delta-like 4

EG-VEGF

Endocrine gland-derived vascular endothelial growth factor

BAMBI

Bone morphogenetic protein and activin membrane-bound inhibitor

Ang-2

Angiopoietin-2

Wnt

Wingless/integrated

Met

Mesenchymal-epithelial transition factor

GDF

Growth differentiation factor

EGFR

Epidermal growth factor receptor

PROKR

Prokineticin receptors

PI3K

Phosphoinositide 3-kinase

ID

Inhibitor of DNA-binding protein

Maspin

Mammary serine protease inhibitor

STAT

Signal transducer and activator of transcription

SOX4

SRY-box transcription factor 4

AKt

Protein kinase B

SNAIL

Snail family transcriptional repressor 1

IGFBP3

Insulin-like growth factor binding protein 3

Slug

Snail family transcriptional repressor 2-like 1

AMIGO

IgG-like domain

JNK

C-Jun N-terminal kinases

c-JUN

Cellular Jun proto-oncogene

ERK

Extracellular signal-regulated kinase

CTGF

Connective tissue growth factor

VEGF

Vascular endothelial growth factor

MAPK

Mitogen-activated protein kinases

N-cadherin

Neural-cadherin

HEY-1

Hes-related family BHLH transcription factor with YRPW motif 1

miR

MicroRNA

ZEB1

Zinc finger e-box binding homeobox 1

IGF-1

Insulin-like growth factor 1

PSMD14

Proteasome 26S subunit, non-ATPase 14

GAB-1

Grb2-associated binder-1

FSTL3

Follistatin-like 3

KISS1

Kisspeptin-1

PIGF

Placenta growth factor

EphrinB2

Ephrin type-b receptor 2

A1AT

Alpha-1 antitrypsin

HTRA1

High temperature requirement A1

HGF

Hepatocyte growth factor

HIF-1α

Hypoxia-inducible factor 1α

Notch1

Neurogenic locus notch homolog protein 1

ETBR

Endothelin receptor type B

TXNIP

Gene-thioredoxin-interacting protein

MGST1

Microsomal glutathione transferase 1

mTOR

Mechanistic target of rapamycin

GSK3β

Glycogen synthase kinase-3beta

MIP-1β

Macrophage inflammatory protein-1β

ADM2

Adrenomedullin 2

uPAR

Urokinase-type plasminogen activator receptor

uPA

Urokinase-type plasminogen activator

IL

Interleukin

ETBR

Endothelin receptor type B

ER

Endoplasmic reticulum

ROS

Reactive oxygen species

Rac1

Ras-related C3 botulinum toxin substrate 1

CCL

C-C motif chemokine ligand

CTHRC1

Collagen triple helix repeat containing-1

Gal-9

Galectin-9

HA

Hyaluronic acid

CCR

Chemokine receptor

CD44

Cluster of differentiation 44

eNOS

Endothelial nitric oxide synthase

IL

Interleukin

IL-1R

Interleukin 1 receptor

CXCR

CXC chemokine receptor

EMT

Epithelial-to-mesenchymal transition

N-cadherin

Neural-cadherin

FOXC2

Forkhead box C2

Gal-14

Galectin-14

G-CSF

Granulocyte colony-stimulating factor

G-CSFR

Granulocyte colony-stimulating factor receptor

dMϕ

Abnormal polarization of decidual macrophages

M2

Alternatively activated macrophages

Shh

Sonic hedgehog

Wnt

Wingless/integrated

Gli1

Glioma-associated oncogene homolog 1

PPARs

Peroxisome proliferator-activated receptors

RXR-α

Retinoid X receptor-α

SAA

Serum amyloid A

TLR4

Toll-like receptor 4

E2F1

E2F transcription factor 1

ADAMTS-7

A disintegrin and metalloproteinase with thrombospondin type 1 motif 7

NF-κB

Nuclear factor-kappa B

PAS

Placenta accrete spectrum disorders

CV-MSC

Chorionic villous mesenchymal stem cell

hUCMSC

Human umbilical cord mesenchymal stem cell

DSC

Decidual stromal cell

LRP6

LDL receptor related protein 6

TFCP2

Transcription factor like transcription factor CP2

p57KIP2

P57 kinase inhibitory protein(KIP)2

Lefty

Left-right determination factor

CLDN3

Claudins3

ANXA4

Annexin A4

JAK

Janus kinase

Rab7

Ras-related protein 7

SAPK

Stress-activated protein kinases

miRNAs

MicroRNAs

BRD4

Bromodomain containing 4

AOC1

Amine oxidase copper containing 1

Wnt

Wingless/integrated

ALKBH5

AlkB homolog 5

SIRT2

Sirtuin 2

m6A

N6-methyladenosine

SMADs

SMAD family members

ITGA1

Integrin subunit alpha 1

ACKR2

Atypical chemokine receptor 2

circRNAs

Circular RNAs

circHIPK3

Histone deacetylase circular RNA homeodomain interacting protein kinase 3

KCMF1

Potassium channel modulatory factor 1

circ-FURIN

CircRNA furin, paired basic amino acid cleaving enzyme

TFAP2A

Transcription factor AP-2 alpha

lncRNAs

Long non-coding RNAs

MALAT1

Metastasis-associated lung adenocarcinoma transcript-1

FBXW7

F-box and WD repeat domain–containing 7

TGF-β

Transforming growth factor

CRY2

Cryptochrome 2

EPHB4

Ephrin receptor B4

ALK7

Activin receptor-like kinase 7

THBS2

Thrombospondin 2

sFlt-1

Soluble fms-like tyrsine kinase-1

β-TrCP

E3 ubiquitin-containing protein ligases

GATA2

GATA-binding protein 2

CD97

Cluster of differentiation 97

L. crispatus

Lactobacillus crispatus

GM

Gut microbiota

F. nucleatum

Fusobacterium nucleatum

Am

Akkermansia muciniphila

Am-EVs

Akkermansia muciniphila extracellular vesicles

ROR1

Receptor tyrosine kinase-like orphan receptor 1

STX2

Syntaxin2

EID1

EP300 interacting inhibitor of differentiation 1

EIF5A1

Eukaryotic translation initiation factor 5A1

NCOA6

Nuclear receptor coactivator 6

MARVELD1

MARVEL domain-containing 1

FtMt

Mitochondrial ferritin

MSCs

Mesenchymal stem cells

DSCs

Decidual stromal cells

HDSCs

Human Decidual stromal cells

PACAP

Pituitary adenylate cyclase-activating polypeptide

VCTs

Villous cytotrophoblasts

DSCs

Decidual stromal cells

PTPRO

Protien tyrosine phosphatase receptor type O

TIPE2

DTumor necrosis favtor-ɑ-induced protein-8 like-2

Author contribution

YY contributed to the study’s conception and design. All authors wrote the manuscript. YY, ZL, SW, YJ, and ZC completed the review editing. ZL and SW conducted the figure presentation. All authors revised the article and approved the final version.

Funding

This study is funded by the Research Foundation of Pediatrics College in Guangzhou Medical University (2023ekky001), and the Research Foundation of Guangzhou Medical University (2022A151).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The authors confirm that (1) the work described has not been published before; (2) it is not under consideration for publication elsewhere; (3) all co-authors have approved its publication; and (4) the responsible authorities have approved its publication at the institution where the work is carried out.

Competing interests

The authors declare no competing interests.