Molecular and immunological developments in placentas

Abstract

Cytotrophoblasts differentiate in two directions during early placentation: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). STBs face maternal immune cells in placentas, and EVTs, which invade the decidua and uterine myometrium, face the cells in the uterus. This situation, in which trophoblasts come into contact with maternal immune cells, is known as the maternal-fetal interface. Despite fetuses and fetusderived trophoblast cells being of the semi-allogeneic conceptus, fetuses and placentas are not rejected by the maternal immune system because of maternal-fetal tolerance. The acquired tolerance develops during normal placentation, resulting in normal fetal development in humans. In this review, we introduce placental development from the viewpoint of molecular biology. In addition, we discuss how the disruption of placental development could lead to complications in pregnancy, such as hypertensive disorder of pregnancy, fetal growth restriction, or miscarriage.

1. Introduction

Blastocysts are composed of the trophectoderm (TE), the outer layer of the blastocyst, and the inner cell mass, from which the fetus arises. Placental development starts in the uterus after the attachment of the trophectoderm to the decidualized endometrium in a low-nutrient and hypoxic environment [1]. Early placentation is supported by cytokines, including inflammatory cytokines, and growth factors produced by the endometrial glands. The trophectoderm then starts to proliferate and differentiate into proliferative cytotrophoblasts (CTBs). CTBs, which form cell columns at the tips of villi, are able to differentiate in two directions: into syncytiotrophoblasts (STBs) on the fetal side and into extravillous trophoblasts (EVTs) on the uterine side (Figure 1) [2]. Early placentation occurs under hypoxic conditions, with approximately 2% oxygen tension [3]. This protects the fetus from oxidative stress as it does not yet have a redox system. Then, the oxygen concentration increases to 7% oxygen tension by approximately the 13^th week of gestation, at which point the trophoblastic plug disintegrates away from the spiral arteries.

Figure 1. Differentiation of trophoblasts.

Cytotrophoblasts (CTBs) differentiate in two directions: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). STBs are formed by fusion of CTBs on the maternal side. On the other hand, EVTs detach from CTBs in the cell column, and invade the maternal decidua and myometrium. At the decidua, EVTs contact with maternal immune cells. Endovascular EVTs, which replace the endothelium and tunica media in the spiral artery, help maintain maternal blood flow into the placenta. dNK; decidual NK cells, DC; dendritic cells, Mϕ; macrophages.

STBs are generated by the fusion of CTBs via syncytialization (Figure 1). Syncytialized STBs form villi, which are involved in exchanging glucose, amino acids, organic ions, and oxygen between maternal and fetal blood. In humans, villi are composed of two layers, which are covered with a multinucleated and fused STB layer underpinned by CTBs. Nutrient exchange in the villi occurs via STBs, preventing direct contact between maternal and fetal blood. Since STBs do not express MHC class I or II, and they are not recognized by CD8⁺ cytotoxic T cells, they are able to escape from maternal immune cells [4]. The other direction of CTB differentiation involves the generation of EVTs. EVTs invade the endometrium, reaching one-third of the depth of the myometrium, and can be classified into two types according to the direction of differentiation: endovascular EVTs toward the spiral arteries, and interstitial EVTs into the myometrium (Figure 1) [5]. Endovascular EVTs are involved in the vascular remodeling of the spiral arteries by replacing themselves, resulting in an adequate supply of blood and nutrients into the placenta. This process is also supported by uterine natural killer (uNK) cells, by inducing apoptosis in vascular smooth muscle cells [6]. By contrast, the role of interstitial EVTs remains somewhat obscure. It is generally accepted that EVTs anchor the placenta with the uterus. Interstitial EVTs first encounter a variety of maternal immune cells—including uNK cells, macrophages, T cells, and stromal cells—during invasion (Figure 1). Upon cell-to-cell or cell-to-matrix contact, EVTs produce numerous cytokines, chemokines, and matrix metalloproteinases to complete the process. However, it remains to be fully elucidated why the invasion of EVTs is stopped after one-third the depth of the myometrium. Clarifying this mechanism is of great clinical importance since the development of a technique for regulating EVT invasion in the uterus may reduce the occurrence of massive bleeding-related maternal death due to placenta percreta. Inhibition of EVT invasion leads to preeclampsia. In addition, disruption of trophoblast differentiation, either STBs or EVTs, could result in multiple pregnancy complications, including miscarriage, fetal growth restriction (FGR), or preeclampsia.

2. Markers of trophoblast differentiation

2.1. Trophoblast stem cells

Although evidence for early embryogenesis in humans is increasing, placentation research often leverages animal models over human ones because of the difficulties associated with obtaining human samples. However, due to the differences between the placental structures of humans and other species, it is difficult to deduce phenomena in human placentas from data obtained using animal models. An alternative approach for the study of human placental development involves the use of trophoblast cell lines and primary human trophoblasts [7]. However, the molecular mechanisms in the former, especially those underlying cellular survival, are often modified or disrupted compared with normal trophoblasts. In the latter, culturing cells for a long time is often not possible. During the development of trophoblast stem cell research, mouse trophoblast stem (mTS) cells were first established from blastocysts and extra-embryonic ectoderm [8]. Human trophoblastic stem (hTS) cells were constructed 20 years later [9]. hTS cells stemness is maintained by culturing with activated wingless/integrated and epidermal growth factor, combined with the inhibition of transforming growth factor-β, histone deacetylase, and Rho-associated protein kinase. In hTS cells fibroblast growth factor receptor 2B (FGFR2B), but not FGFR2C, is highly expressed. In mTS cells on the other hand, caudal type homeobox 2 (CDX2), eomesodermin, estrogen-related receptor β, and SRY-box transcription factor 2—which maintain stemness—are highly expressed; these are very low in hTS cells. Research on human placental differentiation is expected to advance new uses of hTS cells.

2.2. Differentiation to STBs

CDX2 is a highly specific TE marker. CDX2 is temporally expressed in human and mouse TEs [10]. The expression of CDX2 was observed in most cells in TE at 5 days post-fertilization, but only a few cells were observed after 6 days. In spite of the differential stages, trophoblasts commonly express GATA-binding protein (GATA) 2, GATA3, transcription factor AP-2 gamma (TFAP2C), keratin 7, and keratin 19, but not the major histocompatibility complex HLA-A, HLA-B, Thy-1 cell surface antigen (also known as CD90), vimentin [9, 11–13]; the reverse is true in the stromal cells of placentas (Figure 2). Previously, STBs were found to lack GATA3 and TFAP2C expression [14]. Proliferative CTBs express tumor protein 63 (TP63)—a DNA-binding transcription factor that regulates proliferation, differentiation, cell adhesion and apoptosis, E74-like ETS transcription factor 5, and TEA domain transcription factor 4 [9, 14]. TP63 in the third trimester of CTBs was found to be lower than in the first trimester, consistent with the lower proliferative capacity of the third trimester CTBs. For the differentiation of hTS cells into STBs, forskolin, a cyclin AMP activator, enhances cellular fusion, resulting in the formation of multinucleated STBs [9]. These cells express cell surface markers specific to STBs, including syndecan 1 (also known as CD138), human chorionic gonadotropin (hCG), and human placental lactogen (hPL). In pregnant women, hCG expression rises, peaks at 10 weeks of gestation, and gradually decreases. Meanwhile, the serum hPL concentration, which increases linearly during pregnancy, had been used to evaluate placental growth in clinical settings. Syncytialization in primary trophoblasts is induced by a Rho-associated protein kinase inhibitor, which is required for maintaining stemness in hTS cells; whereas in BeWo cells a choriocarcinoma cell line—frequently used as an in vitro syncytialization model—is induced by forskolin [7, 15]. Although hCG and hPL are often used to detect STBs, EVTs also express these hormones [16, 17]. Therefore, these placental hormones are not specific to STBs.

Figure 2. Molecular markers of trophoblast-differentiation.

Cytotrophoblasts (CTBs) differentiate into syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). Common markers and differentiation markers are shown. GCM1 (glial cells missing transcription factor 1) is involved in differentiation to STBs, and hypoxia enhances differentiation to EVTs. CK7: cytokeratin 7, CK19; cytokeratin 19, ELF5: E74-like ETS transcription factor 5, hCG; human chorionic gonadotropin, hPL; human placental lactogen, TEAD4; TEA domain transcription factor 4, TP63; tumor protein 63, SDC1; syndecan 1.

2.3. Differentiation to EVTs

Neuregulin-1 and A83–01, an anaplastic lymphoma kinase inhibitor, are required for the differentiation of hTS cells into EVTs on Matrigel (extracellular matrix-based hydrogel). These differentiated EVTs express high levels of HLA-G, but not integrin α6 or cadherin-1 (also known as E-cadherin), which are markers of CTBs; syndecan-1, a marker of STBs; or vimentin, a marker of stromal cells (Figure 2). Inducible EVTs from hTS cells showed similar expression patterns to EVTs isolated from human placental tissues. Combined analysis with gene editing in these cells is expected to enhance the study of human placentation. Induced trophoblast stem cells in mice were established by introducing TFAP2C, GATA3, eomesodermin, and ETS proto-oncogene 2 [18, 19]. Human trophoblast organoids are generated; STBs form a villous structure, and HLA-G-positive EVTs invade the Matrigel three-dimensionally [20].

3. Functions and differentiation in trophoblasts

3.1. Human endogenous retrovirus and STB differentiation

The surface area of expanded and fused STBs amounts to 12–14 m² in the normal term placenta, contributing to nutrient exchange, pathogen prevention, and hormonal production [21]. Placental growth restriction, which is commonly complicated in severe preeclampsia, largely connects to FGR due to inadequate nutrient exchange. Hypomethylation in human and mouse placentas, which are compared to other somatic cells, is thought to contribute to STB differentiation through the expression of human endogenous retroviruses (HERV) in placentas [22]. The HERV families are important for placental development, including ERV-W and ERVFRD, which correspond to syncytin-1 and syncytin-2, respectively, which are predominantly taken into the placenta from retroviruses during evolution [23]. As the syncytialization of STBs from CTBs proceeds unidirectionally and continuously during pregnancy, the differentiation of STBs is stringently regulated by HERV. During development, syncytin-1 and syncytin-2 are differentially regulated by their methylation status, depending on the developmental stage [24]. Cellular fusion defects in CTBs have been previously found to result in embryonic lethality between 11.5 and 13.5 days of gestation in syncytin-A, corresponding to syncytin-1 knockout mice [25]. This syncytin-A-deficient placenta showed disruptions in the labyrinth layer, with decreased vascularization. Some studies have reported downregulated syncytin-1 mRNA levels in the placenta of humans with preeclampsia, accompanied by hypermethylation of syncytin-1 promoter lesions [26, 27]. Syncytin-1 is transcriptionally regulated by glial cells missing transcription factor 1 (GCM1), a master regulator of STB differentiation, in trophoblast cells [28]. In mice, GCM1 mutations led to failure of the labyrinth layer to develop, resulting in embryonic lethality by embryonic day E10 [29]. GCM1 is upregulated by acute hypoxia [30] but is inhibited by chronic hypoxia via hypoxia-inducible factor 2α [31]. Humans with preeclamptic placenta suffer from chronic hypoxia, which may induce downregulation of GCM1 [32]. Syncytin-2, specifically expressed in CTBs, binds to the major facilitator superfamily domain-containing protein 2A (MFSD2A), which is expressed in STBs [33]. The placenta of syncytin-B knockout mice indicate that syncytin-B is essential for the formation of STB layer-II, resulting in FGR and a reduction in fetal number [34]. In addition, an anti-syncytin protein, suppressyn, has also been discovered [35]. Suppressyn binds to the syncytin-1 receptor and inhibits syncytin-mediated trophoblast fusion. However, the precise mechanism for this suppression during STB differentiation is yet to be elucidated.

3.2. Cellular senescence and STB differentiation

Cellular senescence is a characteristic of aging, recognized as a negative change in cellular functions, inducing a senescence-associated secretory phenotype (SASP) in cancers or aging [36, 37]. Non-trophoblast cells, syncytin-1-induced fusion IMR-90 cells, and normal human diploid fibroblasts all show features of senescent cells, confirmed by SA-β-gal, as well as the cyclin kinase inhibitors p16, p21, and p53, accompanied by SASP phenotypes [38]. This response is due to DNA damage induced by an increase in reactive oxygen species. In the STBs of term placentas, p16, p21, and p53 are considered senescence markers. The expression of p21 in STBs in third trimester placentas has been reported by a research group [39]. However, p21 in STBs is required for the function and differentiation of STBs. p21 forms a complex with GCM1 and transactivates syncytin-2 in G0-phase CTBs, preventing pathogen invasion and reduction of progesterone production [40]. STBs induced to fuse by syncytin-2 overexpression (not at the G0-phase) exhibited compromised functions. Defects in the progesterone production of STBs are consistent with defects in mitochondrial remodeling during STB differentiation, confirmed by the stability of cytochrome P450 family 11 subfamily A member 1, and an increase in small and dense mitochondria [41, 42]. The need for cellular senescence has also been confirmed in p16^−/−/p53^−/− mice [43], where p16^−/−/p53^−/− placentas exhibited trophoblast hyperplasia and collapsed vasculature in the labyrinth layer because of continuous cellular proliferation. Differentiated primary STBs also show a tendency toward senescence, hormonal production, and SASP, accompanied by a reduction in gelatinase activity by matrix metallopeptidase (MMP) 2 and MMP-9. Reduced activity is observed in human placentas with FGR, and MMP-9 knockout mice exhibit characteristics associated with preeclampsia and FGR [44]. Thus, senescence in STBs is thought to be functionally required for two reasons: (i) for resistance against apoptosis via B-cell lymphoma 2 expression, and (ii) to enlarge cells for efficient nutrient exchange [45, 46]. In addition, senescence seems to reflect trophoblast invasion in EVTs, as well as STB differentiation.

3.3. Hypoxic conditions and EVT differentiation and functions

Although hypoxia inhibits the differentiation of STBs and reduces hCG secretion and hPL expression [47], hypoxia augments CTBs isolated with high levels of epidermal growth factor receptor expression to differentiate into EVTs rather than STBs [48]. This is also shown by the trophoblast differentiation model of human pluripotent stem cells, in which the aryl hydrocarbon receptor nuclear translocator (also known as HIF-1 β) plays a central role in EVT differentiation [49]. An oxygen tension of 2% transcriptionally enhances HLA-G, an EVT marker; but not hCG, an STB marker, in CTBs. As a mechanism by which EVTs outgrow the cell column, the expression of lysyl oxidase—which is induced by 1% oxygen but not 20% oxygen concentration—is required in villous explant cultures [50]. An in vitro study found side-population trophoblasts to potentially differentiate STBs or EVTs [51]. Fewer side-population trophoblasts were detected in the FGR placenta compared with normal term placentas, suggesting that trophoblast stem cells maintain placental growth until term. Continuous EVT differentiation, even in the third trimester, may be related to continuous placental growth.

Gene ontology analysis has shown that EVT cells are divided into three subtypes at 8 weeks of gestation and two types at 24 weeks of gestation [52]. EVTs in the cell column express ribonucleotide reductase regulatory subunit M2 which is involved in DNA replication; EVTs in the distal site are identified by the expression of plasminogen activator inhibitor-1, suggestive of SERPINE1-mediated invasiveness in distal EVTs. In other methods for distinguishing distal EVTs, interstitial EVTs and endovascular EVTs are differentially detected by placenta-specific protein 8 (PLAC8) expression. PLAC8, which is induced by hypoxia, is involved in the differentiation of interstitial EVTs. Since PLAC8 is highly expressed in preeclamptic placentas compared to normal placentas [53], the balance of differentiation toward interstitial or endovascular EVTs may contribute to normal placentation. At 24 weeks of gestation, tachykinin-3—which plays roles in gonadotropin-releasing hormone and luteinizing hormone secretion and normal follicular development in healthy women [54]—is divided into two types: one showing the characteristics of wounding, digestion, and the negative regulation of the immune system, and the other associated with growth regulation and gonadotropin secretion. Notch Receptor 1 (Notch1) maintains the stemness of the progenitor cells of EVTs via two regulators controlling self-renewal of villous CTBs, namely TP63 and TEAD4 [55]. The activation of Notch1 has also been confirmed in integrin α2-positive cells and proliferative CTBs in the cell column in the first trimester [56]. On the other hand, YAP, which controls organ size and inhibits cancer cell proliferation, is responsible for stemness maintenance and repression of syncytialization of STBs in combination with TEAD4 [57].

Since hypoxic effects can vary temporally and spatially in the placenta, hypoxia has a bifocal effect on pregnancy. A typical pathological placenta, such as preeclampsia or FGR, arises from a maternal-fetal interface with poor perfusion and chronic hypoxia [58, 59]. HLA-G, which not only is a marker of EVTs but also is involved in fetal tolerance [60], is downregulated by hypoxia via miR-365 [61, 62]. Meanwhile, aberrant transcriptional regulation of CTBs in the smooth chorion of the fetal membrane has been reported in severe preeclamptic placentas. Smooth chorion CTBs in the third trimester, which express cadherin-1 and HLA-G-like CTBs in the early second trimester, retain their proliferative and invasive characteristics [63]. These findings suggest that the undisrupted progress of trophoblast differentiation is required for normal placentation. Intact trophoblast differentiation is also sustained by endometrial and decidual tissues before and during placentation. Since decidual cells secrete pro-invasive cytokines and chemokines, including IL-1β, IL-5, IL-6, IL-8, and CCL11, anti-invasive cytokines IL-10, IL-12, and vascular endothelial growth factor (VEGF), these cytokines are involved in balancing the invasiveness of EVTs in a manner dependent on the gestational stage [64]. Normal decidualization is hampered by age; hormonal receptors, including the estrogen receptor α and progesterone receptor, are aberrantly expressed in epithelial and stromal cells in aged mice, resulting in poor placentation due to immature or delayed decidualization [65]. Furthermore, human endometrial stromal cells obtained from non-pregnant women with preeclampsia during the previous pregnancy failed to decidualize in an in vitro differentiation model assay [66]. This indicates that the conditioned medium obtained from the stromal cells did not enhance EVT invasion, due to a reduction in the secretion of prolactin and insulin-like growth factor binding protein 1 (IGFBP)-1.

3.4. The role of autophagy in the differentiation and function of EVTs

Recently, the role of autophagy in oocytogenesis, embryogenesis, implantation, and placentation has been explored [67]. Autophagy is activated by hypoxia in primary trophoblasts in vitro; the activation is observed in interstitial EVTs at 8 weeks of gestation in normal pregnant tissues [68]. The activation of autophagy is observed more frequently in distal EVTs than in proximal EVTs. An in vitro study showed that an autophagy-deficient EVT cell line showed reduced function, invasiveness, and vascular remodeling under hypoxia. Although hypoxia is required for trophoblast differentiation, chronic hypoxia induces inflammation and endoplasmic reticulum stress via pyroptosis in trophoblasts [69]. The autophagy-related proteins Atg16L1 and FIP200 mediate normal decidualization and uterine receptivity [70, 71]. Moreover, autophagy activation in decidual stromal cells improves implantation failure by increasing NK cell residence [72]. However, the role of autophagy in normal placentation remains controversial. Autophagy suppression inhibits trophoblast invasion via decidual NK cells in cases of recurrent miscarriage [73]. On the other hand, a decrease in mitofusin-2 activates autophagy in trophoblasts, resulting in unexplained miscarriage [74]. It is unknown whether the inhibition or overactivation of autophagy contributes to the pathophysiology of preeclampsia [68, 75, 76]. To verify the role of autophagy in placentation, placenta-specific atg7 knockout mice, in which atg7 was deleted in the trophectoderm but not in the inner cell mass, were established. In pseudo-pregnant mice, which possessed intact autophagy function, blastocysts were implanted into the uterus, and the resulting fetuses and placentas were analyzed. During pregnancy, a significant increase in blood pressure was observed in the dams, and placental growth was inhibited due to a reduction in the spongiotrophoblast layer [77]. This reduction could be due to the increase in apoptotic cells in the spongiotrophoblast layer, because atg7 is involved in preventing cell death against metabolic stress via p53 activation [78]. Although the invasion of mouse trophoblasts is shallower than in humans, invasion and vascular remodeling—two necessary characteristics of EVTs for normal placentation—were also impaired in this model. The accumulation of p62 was more frequently observed in spongiotrophoblasts and giant trophoblast cells, corresponding to CTBs and EVTs in humans, than in STBs in the labyrinth layer. Thus, autophagy impairment is likely to affect EVT function and differentiation. Another murine model of autophagy inhibition, labyrinth layer-specific atg7 deficient mice, showed FGR [79]. Thus, placental autophagy is involved not only in placental growth, but also in fetal growth. Moreover, the observed findings in autophagy-deficient placentas are similar to those in the placenta with severe preeclampsia (Figure 3).

Figure 3. Common features between the autophagy-knockout placenta and human placentas with severe preeclampsia.

Invasion failure and vascular remodeling failure, resulting in small placentas, are common features. Though accumulation of p62 and TFEB downregulation are related to autophagy inhibition, these findings are also observed in both the autophagy-knockout placenta and human preeclampsia placentas. However, fetal size is not decreased in the autophagy-knockout placenta, though the blood pressure is significantly increased in dams.

Autophagy impairment, which leads to the deposition of aggregated proteins in the central nervous system, causes neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease [80]. Deposition induced by autophagy impairment has also been observed in preeclamptic placentas [81]. This is related to the inhibition of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, via sera from preeclamptic women [82]. The loss of TFEB in placentas resulted in an aberrant structure in the labyrinth layer due to a reduction in VEGF [83]. The mRNA levels of placental growth factor and the protein levels of TFEB were decreased in placenta-specific atg7 knockout mice and labyrinth-specific atg7 knockout mice, respectively, suggesting that TFEB regulates normal placental development. In addition, excessive endoplasmic reticulum stress, which is observed in preeclamptic placentas [69], also inhibits autophagy in trophoblasts by impairing lysosomes [84]. Consistent with lysosomal impairment in preeclamptic placentas, the concentration of β-galactosidase—a hydrolase in lysosomes, which are detectable in human sera—was found to be significantly lower in the sera of women with preeclampsia than in normal pregnant women, suggesting that it could play a role in placental growth restriction.

4. Immune cells

In the uterus, the growing fetus needs to exercise a degree of immunological tolerance to the mother. Otherwise, the maternal immune cells may attack the fetus, resulting in miscarriage during early pregnancy or preeclampsia in later stages. Various mechanisms in immune cells contribute to the induction of maternal tolerance.

4.1. Natural killer cells

NK cells with fewer granules are dramatically increased during the secretory phase in the endometrium, in response to increased progesterone levels. Further increases in NK cells occur in the uterus upon pregnancy. Peripheral blood lymphocytes are mainly composed of T-cells. However, NK cells constitute the main population of decidual lymphocytes. Uterine immune cells comprise approximately 70% uNK cells, 20% macrophages, and 10% T cells. When comparing peripheral blood NK and uNK cells, peripheral blood NK cells, which express CD16⁺CD56^dim, show high cytotoxicity by introducing cytotoxic granules to target cells. Meanwhile, uNK cells (also called decidual NK cells), which are characterized by CD16-CD56^bright, produce a variety of cytokines or angiogenic factors and show low cytotoxicity [85–87]. Although the number of leukocytes, especially macrophages and dendritic cells (DCs), is decreased at the maternal-fetal interface with aging, the maturity and abundance of uNK cells is sustained in the uterus of pregnant women despite the reduction of peripheral NK cells [65]. Although the peripheral blood consists of approximately 10% CD56^bright NK cells, these peripheral cells are characteristically different from CD56^bright uNK cells [88]. Their numbers gradually decrease after mid-gestation, and only a few can be found in term placentas. Thus, uNK cells are more important for maintaining pregnancy in the early—rather than the mid to late—gestational periods. Although increases in CD16⁺CD56^dim uNK cells, as observed in miscarriages, are thought to induce fetal rejection [89], CD16-CD56^bright uNK cells—which possess cytotoxic granules—directly attack trophoblasts and induce apoptosis in vitro [90]. Therefore, CD16^-CD56^bright uNK cells may exert a two-fold effect during pregnancy.

uNK cells accumulate at the implantation site and are involved in decidualization to induce immune tolerance and vascular remodeling. During placental development, uNK cells are thought to participate in spiral artery remodeling via VEGF during early human pregnancy [6, 91]. An in vitro study showed that IL-6 and CXCL8 produced by endovascular EVTs are involved in the accumulation of uNK in the spiral artery, initiating vascular remodeling via chemokines, CCL14 and CXCL6 [92]. Like trophoblast senescence in STB formation, senescence is also induced in NK cells in response to soluble HLA-G from trophoblasts via CD158d [93]. Despite the SASP phenotype in senescent NK cells, NK cells promote remodeling of the maternal spiral arteries during early pregnancy. Since senescent cells have been reported to express NKG2D ligands, senescent EVTs may attract uNK cells expressing NKG2D receptors to the maternal-fetal interface in humans [94]. In fact, reduced numbers of uNK cells have been observed in placental bed biopsy samples of FGR or pre-eclamptic placentas [95]. In a pregnant mouse model, the administration of IL-11 induces features resembling preeclampsia, accompanied by a reduction in uNK cells and shallow trophoblast invasion [96]. Thus, uNK and trophoblast invasion are required for normal placental development.

4.2. T cells and dendritic cells

As for maternal immune tolerance, regulatory T-cells (Treg cells), which are identified as CD4⁺CD25⁺Foxp3⁺, are increased in the uterus, and preferentially maintain allogenic pregnancy to accept the paternal antigens expressed by the fetus [4]. Treg cells in humans also increase systemically and locally during pregnancy [97]. In a mouse model, uterine CD11c⁺ antigen-presenting cells in the seminal fluid induce paternal antigen-specific Treg cells [98]. Uterus-derived DCs migrating into the draining lymph nodes highly express programmed cell death ligand-2 (PD-L2), which mediates T-cell suppression via the engagement of its receptor, PD-1 [99]. In humans, clonally expanded CD8⁺ effector memory T-cells in the decidua—which are assumed to recognize paternal antigens—express PD-1 in normal pregnancies [100]. These results indicate that PD-L2-expressing cells are involved in the inhibition of CD8⁺ effector memory T cells via PD-L2/PD-1 interaction. In human Treg cells, clonally expanded Tregs—which are also assumed to recognize paternal antigens— were expanded in the decidua, but not in the periphery during early pregnancy [101]. The proportion of clonally expanded decidual Treg cells gradually increased during gestational weeks, and the proportion in normal pregnancy was significantly higher than that in preeclampsia. The same clones of decidual effector Treg cells were maintained between past and subsequent pregnancies in individuals with normal pregnancies. Although preeclampsia is likely to occur in women who are pregnant for the first time, the maintenance or induction of clonal Treg cells, which do recognize paternal antigens, may reduce the occurrence of preeclampsia in the following pregnancy. On the other hand, HLA-G⁺ EVTs in the first trimester induce Treg cells which express high PD-1 [60]. This ability was also found to be stronger in EVTs obtained from pregnant women with a male fetus than those with a female fetus.

5. Conclusions

Normal placentation depends on the intact development of trophoblasts. In addition, the development and function of trophoblasts are regulated not only by endogenous factors, transcriptional factors, and miRNAs, but also by exogenous factors, growth factors, cytokines, and chemokines. Owing to the development of tools for human trophoblast differentiation in in vitro models by hTS in recent studies, further progress is expected in this field. As discussed in this review, placenta-specific gene manipulation may be more useful than a systemic gene knockout mouse model for the investigation of placental development. This technique has shown that defects in autophagy in placentas affect placental growth rather than fetal growth, as well as inducing hypertension in dams. Future studies should aim to investigate the interconnection between endogenous mechanisms in trophoblasts and local immune reactions at the maternal-fetal interface for the development of new therapies for preeclampsia, FGR, and recurrent miscarriages.

Abbreviations:

CDX2

caudal type homeobox 2

CTB

cytotrophoblast

EVT

extravillous trophoblast

FGR

fetal growth restriction

GCM1

glial cells missing transcription factor 1

GATA

GATA binding protein

hCG

human chorionic gonadotropin

HERV

human endogenous retrovirus(es)

HLA

major histocompatibility complex

hPL

human placental lactogen

hTS

human trophoblastic stem

mTS

mouse trophoblast stem

Notch1

Notch Receptor 1

PLAC8

placenta-specific protein 8

SASP

senescence-associated secretory phenotype

STB

syncytiotrophoblast

TE

trophectoderm

TEAD4

TEA domain transcription factor 4

TFAP2C

transcription factor AP-2 gamma

TFEB

transcription factor EB

TP63

tumor protein 63

uNK

uterine NK

VEGF

vascular endothelial growth factor