**6.1 Soluble fms-like tyrosine kinase 1 (sFlt-1)**

Soluble Flt-1 is an anti-angiogenic form of VEGF receptor 1. sFlt-1 acts as a potent scavenger of VEGF and PlGF (**Figure 4**), thus preventing their interaction with endothelial receptors on the cell surface, and subsequently induces endothelial dysfunction. Elevated concentration of sFlt-1 has been as early as 5 weeks before the diagnosis of preeclampsia and correlates with severity of disease [17, 18]. Some other studies also support this sFlt-1 role in the pathogenesis of preeclampsia [19–21].

### **6.2 Placental growth factor**

Placental growth factor (PlGF) is a prominent angiogenic factor in the development of the placental vascular system [22, 23]. During normal pregnancy, PlGF can

#### **Figure 4.**

*Circulating sFlt-1 in the maternal blood leads to a net decrease in PlGF and VEGF in the vasculature, which are necessary for normal placental angiogenesis. In PE angiogenic balance is disturbed and may result in endothelial dysfunction.*

be detected in the maternal circulation from 8 weeks gestation, reaching a maximal concentration toward the end of second trimester and declining thereafter until delivery [24]. In line with its pro-angiogenic function, reduced levels of PlGF were found in preeclampsia [18, 25, 26].

The commercial kits available for determination of PlGF are mostly using sandwich enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics International, Thermo Fisher Scientific, IBL International, Abcam) or fluoroimmunometricassay (PerkinElmer). In a multicenter, prospective study PROGNOSIS the Elecsys (Roche) sFlt-1/PlGF ratio proved to be a helpful tool in enabling clinicians to rule out the occurrence of preeclampsia for 1 week at cutoff of 38 or lower in women in whom the syndrome is suspected clinically. A ratio more than 38 indicates an increased risk of developing preeclampsia in the next 4 weeks [27].

#### **6.3 sEndoglin**

Endoglin (Eng) is a type I membrane glycoprotein localized to the cell membrane where it constitutes the transmembrane co-receptor for TGF beta receptor complex (TGF-β1 and TGF-β3) [28]. Circulating sEng was found to be high in preeclamptic women even prior to the disease manifestations correlating with disease severity and falls after delivery [17, 29], making it a reliable predictor of patients destined to develop severe early-onset preeclampsia [30].

Research has shown that near the time of delivery there is a rise in antiangiogenic factors including [31, 32] soluble endoglin (sEng) [33], a drop in the pro-angiogenic placental growth factor (PlGF) [17], and slight changes in the vascular endothelial growth factor (VEGF) [34]. These have been associated with increases in the anti-angiogenic sFlt-1/PlGF ratio [35] and a decrease in the proangiogenic PlGF/(sFlt-1 + sEng) ratio [36, 37].

Other studies have reported increases in inhibin A [38] and placental protein 13 (PP13) [39] near delivery. The elevated tumor necrosis factor alpha (TNFα) has been detected in preterm delivery [40] and also in FGR [41].

#### **6.4 Placental protein 13 (PP13)**

PP13 is a member of the galectin family, predominantly expressed by the syncytiotrophoblast, during placental vascular development [42, 43]. Serum concentrations of PP13 are significantly lower in women who later develop preeclampsia, FGR, and preterm birth [39, 44]. Combining first trimester PP13 with other parameters may further improve predictive performance.

#### **6.5 Pregnancy-associated plasma protein A (PAPP-A)**

PAPP-A is a peptidase produced by syncytiotrophoblast with hydrolytic activity for insulin-like growth factor-binding proteins [45, 46]. Decreased levels of PAPP-A in the first trimester have been associated with increased risk of preeclampsia [47], not a good predictor of late-onset preeclampsia [48].

#### **6.6 Free fetal nucleic acids**

The examination of fetal cells, specifically erythroblasts, and of cell-free fetal DNA from the blood of pregnant women is a subject of intense research, with the aim of developing new risk-free methods for prenatal diagnosis [49, 50]. In preeclamptic pregnancies [51], cell-free fetal DNA is elevated long before the

**67**

*Clinical, Biochemical, and Biophysical Markers of Angiogenesis in Preeclampsia*

**Biomarker The characteristics for prediction (95% Cl)**

Specificity 0.92 (0.92–0.92)

Specificity 0.90 (0.89–0.91)

Specificity 0.88 (0.87–0.89)

Specificity 0.89 (0.89–0.89)

*The pooled sensitivity and specificity of the separate meta-analyses for some biomarkers.*

clinical onset of the disease [52, 53]. Total free DNA has also been used and has been reported to be increased in women who subsequently develop preeclampsia [54]. New methods based on immunodiagnostics that measure the level of biomarkers as well as sonographic devices that measure the uterine artery blood flow have emerged as promising avenues that can lead to more accurate differential diagnoses.

Biophysical markers have also been developed to evaluate blood flow through the uterine arteries to the placenta. In the case of preeclampsia, an abnormal placentation results in decreased penetration of maternal spiral arteries in the junctional zone myometrium by cytotrophoblast cells. The consequence is that high blood flow and low-resistance vessels do not occur. Doppler ultrasonography has been evaluated as a potential predictive test for preeclampsia. Parameters such as the resistance index to the flow (RI), the average pulsatility index (PI), and the peak

Angiogenic factors and biophysical markers may be combined for predicting preeclampsia. The combinations which give us best results are biochemical markers sFlt-1 and PlGF with Doppler [59, 60] and additional sEng [36] or PP13 [36, 61–63] and PAPP-A [63–66]. The pooled sensitivity of all single biomarkers for PE was 0.40 (95% Cl 0.39–0.41) at a false-positive rate of 10%. The area under the summary of receiver operating characteristic curve (SROC) was 0.786. The pooled sensitivity and specificity of the separate meta-analyses for some biomarkers are shown in **Table 4**. Wu et al. in their study got a pooled sensitivity of 0.91 (95% Cl: 0.90–0.91) and SROC of 0.893 for a combination of clinical characteristics, biomarkers, and

Nowadays, various high-throughput techniques have evolved, thus allowing us simultaneous examination of thousands of genes (genomics), gene transcripts (transcriptomics), proteins (proteomics), metabolites (metabolomics), protein interaction (interactomics), and chromatin modifications (epigenomics) in single experiments. mRNA-circulating placenta-specific mRNA in serum from preeclamptic women might be useful for the prediction of preeclampsia. In this study inhibin A,

*DOI: http://dx.doi.org/10.5772/intechopen.85732*

PAPP-A Sensitivity 0.30 (0.29–0.32)

Inhibin A Sensitivity 0.32 (0.25–0.39)

PP13 Sensitivity 0.37 (0.33–0.41)

PlGF Sensitivity 0.65 (0.63–0.67)

**6.7 Biophysical markers**

**Table 4.**

**6.8 Combination of tests**

Doppler pulsatility indexes [67].

**7. Novel methods of diagnosis**

systolic flow (PSF) have been identified [55–58, 76].

*Clinical, Biochemical, and Biophysical Markers of Angiogenesis in Preeclampsia DOI: http://dx.doi.org/10.5772/intechopen.85732*


#### **Table 4.**

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

found in preeclampsia [18, 25, 26].

next 4 weeks [27].

**6.3 sEndoglin**

be detected in the maternal circulation from 8 weeks gestation, reaching a maximal concentration toward the end of second trimester and declining thereafter until delivery [24]. In line with its pro-angiogenic function, reduced levels of PlGF were

The commercial kits available for determination of PlGF are mostly using sandwich enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics International, Thermo Fisher Scientific, IBL International, Abcam) or fluoroimmunometricassay (PerkinElmer). In a multicenter, prospective study PROGNOSIS the Elecsys (Roche) sFlt-1/PlGF ratio proved to be a helpful tool in enabling clinicians to rule out the occurrence of preeclampsia for 1 week at cutoff of 38 or lower in women in whom the syndrome is suspected clinically. A ratio more than 38 indicates an increased risk of developing preeclampsia in the

Endoglin (Eng) is a type I membrane glycoprotein localized to the cell membrane where it constitutes the transmembrane co-receptor for TGF beta receptor complex (TGF-β1 and TGF-β3) [28]. Circulating sEng was found to be high in preeclamptic women even prior to the disease manifestations correlating with disease severity and falls after delivery [17, 29], making it a reliable predictor of patients

Research has shown that near the time of delivery there is a rise in antiangiogenic factors including [31, 32] soluble endoglin (sEng) [33], a drop in the pro-angiogenic placental growth factor (PlGF) [17], and slight changes in the vascular endothelial growth factor (VEGF) [34]. These have been associated with increases in the anti-angiogenic sFlt-1/PlGF ratio [35] and a decrease in the pro-

Other studies have reported increases in inhibin A [38] and placental protein 13 (PP13) [39] near delivery. The elevated tumor necrosis factor alpha (TNFα) has

PP13 is a member of the galectin family, predominantly expressed by the syncytiotrophoblast, during placental vascular development [42, 43]. Serum concentrations of PP13 are significantly lower in women who later develop preeclampsia, FGR, and preterm birth [39, 44]. Combining first trimester PP13 with

PAPP-A is a peptidase produced by syncytiotrophoblast with hydrolytic activity for insulin-like growth factor-binding proteins [45, 46]. Decreased levels of PAPP-A in the first trimester have been associated with increased risk of preeclampsia [47],

The examination of fetal cells, specifically erythroblasts, and of cell-free fetal DNA from the blood of pregnant women is a subject of intense research, with the aim of developing new risk-free methods for prenatal diagnosis [49, 50]. In preeclamptic pregnancies [51], cell-free fetal DNA is elevated long before the

destined to develop severe early-onset preeclampsia [30].

been detected in preterm delivery [40] and also in FGR [41].

other parameters may further improve predictive performance.

**6.5 Pregnancy-associated plasma protein A (PAPP-A)**

not a good predictor of late-onset preeclampsia [48].

angiogenic PlGF/(sFlt-1 + sEng) ratio [36, 37].

**6.4 Placental protein 13 (PP13)**

**6.6 Free fetal nucleic acids**

**66**

*The pooled sensitivity and specificity of the separate meta-analyses for some biomarkers.*

clinical onset of the disease [52, 53]. Total free DNA has also been used and has been reported to be increased in women who subsequently develop preeclampsia [54].

New methods based on immunodiagnostics that measure the level of biomarkers as well as sonographic devices that measure the uterine artery blood flow have emerged as promising avenues that can lead to more accurate differential diagnoses.

#### **6.7 Biophysical markers**

Biophysical markers have also been developed to evaluate blood flow through the uterine arteries to the placenta. In the case of preeclampsia, an abnormal placentation results in decreased penetration of maternal spiral arteries in the junctional zone myometrium by cytotrophoblast cells. The consequence is that high blood flow and low-resistance vessels do not occur. Doppler ultrasonography has been evaluated as a potential predictive test for preeclampsia. Parameters such as the resistance index to the flow (RI), the average pulsatility index (PI), and the peak systolic flow (PSF) have been identified [55–58, 76].

#### **6.8 Combination of tests**

Angiogenic factors and biophysical markers may be combined for predicting preeclampsia. The combinations which give us best results are biochemical markers sFlt-1 and PlGF with Doppler [59, 60] and additional sEng [36] or PP13 [36, 61–63] and PAPP-A [63–66]. The pooled sensitivity of all single biomarkers for PE was 0.40 (95% Cl 0.39–0.41) at a false-positive rate of 10%. The area under the summary of receiver operating characteristic curve (SROC) was 0.786. The pooled sensitivity and specificity of the separate meta-analyses for some biomarkers are shown in **Table 4**. Wu et al. in their study got a pooled sensitivity of 0.91 (95% Cl: 0.90–0.91) and SROC of 0.893 for a combination of clinical characteristics, biomarkers, and Doppler pulsatility indexes [67].

#### **7. Novel methods of diagnosis**

Nowadays, various high-throughput techniques have evolved, thus allowing us simultaneous examination of thousands of genes (genomics), gene transcripts (transcriptomics), proteins (proteomics), metabolites (metabolomics), protein interaction (interactomics), and chromatin modifications (epigenomics) in single experiments.

mRNA-circulating placenta-specific mRNA in serum from preeclamptic women might be useful for the prediction of preeclampsia. In this study inhibin A, p-selectin, and VEGF receptor mRNA values were higher in preeclampsia, whereas human placental lactogen, KISS-1, and plasminogen activator type 1 were lower, both compared to normotensive controls [68]. Similar results were reported from some other studies also [69, 70], where circulating cells of fetal/ placental origin were a source of mRNA. mRNAs were increased in women with preeclampsia, and there was a direct correlation between expression levels and the severity of the disease.

Protein, a functional product of gene expression can be measured. A set of differently expressed proteins which are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity, and acute phase responses can be measured. A different pattern of proteins between the group of women who subsequently developed preeclampsia on one side and without preeclampsia on the other side [71] was reported. It is also reported that women with severe preeclampsia have a unique urine proteomic pattern [72] and that this proteomic profile appeared more than 10 weeks before the clinical manifestations, and this distinguished preeclampsia from other hypertensive or proteinuric disorders in pregnancy [73].

Some studies revealed that metabolomic strategies might be appropriate for investigating the metabolic function of trophoblast or placental tissue, and it was found that preeclamptic pregnancies have a different metabolomic profile when compared to normal pregnancies [74, 75].

These novel technologies in preeclampsia appear quite promising. The number of studies is growing, and the results suggest that the use of transcriptomic, proteomic, and metabolomic profiles may be predictive for preeclampsia. These techniques open new possibilities to find a new set of biomarkers for preeclampsia. Future studies are needed, with the collaborative efforts of bioinformatics, biostatistics, researchers, and clinicians.

#### Key points


### **8. Conclusions**

Many studies demonstrate the importance of optimal management of blood pressure in pregnancy hypertension. The use of angiogenic biomarkers gives us promising results for the prediction and diagnosis of preeclampsia, but there is still a lack of specific and reliable biomarkers to predict preeclampsia, particularly in the first trimester of pregnancy. New methods to isolate and characterize markers outside the protein field (lipids, nucleic acids, etc.) from serum/plasma/urine/ saliva are useful.

**69**

**Author details**

provided the original work is properly cited.

Osredkar Joško1,2\* and Kumer Kristina1

Biochemistry, Ljubljana, Slovenia

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 University Medical Centre Ljubljana, Clinical Institute of Clinical Chemistry and

2 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to: josko.osredkar@kclj.si

*Clinical, Biochemical, and Biophysical Markers of Angiogenesis in Preeclampsia*

*DOI: http://dx.doi.org/10.5772/intechopen.85732*

*Clinical, Biochemical, and Biophysical Markers of Angiogenesis in Preeclampsia DOI: http://dx.doi.org/10.5772/intechopen.85732*
