**2. Characteristics of the groups and study design**

**1. Introduction**

uteroplacental blood flow [4–8].

and their tissue inhibitors (TIMPs) [11–14].

tors of matrix metalloproteinases (TIMPs) [22–24].

the vasculature [9, 10].

Adequate formation of uteroplacental and fetal placental blood flow is the determining factor of physiological pregnancy and fetal development. Successful uterine‐placental vascular morphogenesis and embryonic morphogenesis of fetal blood system are the basis of these processes. There are two stages of vascular morphogenesis: vasculogenesis—primary forma‐ tion and development of blood vessels *de novo* from committed mesodermal cells—and angio‐ genesis—formation of new blood vessels from existing vascular structures, which reflect the

"Early pregnancy" period includes several time intervals when the most significant for angio‐ genesis events occur, determining further course and outcome of pregnancy. During gestation up to 6 weeks, the primary fetal circulatory system and placental bed with the development of villi are formed, and extensive vascularization of placental villous tree occurs. The 6–8th weeks of pregnancy are marked by the start of transition to the placental circulation, as well as by the most expressed invasion of extravillous trophoblast into maternal spiral arteries (first wave of trophoblast invasion). The period of 11–13 weeks is considered to be borderline and is characterized by the completion of embryogenesis, the starting period of fetal develop‐ ment, fading of the first wave of trophoblast invasion and further increase in the volume of

During trophoblast invasion into endometrium, interactions with components of the extracel‐ lular matrix, which is mediated by cell adhesion molecules, occur. Proteinases participate in the degradation of extracellular matrix and cell migration deep into the myometrium through the uterine spiral arteries. Cytotrophoblast cells change their phenotype from epithelial to endothelial, producing a large number of soluble factors contributing to the development of

Growth factors play the key role in vessels formation. They serve as cell mitogens, as attrac‐ tants in the formation of vascular architectonics, and most important, as morphogens. The main regulators of angiogenesis are members of the family of vascular endothelial growth factor (VEGF). In addition to direct activators of angiogenesis, there is a large group of factors, whose effect on angiogenesis is nonspecific. It includes matrix metalloproteinases (MMPs)

Decidual NK‐cells in early pregnancy, at the stage preceding the invasion of trophoblast into the maternal arteries, produce VEGF, placental growth factor (PLGF) and matrix metallopro‐ teinases (MMPs), in particular MMP‐2 and MMP‐9 [15–17]. These MMPs are collagenases of IV type which specifically hydrolyze the collagen of basement membranes and thereby facilitate cell invasion through the basement membranes and stimulate angiogenesis [18–21]. Decidual NK‐cells are the main source of MMP‐2 and TIMP‐2 from the group of tissue inhibi‐

Trophoblast also produces factors regulating processes of vessels formation. Particularly, MMP‐9, TIMP‐1, TIMP‐2 and TIMP‐3 are produced by the cells of extravillous tropho‐ blast. Villous cytotrophoblast cells and invasive endovascular trophoblast produce MMP‐2

formation of vascular system of a fetus and placenta during pregnancy [1–4].

78 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

The prospective study included 66 patients with early stage pregnancy.

The control group consisted of 20 patients with normal pregnancy. All the patients had one or two previous pregnancies uneventfully completed at term.

The main subgroup A consisted of 16 patients with the history of miscarriage and current threatening miscarriage. All patients from the main subgroup A gave live births at term. The main subgroup B included 30 patients whose pregnancy ended as «missed abortion».

In patients of the control group and of the main subgroup A, the study of angiogenesis‐ related factors in peripheral blood samples was performed within 6 weeks, at 7–8 weeks and at 11–14 weeks of pregnancy. In the main subgroup B, the investigation was conducted at the time of diagnosis of «missed abortion» (8 patients were examined before 6 weeks, 14 patients—at 7‐8 weeks and 8 patients—at 11‐14 weeks).

The criteria for inclusion into the main groups were as follows: history of two and more early pregnancy losses, no childbirth in current marriage, singleton natural pregnancy.

Exclusion criteria were endometriosis, POS, uterine fibroids, induced pregnancy and/or extragenital conditions (diabetes mellitus, psoriasis and systemic autoimmune diseases, and malignancies), FV L and FII G20210A gene mutations, and activation of bacterial viral infec‐ tions. Groups were matched for the patient's age.

Diagnosis of recurrent miscarriage was established according to the International Classification of Diseases 10th Edition (ICD‐10‐CM). Pregnancy was diagnosed based on the ultrasonogra‐ phy study and HCG.

The samples of peripheral blood for investigation of angiogenesis‐related factors were obtained from the cubital vein not later than 5 days after ultrasonic detection of cessation of pregnancy development in the patients of the main subgroup B in case of intact chorion (absence of retroplacental and/or retro amniotic hematomas, vaginal tract bleedings). Prior to investigation, the serum was stored at the temperature of «‐80°C».

## **3. Methods**

Determination of serum levels of VEGF, VEGF‐R1 (sFlt‐1), VEGF‐R2 (sKDR), MMP‐2, MMP‐9, TIMP‐1, TIMP‐2 and PLGF was performed by enzyme‐linked immunosorbent assay (ELISA) using standard test systems: Bender MedSystems GmbH (Austria) and R&D Systems (USA). The optical density was measured using the plate reader BioTek (USA) at the wavelength of 450 nm. Construction of the calibration curve and calculation of the concentrations of VEGF, sFlt‐1, sKDR, MMP‐2, MMP‐9, TIMP‐1, TIMP‐2 and PLGF were performed by linear regres‐ sion equation in logarithmic coordinates.

The statistical processing was carried out using statistical analysis package for Microsoft Office Excel 2007 and software package Statistica for Windows 7.0, Statsoft Inc. (USA). Control of the normality of obtained parameters in studied groups was performed with Shapiro‐Wilk W test. The significance of differences of mean values of the measured parameters was evalu‐ ated using unpaired t‐test with different dispersions. Differences were considered significant at a significance level *p* < 0.05.

## **4. Results**

Results of the study of angiogenesis‐related factors are presented in **Tables 1** and **2**.

#### **4.1. Time course of angiogenesis‐related factors in patients with normal pregnancy**

It was found that VEGF had the maximum concentration in blood at terms up to 6 weeks of gestation: Contents of this factor was significantly higher (more than 10 times) compared with further points of study. Concentration of PLGF at terms up to 6 weeks and at 7–8 weeks remained unchanged but increased more than two times by 11–14 weeks; this is consistent with the published data [24]. This trend in changing of VEGF and PLGF during early preg‐ nancy probably provides pro‐angiogenic effect because under the decrease in VEGF, PLGF is able to synergistically enhance angiogenesis promoted by VEGF [26].

Level of sVEGF‐R1 was minimal at the starting point of the study but significantly increased reaching maximum values at 11‐14 weeks. In contrast, the concentration of sVEGF‐R2 was decreased only at 7‐8 weeks of pregnancy. It is known that VEGF interacts with receptors VEGF‐R1 and VEGF‐R2, while VEGF‐R1 is the only receptor for PLGF [27]. These factors compete for binding with VEGF‐R1. Under increased secretion of VEGF and PLGF, this leads to exhaustion of VEGF‐R1 and predominance of VEGF‐R2 in circulation. PLGF is also supposed to be able to replace VEGF in the VEGF/VEGF‐R1 complex, activating the


**\***Comparison with the control group;

The samples of peripheral blood for investigation of angiogenesis‐related factors were obtained from the cubital vein not later than 5 days after ultrasonic detection of cessation of pregnancy development in the patients of the main subgroup B in case of intact chorion (absence of retroplacental and/or retro amniotic hematomas, vaginal tract bleedings). Prior to

Determination of serum levels of VEGF, VEGF‐R1 (sFlt‐1), VEGF‐R2 (sKDR), MMP‐2, MMP‐9, TIMP‐1, TIMP‐2 and PLGF was performed by enzyme‐linked immunosorbent assay (ELISA) using standard test systems: Bender MedSystems GmbH (Austria) and R&D Systems (USA). The optical density was measured using the plate reader BioTek (USA) at the wavelength of 450 nm. Construction of the calibration curve and calculation of the concentrations of VEGF, sFlt‐1, sKDR, MMP‐2, MMP‐9, TIMP‐1, TIMP‐2 and PLGF were performed by linear regres‐

The statistical processing was carried out using statistical analysis package for Microsoft Office Excel 2007 and software package Statistica for Windows 7.0, Statsoft Inc. (USA). Control of the normality of obtained parameters in studied groups was performed with Shapiro‐Wilk W test. The significance of differences of mean values of the measured parameters was evalu‐ ated using unpaired t‐test with different dispersions. Differences were considered significant

Results of the study of angiogenesis‐related factors are presented in **Tables 1** and **2**.

**4.1. Time course of angiogenesis‐related factors in patients with normal pregnancy**

able to synergistically enhance angiogenesis promoted by VEGF [26].

It was found that VEGF had the maximum concentration in blood at terms up to 6 weeks of gestation: Contents of this factor was significantly higher (more than 10 times) compared with further points of study. Concentration of PLGF at terms up to 6 weeks and at 7–8 weeks remained unchanged but increased more than two times by 11–14 weeks; this is consistent with the published data [24]. This trend in changing of VEGF and PLGF during early preg‐ nancy probably provides pro‐angiogenic effect because under the decrease in VEGF, PLGF is

Level of sVEGF‐R1 was minimal at the starting point of the study but significantly increased reaching maximum values at 11‐14 weeks. In contrast, the concentration of sVEGF‐R2 was decreased only at 7‐8 weeks of pregnancy. It is known that VEGF interacts with receptors VEGF‐R1 and VEGF‐R2, while VEGF‐R1 is the only receptor for PLGF [27]. These factors compete for binding with VEGF‐R1. Under increased secretion of VEGF and PLGF, this leads to exhaustion of VEGF‐R1 and predominance of VEGF‐R2 in circulation. PLGF is also supposed to be able to replace VEGF in the VEGF/VEGF‐R1 complex, activating the

investigation, the serum was stored at the temperature of «‐80°C».

80 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**3. Methods**

sion equation in logarithmic coordinates.

at a significance level *p* < 0.05.

**4. Results**

**\*\***comparison with the main subgroup A;
