**3. Placenta transcriptome in pathological pregnancy**

The alteration of gene expression pattern in placenta tissue may reflect metabolic disorders associated with pregnancy such as gestational diabetes mellitus or pathologies that compromise pregnancy success such as preeclampsia [40]. We will summarize main findings reported in preeclampsia and gestational diabetes mellitus that represent the two pathologies mainly investigated by transcriptomic analyses.

### **3.1. Preeclampsia**

Preeclampsia is a severe placenta disease that occurs in 3–5% of pregnant women and is a source of complications for mother and fetus [41–43]. The preeclampsia is initiated at the time of trophoblast invasion and remodeling of the spiral arteries during the first and early second trimester of pregnancy [41, 44, 45]. Initial studies have shown that elevated levels of various placental proteins in maternal blood such as FLT1 (fms-related tyrosin kinase 1), sENG (soluble engolin) and PGF (placental growth factor) in early pregnancy may be predictive of preeclampsia development [46] but the presence of false negative rates limits their clinical use [47]. The use of high throughput methods such as microarray is likely useful to identify new biomarkers and potential therapeutic targets. **Table 4** summarizes transcriptional studies of


**Species Placenta and** 

Human Placenta with

Human Placenta with

Human Placenta with

Human Placenta with

Human Placenta with

Human Placenta with

Human Placenta with

preeclampsia or from healthy donors (mean 34.6 and 38.6 weeks, respectively)

preeclampsia or from healthy donors (34–37 weeks)

**gestational age**

preeclampsia or from healthy donors (39 weeks)

preeclampsia or from healthy donors (35–39 weeks)

preeclampsia (31–39 weeks), preterm labor (24–33 weeks) or from healthy donors (37–39 weeks)

preeclampsia (mean 34.2 weeks), with small for gestational age (mean 34.5 weeks) or both (mean 33.9 weeks)

preeclampsia or from healthy donors (mean 37.5 and 39.8 weeks, respectively)

**Technique Results References**

Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies

• Perturbation of pathways involved in vascular function and immunological balance in

[55]

35

http://dx.doi.org/10.5772/intechopen.80551

[56]

[57]

[58]

[59]

[60]

[61]

• Some identified-genes have been previously reported (e.g. leptin) or not previously associ-

immune system, inflammation, oxidative stress, signaling, growth and development

modulated in preeclampsia and preterm group

• Functional analysis shows a common activity of these genes associated to cellular activities

• Some genes identified have been previously reported (leptin) or not previously associated with preeclampsia (CYP11A and

RNA-Seq • 53 differently expressed genes are modulated

preeclampsia group

ated with preeclampsia

Microarray • 58 genes are modulated and associated with

Microarray • 20 miRNAs and 120 mRNAs are differentially

compared to healthy donors

Microarray • No significant difference between preeclampsia and small-for gestational-age groups • Increased anti-angiogenic gene expressions are

Microarray • Differentially expressed genes associated with

Microarray • 896 differentially expressed genes are found

• Decreased of NOX4 gene

Microarray • 2109 differentially expressed genes are found • Down-modulation of CD4

SIAE and ENG

motivity are found

inflammation, immune regulation and cell

• Up-regulation of HTRA4, LHB and β-hCG

• Up-regulation of LEP, FLT1, PAPPA2, INHA,

pathways

CDKN1C)

observed


**Species Placenta and** 

34 Placenta

**gestational age**

Human At term preeclampsia or healthy

Human At term preeclampsia or healthy

Human At term (>37 weeks) preeclampsia or healthy

Human Placenta with

Human Placenta with

Human Placenta with

preeclampsia or from healthy donors (253–273 days)

preeclampsia or from healthy donors (<32 weeks)

preeclampsia, increased vascular resistance (notch) or from healthy donors

**Technique Results References**

RQ genes, is up-regulated in placenta tissue

[48]

[49]

[50]

[51]

[52]

[53]

[54]

• HLA-A and HLA-DRB1 expression are related to the reduction of birthweight but not placenta

preeclampsia group compared to controls

group compared to controls, 35% present an

• Up-regulated genes are associated with cell-

• Up-regulation of immune-system activation-

• No differences of MHC complex in the two

• Genes involved in transcriptional regulation, vaso-regulative pathways. Hypothetical protein and gene sequences with unknown functions

regulation, lipid biosynthesis, protein biosynthesis and transport, signal transduction are

• Genes previously involved in preeclampsia such as Flt-1, leptin, HTRA1 and SIGLEC6 are

related protein, obesity-related protein, transcription factor, immunological factor, protease inhibitor, neuro-mediator, endocrine-related protein, oncogenic factor and growth factor • Obese gene is the most up-modulated gene

chemotaxis, NF-kappa B pathway are found in preeclampsia group compared to notch • Down-modulated genes associated with antigen processing and presentation (human leukocyte antigen B) are found in preeclampsia

Microarray • HLA-DRB1, but not HLA-A RQ and CSTM2

Microarray • Increased glycogen phosphorylase gene in

Microarray • Among 368 regulated genes in preeclampsia

expression higher than 2.0

cycle or apoptosis functions

Microarray • Genes involved in cell proliferation, immune

Microarray • Modulation of genes involved in cell adhesion-

• Up-modulated genes associated with

• Results suggest that progression of preeclampsia from notching is dependent of the

development of inflammation

group compared to notch

related genes

groups

expressed

up-modulated

modulated

Microarray • 148 genes are altered

Human Term (32–40 weeks) Microarray • A few numbers of genes (21) are differentially

weight

from women with pre-eclampsia


Gene associated with hypoxia and oxidative stress are frequently found in placenta from women with preeclampsia. The placental hypoxia associated to preeclampsia is due to shallow implantation, impaired trophoblast invasion or vascularization of placenta arteries [40, 74]. The hypoxia leads to low birth weight and newborn diseases [75]. The gene encoding leptin has emerged as a potential biomarker of preeclamptic placentas from transcriptomic analyses [52, 56, 76]. This gene is known to be up-modulated in placenta from patients who experience chronic hypoxic ischemia [77]. Other genes involved in hypoxia are down-modulated in preeclampsia: this is the case for the gene encoding a glutathione reductase, an antioxidant protein, whereas the gene encoding the thioredoxin peroxidase is up-regulated [78]. In contrast, Zhang et al. found no modulation of genes encoding enzymes involved in oxidative

Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies

http://dx.doi.org/10.5772/intechopen.80551

37

The transcription analysis enables the identification of several up-modulated genes including genes involved in apoptosis processes [50], activin-A, inhibin A [80], soluble sENG [61], soluble sFlt-1 [61] and placental growth factor PGF [81]. In these studies, three angiogenic genes are found differently expressed: they include sFLT1 (also known as vascular endothelial growth factor, VEGF), PGF and sENG that are up-modulated in preeclampsia and associated with severe preeclampsia [61, 62, 82, 83]. In a case-control clinical study, the level of sFLT1 increases and that of PGF decreases during normal pregnancy; this response is more pronounced in women who develop preeclampsia [46]. Thus, it has been proposed that sFLT1 could be used as biomarker for predicting the development of preeclampsia [84]. However, these genes are not found in all transcriptomic studies of preeclamptic placentas [85]. This discrepancy may be explained by the clinical heterogeneity of preeclampsia, placenta sampling sites, gestational weeks, sex of the child, labor and the method of delivery. These parameters restrict the use of these genes as biomarkers for diagnosis or prognosis of

Placenta is an endocrine organ that provides glucose to fetus. A pathological state of insulin resistance leading to glucose intolerance is called gestational diabetes mellitus (GDM) [86]. The mother may develop hemorrhage, hypertension, infection, difficulty in labor and increased risk of mortality [87]. The placentas from GDM patients exhibit histological alterations and elevation of its size and weight at third trimester. This may be due to insufficient production of placental hormones [88]. To better understand the role of placenta in the development of GDM, transcriptomic analyses of placenta from pregnant women with a GDM or mice model of diabetes have been conducted in some studies (**Table 5**). Few genes are differentially modulated and they are predominantly related to extracellular matrix remodeling, immune

Among the immune response category, genes such as TNF are up-modulated. TNF is known to be involved in insulin resistance, obesity and diabetes [95, 96]. Although TNF levels are increased in GDM and type 1 diabetes [97], no relation was found between placenta TNF mRNA amounts and the levels of this cytokine in maternal blood with GDM

stress as compared to controls [79].

**3.2. Gestational diabetes mellitus**

response and regulation of apoptosis categories.

preeclampsia.

[98–100].

**Table 4.** Transcriptomic analysis of pregnancy with preeclampsia.

placenta from women with preeclampsia. Most studies use at term placentas and report a number of modulated genes varying from 20 to more than 2000.

Among the up-modulated genes, those associated with the regulation of the immune response are most frequently reported in transcriptomic analysis of preeclampsia placentas (**Table 4**). These findings may be related to the up-regulated gene expression profile of circulating cells from pregnant women with preeclampsia [65–67] and to the observation of increased levels of inflammatory cytokines including IL-6, IL-8 and TNF [68, 69]. Indeed, using microarray approach on blood samples, we observed a specific transcriptional signature of genes upmodulated in severe preeclampsia including genes associated with ribosome and complement functions [67]. In addition, we had identified VSIG4 (V-set and immunoglobulin domain containing 4) as a biomarker of severe preeclampsia.

Among genes associated with immune response, three studies reported the modulation of human leukocyte antigen (HLA) genes [48, 54, 59]. HLA-A, HLA-B and HLA-DRB1 are upmodulated in placentas from preeclampsia as compared to control group. The molecules, HLA-A and HLA-DRB1 have been associated with preeclampsia outcome [48, 70, 71] and have been related to reduced birth weight but not to placenta weight. Clinical observations show that both birth weight [72] and placenta weight [73] are decreased in pregnant women with preeclampsia.

Gene associated with hypoxia and oxidative stress are frequently found in placenta from women with preeclampsia. The placental hypoxia associated to preeclampsia is due to shallow implantation, impaired trophoblast invasion or vascularization of placenta arteries [40, 74]. The hypoxia leads to low birth weight and newborn diseases [75]. The gene encoding leptin has emerged as a potential biomarker of preeclamptic placentas from transcriptomic analyses [52, 56, 76]. This gene is known to be up-modulated in placenta from patients who experience chronic hypoxic ischemia [77]. Other genes involved in hypoxia are down-modulated in preeclampsia: this is the case for the gene encoding a glutathione reductase, an antioxidant protein, whereas the gene encoding the thioredoxin peroxidase is up-regulated [78]. In contrast, Zhang et al. found no modulation of genes encoding enzymes involved in oxidative stress as compared to controls [79].

The transcription analysis enables the identification of several up-modulated genes including genes involved in apoptosis processes [50], activin-A, inhibin A [80], soluble sENG [61], soluble sFlt-1 [61] and placental growth factor PGF [81]. In these studies, three angiogenic genes are found differently expressed: they include sFLT1 (also known as vascular endothelial growth factor, VEGF), PGF and sENG that are up-modulated in preeclampsia and associated with severe preeclampsia [61, 62, 82, 83]. In a case-control clinical study, the level of sFLT1 increases and that of PGF decreases during normal pregnancy; this response is more pronounced in women who develop preeclampsia [46]. Thus, it has been proposed that sFLT1 could be used as biomarker for predicting the development of preeclampsia [84]. However, these genes are not found in all transcriptomic studies of preeclamptic placentas [85]. This discrepancy may be explained by the clinical heterogeneity of preeclampsia, placenta sampling sites, gestational weeks, sex of the child, labor and the method of delivery. These parameters restrict the use of these genes as biomarkers for diagnosis or prognosis of preeclampsia.

## **3.2. Gestational diabetes mellitus**

placenta from women with preeclampsia. Most studies use at term placentas and report a

proteolysis.

**Technique Results References**

[62]

[63]

[64]

• Differentially expressed genes are observed between early and late onset preeclampsia • Up-regulation of FLT1, PAPPA2, CGB5, LEP

• Down-modulation of PDGFD, BHLHB3 and

• Differential expression of autophagy-associated genes is found in microarray datasets from

Microarray • 213 and 82 genes are found up- and downmodulated, respectively

separate published studies

• The most highly affected pathways in preeclampsia placenta are Wnt, ErbB, PPAR, Hedgehog signaling pathways, mRNA surveillance pathway and ubiquitin mediated

• Identification of specific genes for preeclampsia: LEP, HTRA4, SPAG4, LHB, TREM1, FSTL3,

CGB, INHA, PROCR and LTF genes

and INHBA

BMP5

Among the up-modulated genes, those associated with the regulation of the immune response are most frequently reported in transcriptomic analysis of preeclampsia placentas (**Table 4**). These findings may be related to the up-regulated gene expression profile of circulating cells from pregnant women with preeclampsia [65–67] and to the observation of increased levels of inflammatory cytokines including IL-6, IL-8 and TNF [68, 69]. Indeed, using microarray approach on blood samples, we observed a specific transcriptional signature of genes upmodulated in severe preeclampsia including genes associated with ribosome and complement functions [67]. In addition, we had identified VSIG4 (V-set and immunoglobulin domain

Among genes associated with immune response, three studies reported the modulation of human leukocyte antigen (HLA) genes [48, 54, 59]. HLA-A, HLA-B and HLA-DRB1 are upmodulated in placentas from preeclampsia as compared to control group. The molecules, HLA-A and HLA-DRB1 have been associated with preeclampsia outcome [48, 70, 71] and have been related to reduced birth weight but not to placenta weight. Clinical observations show that both birth weight [72] and placenta weight [73] are decreased in pregnant women

number of modulated genes varying from 20 to more than 2000.

**Table 4.** Transcriptomic analysis of pregnancy with preeclampsia.

analysis on microarray experiment in published studies

analysis on microarray experiment in published studies

containing 4) as a biomarker of severe preeclampsia.

with preeclampsia.

**Species Placenta and** 

36 Placenta

**gestational age**

preeclampsia or from healthy donors

Human Variable Meta-gene

Human Term Meta-gene

Human At term placenta with

Placenta is an endocrine organ that provides glucose to fetus. A pathological state of insulin resistance leading to glucose intolerance is called gestational diabetes mellitus (GDM) [86]. The mother may develop hemorrhage, hypertension, infection, difficulty in labor and increased risk of mortality [87]. The placentas from GDM patients exhibit histological alterations and elevation of its size and weight at third trimester. This may be due to insufficient production of placental hormones [88]. To better understand the role of placenta in the development of GDM, transcriptomic analyses of placenta from pregnant women with a GDM or mice model of diabetes have been conducted in some studies (**Table 5**). Few genes are differentially modulated and they are predominantly related to extracellular matrix remodeling, immune response and regulation of apoptosis categories.

Among the immune response category, genes such as TNF are up-modulated. TNF is known to be involved in insulin resistance, obesity and diabetes [95, 96]. Although TNF levels are increased in GDM and type 1 diabetes [97], no relation was found between placenta TNF mRNA amounts and the levels of this cytokine in maternal blood with GDM [98–100].


of genes associated with inflammation

Transcriptional studies reveal strong association of leptin (LEP) with GDM [90, 101, 102]. LEP is up-modulated in placenta and could be a cause or a result of glucose uptake in placentas from women with GDM [102, 103]. Elevated circulating levels of LEP are found in GDM but LEP is also found in others pathological pregnancies such as preeclampsia. The changes in

Finally, common modulated genes are found in transcriptome analyses of placental gene expression in term pregnancy complications, suggesting that they could be associated more to a dysfunction of the placenta than to a specific complication. As examples, FLT1 (fms-related tyrosine kinase 1) or PAPPA2 (Pappalysin-2) are commonly modulated in placenta disorders

Some investigations highlight dysregulation of microRNAs in preeclampsia and GDM, suggesting their role in pathological pregnancies [104]. In preeclampsia, the upregulation of miR-210, miR-20b, miR-29b, miR-16, miR-155 and miR-675 is associated with angiogenesis and trophoblast invasion [44, 105–110]. In contrast, down-modulation of miR-378a-5p, miR-376 and miR-195 is related to the promotion of trophoblast invasion and proliferation [39, 111]. However, some discrepancies are observed according to transcriptomic studies. For example, several studies showed that miR-210 represents the most up-regulated microRNAs in preeclampsia, whereas other studies found no change in placenta from preeclampsia compared to healthy donors [57].

LEP concentration are associated with the modulation of cytokines such as TNF [102].

**Table 5.** Transcriptomic analysis of pregnancy with gestational diabetes mellitus.

and could be used as biomarkers.

**Species Placenta and** 

Human Placenta with

Human Placenta with

GDM (23–41 weeks)

Human At term

**gestational age**

placenta with GDM or from healthy donors

GDM or from healthy donors (38 weeks)

**Transcriptomic technique**

**Results References**

http://dx.doi.org/10.5772/intechopen.80551

[93]

39

[90]

[94]

modulation of miR-27a, miR-9, miR-137, miR-92a, miR-33a, miR-30d, miR-362-5p

• This gene signature targets EGFR, PI3K and AKT genes involved in placental development and fetal growth

Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies

• 18.5% of modulated genes are involved in stress-activated and inflammatory

• Up-regulation of interleukins (IL-1R), leptin, and TNF receptors

• TNF, IL-1β, LEP, IFN-γ and HLA-G are

Microarray • Up-regulation of miR-508-3p and down-

and miR-502-5p

Microarray • 435 genes are modulated

responses

Microarray • 243 genes present an altered expression

differentially modulated • Gene modulation is associated with cytokine-cytokine receptor interaction

**3.3. miRNAs in complicated pregnancy**

#### Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies http://dx.doi.org/10.5772/intechopen.80551 39


**Table 5.** Transcriptomic analysis of pregnancy with gestational diabetes mellitus.

Transcriptional studies reveal strong association of leptin (LEP) with GDM [90, 101, 102]. LEP is up-modulated in placenta and could be a cause or a result of glucose uptake in placentas from women with GDM [102, 103]. Elevated circulating levels of LEP are found in GDM but LEP is also found in others pathological pregnancies such as preeclampsia. The changes in LEP concentration are associated with the modulation of cytokines such as TNF [102].

Finally, common modulated genes are found in transcriptome analyses of placental gene expression in term pregnancy complications, suggesting that they could be associated more to a dysfunction of the placenta than to a specific complication. As examples, FLT1 (fms-related tyrosine kinase 1) or PAPPA2 (Pappalysin-2) are commonly modulated in placenta disorders and could be used as biomarkers.

### **3.3. miRNAs in complicated pregnancy**

**Species Placenta and** 

Human Placenta with

Human Placenta with

Mouse (60% caloriesby-fat diet induced-diabetes) GDM or from healthy donors (mean 37.7– 38.4 weeks)

E 12.5 nCounter

nanostring

GDM (38 weeks)

Mouse

38 Placenta

(Streptozotocin induced-diabetes) **gestational age**

**Transcriptomic technique**

E 10.5 Microarray • 158 genes are modulated in diabetic

**Results References**

[89]

[90]

[91]

[92]

placentas compared to controls (47% down-and 54% up-modulated genes) • Functional category: extracellular matrix, hormones, cell surface receptor, signal transduction, transcription factors, metabolism, channel, cytoskeleton

• Diabetes-induced molecular changes and abnormal differentiation of cells, modification of growth and junctional

involved in markers and mediators of

• Increased expression of genes involved in stress-activated and inflammatory

• Increased expression of genes encoding interleukins, leptin and TNF receptors • Gene modulation is associated with extracellular matrix component and

• Modulated genes are associated with cell functions (activation), immune response, organ development and

• Up-modulation of AQP3, LEP, FLT1, ADFP, CEBPA and MIF genes • AQP3 is a new gene associated with

• Altered gene expression in the fetal

• GDM mice present repressed genes associated with neuro-developmental, cholinergic signaling, IFN/antiviral response, growth, cell cycle regulation

• GDM mice present increased expression of genes associated with inflammation

and RNA binding

zone and labyrinth

angiogenic activators

regulation of cell death • Modulated genes including LEP, CEBPA and MIF have been previously

Microarray • 66 genes are up-modulated in GDM placentas

described

GDM outcome

and apoptosis

brain

Microarray • Increased expression of genes

inflammation

responses

Some investigations highlight dysregulation of microRNAs in preeclampsia and GDM, suggesting their role in pathological pregnancies [104]. In preeclampsia, the upregulation of miR-210, miR-20b, miR-29b, miR-16, miR-155 and miR-675 is associated with angiogenesis and trophoblast invasion [44, 105–110]. In contrast, down-modulation of miR-378a-5p, miR-376 and miR-195 is related to the promotion of trophoblast invasion and proliferation [39, 111]. However, some discrepancies are observed according to transcriptomic studies. For example, several studies showed that miR-210 represents the most up-regulated microRNAs in preeclampsia, whereas other studies found no change in placenta from preeclampsia compared to healthy donors [57].

Altered circulating levels of specific microRNAs have been reported in compromised pregnancies. For example, the low detection of miR-376c level in blood of 16–18 weeks pregnant women is related with the outcome of preeclampsia [112]. In similar conditions, three miRNAs, including miR-132, miR-29a and miR-222, are decreased in blood from women at 16–19 gestational weeks who were diagnosed a GDM at 25–28 weeks of gestation [113]. Thus, despite the promising perspectives to use microRNAs in diagnosis of complicated pregnancies, future studies are needed to provide a proof of concept.

distinct macrophage subsets according to CD11c expression, namely CD11clow and CD11chigh subsets. Both subsets expressed two M2 markers, CD209 (DC-SIGN) and CD206 (mannose receptor). Genes involved in invasion, mobility, inflammatory process and lipid metabolism are enriched in the two subsets, but genes involved in antiapoptotic pathways are found only in CD11high macrophages. In contrast, CD11clow macrophages express genes involved in growth regulation and development, and extracellular communication. These findings are in agreement with those of Svensson et al. who found two macrophage subsets, ICAM-3high and ICAM-3low, in macrophages from first trimester (7–12 weeks) [123]. Both macrophage populations express genes and cytokines associated with M2 profile and are related to CD11c

Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies

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41

The third trimester is associated to a pro-inflammatory environment. This inflammatory state is due to the synchronization of immune-endocrine cross-talk, based on especially estrogens [124], which favors M1 polarization of macrophages [125]. Indeed, a low level of estradiol is sufficient to promote macrophage M1 polarization, as measured by the secretion of inflammatory cytokines such as IL-1β, IL-6 and TNF. The investigation of DNA of at term placenta macrophages reveals the methylation of inflammatory genes including TLR9, IL-1β, IL-12RB2, CD48 and FGR and the hypomethylation of M2 genes including CCL2, CCL13, CCL14 and CD209 [126]. Using microarray analysis, we previously investigated the polarization of macrophages from at term placentas in comparison to macrophage-derived monocytes [127]. We did not find a polarization of at term placenta macrophages. Indeed, both M1 (CXCL9, EDN1, IL-15, IL-15RA and IL-2RA) and M2 (FN1, CTSC and CCL23) genes were up-modulated. Interestingly, we reported for the first time the ability of placental macrophages to formed multinuclear giant cells (MGCs). These MGCs present functional enrichment in genes associated with cytoskeleton reorganization and immune response. In addition, as observed in placental macrophages, MGCs are not polarized. Taking together, although the third trimester is associated with an inflammatory environment, the activation of placental macrophages does

Placental macrophages are likely associated with number of pregnancy complications. Here, we focused on three pathological conditions, preeclampsia, GDM and chorioamnionitis, in

Although alterations of macrophages are suspected in preeclampsia, the studies of placenta macrophages during preeclampsia are controversial [116, 129]. Some studies reported a decreased [115, 130, 131] or an increased [132–135] number of placental macrophages. In addition, they produce inflammatory cytokines and anti-inflammatory cytokines [136, 137]. It has been also reported that the count of M2 macrophages is decreased in the decidua of

expression.

*4.1.2. Third trimester (term placentas)*

not reproduce the classical model of M1/M2 polarization.

which the involvement of macrophages is documented [128] .

*4.1.3. Placenta macrophages in pathological pregnancy*

*4.1.3.1. Preeclampsia*
