the differences within one group in comparison with the data obtained before 6 weeks.

•the differences within one group in comparison with data obtained at 7‐8 weeks (*р* < 0.05).

**Table 2.** Blood levels of matrix metalloproteinases and their tissue inhibitors in the first trimester of pregnancy.

expression of VEGF‐R2 [28]. According to the published data, VEGF‐R2 is a key receptor in angiogenesis [29] although its affinity to VEGF is quite lower than VEGF‐R1 [30].

It is known that normally pro‐angiogenic molecules are less than their inhibitors; neverthe‐ less, intensive vascularization of placenta and of fetal developing organs occurs in pregnancy. This situation can be described as "pro‐angiogenic state." However, it is quite difficult to quantify it, as the described models of angiogenic factors‐receptor interactions do not give guide on the factor/inhibitor ratio, allowing to specify the pro‐angiogenic state or block of angiogenesis [26, 31]. Therefore, trends observed in normal pregnancy may serve as refer‐ ences for challenging cases.

Blood levels of MMP‐2 and TIMP‐2 vary insignificantly in the studied terms of normal preg‐ nancy. Significant decrease in MMP‐9 and TIMP‐1 concentration was found at 7–8 weeks compared with the terms before 6 weeks of pregnancy.

Normally, MMPs blood level is insignificant. Soluble forms of MMPs are present in blood as inactive proenzymes and transfer into active forms after propeptide cleavage under the impact of activation factors including inhibitors. Constant concentrations in the system "protease‐antiprotease" (MMP‐2/TIMP‐2) may indicate extracellular matrix remodeling and vascular morphogenesis which intensively go at these terms; degradation of intersti‐ tial collagens and basement membrane collagens under the participation of these factors is obvious.

#### **4.2. Time course of angiogenesis‐related factors in patients of the main subgroup A**

Blood levels of PLGF and sVEGF‐R1 in patients of main subgroup A significantly increased with gestational age. In contrast, concentration of VEGF was maximum at the starting point of the study but significantly decreased (more than 30 times) by 7‐8 weeks and then remained at the same level till 11–14 weeks of gestation.

Lack of significant differences in dynamics of MMP‐2, MMP‐9, TIMP‐2 and sVEGF‐R2 was observed during entire pregnancy. Concentration of TIMP‐1 significantly decreased by 11–14 weeks.

Thus, in the main subgroup A, we found significant fluctuations of certain soluble factors. For several factors (VEGF, PLGF, MMP‐2, TIMP‐2), trends of this levels alterations at the same study points coincided with those observed during normal pregnancy; there was no statisti‐ cally significant differences between the groups and the most studied terms. At the same time, for sVEGF‐R1, sVEGF‐R2, MMP‐9 and TIMP‐1, the trends were significantly different from the control group. However, these differences do not seem sufficient to cause dramatic conse‐ quences in pregnancy. Probably for successful outcome of pregnancy, a balanced production of VEGF and PLGF, key angiogenic factors for pregnancy [14, 24, 32], is necessary, as well as the balance between MMP‐2 and TIMP‐2, because MMP‐2 is the key regulator of trophoblast invasion [17, 19] especially in early pregnancy.

## **4.3. Angiogenesis‐related factors in the main subgroup B**

expression of VEGF‐R2 [28]. According to the published data, VEGF‐R2 is a key receptor in

It is known that normally pro‐angiogenic molecules are less than their inhibitors; neverthe‐ less, intensive vascularization of placenta and of fetal developing organs occurs in pregnancy. This situation can be described as "pro‐angiogenic state." However, it is quite difficult to quantify it, as the described models of angiogenic factors‐receptor interactions do not give guide on the factor/inhibitor ratio, allowing to specify the pro‐angiogenic state or block of angiogenesis [26, 31]. Therefore, trends observed in normal pregnancy may serve as refer‐

Blood levels of MMP‐2 and TIMP‐2 vary insignificantly in the studied terms of normal preg‐ nancy. Significant decrease in MMP‐9 and TIMP‐1 concentration was found at 7–8 weeks

Normally, MMPs blood level is insignificant. Soluble forms of MMPs are present in blood as inactive proenzymes and transfer into active forms after propeptide cleavage under the impact of activation factors including inhibitors. Constant concentrations in the system "protease‐antiprotease" (MMP‐2/TIMP‐2) may indicate extracellular matrix remodeling and vascular morphogenesis which intensively go at these terms; degradation of intersti‐ tial collagens and basement membrane collagens under the participation of these factors is

**4.2. Time course of angiogenesis‐related factors in patients of the main subgroup A**

Blood levels of PLGF and sVEGF‐R1 in patients of main subgroup A significantly increased with gestational age. In contrast, concentration of VEGF was maximum at the starting point of the study but significantly decreased (more than 30 times) by 7‐8 weeks and then remained

Lack of significant differences in dynamics of MMP‐2, MMP‐9, TIMP‐2 and sVEGF‐R2 was observed during entire pregnancy. Concentration of TIMP‐1 significantly decreased by

Thus, in the main subgroup A, we found significant fluctuations of certain soluble factors. For several factors (VEGF, PLGF, MMP‐2, TIMP‐2), trends of this levels alterations at the same study points coincided with those observed during normal pregnancy; there was no statisti‐ cally significant differences between the groups and the most studied terms. At the same time, for sVEGF‐R1, sVEGF‐R2, MMP‐9 and TIMP‐1, the trends were significantly different from the control group. However, these differences do not seem sufficient to cause dramatic conse‐ quences in pregnancy. Probably for successful outcome of pregnancy, a balanced production of VEGF and PLGF, key angiogenic factors for pregnancy [14, 24, 32], is necessary, as well as the balance between MMP‐2 and TIMP‐2, because MMP‐2 is the key regulator of trophoblast

angiogenesis [29] although its affinity to VEGF is quite lower than VEGF‐R1 [30].

82 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

ences for challenging cases.

obvious.

11–14 weeks.

compared with the terms before 6 weeks of pregnancy.

at the same level till 11–14 weeks of gestation.

invasion [17, 19] especially in early pregnancy.

## *4.3.1. Blood levels of angiogenesis‐related factors in patients with missed abortion before 6 weeks of gestation*

Missed abortion before 6 weeks was characterized by significant differences of the levels of angiogenesis‐related factors: sVEGF‐R1, PLGF, MMP‐2, TIMP‐1 and TIMP‐2. Thus, with missed abortion, the level of PLGF was lower than in the control group; however, there was no significant difference with the main subgroup A. Blood level of sVEGF‐R1 did not dif‐ fer from the values of the control group. However, comparison with the main subgroup A showed that in complicated pregnancy which further accomplished with childbirth, level of sVEGF‐R1 was higher than in missed abortion. These results may indicate the existence of subtle mechanisms of regulation in the system "ligand/receptor," where the most important aspect is not a certain level of material content of the molecule, but the impacts of the factors on each other.

The concentrations of MMP‐2 in the main subgroups A and B were significantly higher than in the control group. The maximum level of MMP‐2 in blood was detected in missed abortion. TIMP‐1 and TIMP‐2 levels in the control group and main subgroup A differed insignificantly; in the main subgroup B, the level of TIMP‐1 was significantly lower and the level of TIMP‐2 was significantly higher at these terms.

Thus, more distinct differences specific for missed abortion were found in the group of factors "protease‐antiprotease." The content of the factors in the system MMP‐2–TIMP‐2 at this term is, probably, critical for pregnancy. It is known that MMP‐2 and TIMP‐2 are most important at early terms of gestation during trophoblast invasion into maternal tissues [19]. Constant expression of MMP‐2 and TIMP‐2 was detected in apical layer of the syncytiotrophoblast and decidual NK‐cells [19]. The published data suggest a possibility of syncytium activation by MMP‐2, and the participation of this molecule in the processing of paracrine factors synthe‐ sized by these cells [19, 21]. There is a probability of change in the balance of production of biologically active molecules determining the development of pregnancy, due to the excessive production of MMP‐2 or TIMP‐2.

Pregnancy‐initiated angiogenesis is closely associated with tissue remodeling and vasculariza‐ tion. Changes occurring in the endometrium decidualization include as follows: infiltration of tissues by immune cells (uNK‐cells and macrophages), extracellular matrix remodeling with cell invasion through the matrix and membrane, and lysis of muscle‐elastic tissue elements. In this period, the most important processes are formation of the placental bed and vasculariza‐ tion of the villi [6, 8, 10]. It is known that embryos which stopped to develop at 3–5 weeks had vascular villi, large hydropic dystrophy of the villous stroma and no embryonic blood vessels [33–35]. The disbalance of angiogenic factors, causing autocrine and paracrine effects to each other and to the cells, leads to microenvironment that is not compatible with the development of pregnancy. Excessive protease activity under these conditions may also reflect degenera‐ tive processes in the uterus before abortion.

## *4.3.2. Blood levels of angiogenesis‐related factors in patients with missed abortion at 7–8 weeks*

Pathological changes specific for missed abortion at 7–8 weeks were reflected by the signifi‐ cant increase in VEGF‐R1, VEGF‐R2 and TIMP‐2 levels compared with the control group.

The interval of 7–8 weeks of gestation is marked by the first wave of trophoblast invasion into the mother's arteries and the start of uteroplacental blood flow. Extravillous trophoblast has a high invasive potential and expresses VEGF, PLGF and VEGF‐R1 [36]. The receptor VEGF‐R2 is expressed by the cells of fetoplacental complex [36, 37]. It should be noted that the level of VEGF‐R1 is five times higher and VEGF‐R2 more than two times differ from these factor levels in the control group. According to the published data, soluble forms of VEGF‐R1 and VEGF‐R2 are able to block angiogenesis and adversely affect the migration and proliferation of endothe‐ lial cells [27, 38, 39]. However, comparison with the main subgroup A did not show any signifi‐ cant differences in the studied period. Therefore, excessive levels of soluble forms of VEGF‐R1 and VEGF‐R2 in maternal circulation are not the main cause of this pathology at this term. It is known that adequate angiogenesis of villous tree and chorionic villi are critical stage and con‐ dition for further development of the placenta and fetus [1–4]. Inadequate start of restriction of uterine arteries at these terms initiates a chain of troubles in the system mother‐placenta‐fetus.

The key process of pregnancy is cytotrophoblast invasion into uterine arteries. At the same time, there is arteriolar lumen expansion under the impact of metalloproteinases. Excess of tissue inhibitors at 7–8 weeks, probably, leads to incomplete trophoblast invasion and insuf‐ ficient lumen expansion which under the increased secretion of VEGF‐R1 and VEGF‐R2 can result in vasospasm and enhanced vascular permeability‐specific manifestations of increased contents of these factors [36, 40] which adversely affect the embryo development.

## *4.3.3. Blood levels of angiogenesis‐related factors in patients with missed abortion at 11–14 weeks of pregnancy*

Missed abortion detected at of 11–14 weeks was characterized by a significant decrease in sVEGF‐R1, PLGF, MMP‐2 and MMP‐9 compared with the control group and main sub‐ group A. Level of TIMP‐2 was significantly lower than in the main subgroup A, but close to the control group.

The end of the first trimester of pregnancy is marked by the start of the fetal period of intra‐ uterine human development, fading of the first wave of trophoblast invasion and prepara‐ tion for a second wave at 16‐18 weeks of pregnancy. Significant decrease in soluble forms of VEGF‐R1 and PLGF in the main subgroup B could evidence the role of these factors in the pathologic processes leading to pregnancy loss. PLGF is the main regulatory factor in the first trimester of physiological pregnancy [2, 24] that acts as paracrine regulator of decidual angiogenesis and autocrine regulator of trophoblast function [24]. The synergy of effects of PLGF and VEGF on angiogenesis manifests with the morphogenesis of more mature and stable vasculature [24, 27]. Its effect is more significant in angiogenesis than in vasculogenesis [2]. Besides inhibition of angiogenesis, soluble form of the receptor VEGF‐R1 also provides "support," and thus, the effect depends on the factor blood level. Probably in this situation, levels of sVEGF‐R1 and PLGF are insufficient for adequate angiogenesis.

## **4.4. Ratio of angiogenesis‐related factors**

*4.3.2. Blood levels of angiogenesis‐related factors in patients with missed abortion at 7–8 weeks*

84 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Pathological changes specific for missed abortion at 7–8 weeks were reflected by the signifi‐ cant increase in VEGF‐R1, VEGF‐R2 and TIMP‐2 levels compared with the control group.

The interval of 7–8 weeks of gestation is marked by the first wave of trophoblast invasion into the mother's arteries and the start of uteroplacental blood flow. Extravillous trophoblast has a high invasive potential and expresses VEGF, PLGF and VEGF‐R1 [36]. The receptor VEGF‐R2 is expressed by the cells of fetoplacental complex [36, 37]. It should be noted that the level of VEGF‐R1 is five times higher and VEGF‐R2 more than two times differ from these factor levels in the control group. According to the published data, soluble forms of VEGF‐R1 and VEGF‐R2 are able to block angiogenesis and adversely affect the migration and proliferation of endothe‐ lial cells [27, 38, 39]. However, comparison with the main subgroup A did not show any signifi‐ cant differences in the studied period. Therefore, excessive levels of soluble forms of VEGF‐R1 and VEGF‐R2 in maternal circulation are not the main cause of this pathology at this term. It is known that adequate angiogenesis of villous tree and chorionic villi are critical stage and con‐ dition for further development of the placenta and fetus [1–4]. Inadequate start of restriction of uterine arteries at these terms initiates a chain of troubles in the system mother‐placenta‐fetus.

The key process of pregnancy is cytotrophoblast invasion into uterine arteries. At the same time, there is arteriolar lumen expansion under the impact of metalloproteinases. Excess of tissue inhibitors at 7–8 weeks, probably, leads to incomplete trophoblast invasion and insuf‐ ficient lumen expansion which under the increased secretion of VEGF‐R1 and VEGF‐R2 can result in vasospasm and enhanced vascular permeability‐specific manifestations of increased

contents of these factors [36, 40] which adversely affect the embryo development.

levels of sVEGF‐R1 and PLGF are insufficient for adequate angiogenesis.

*of pregnancy*

the control group.

*4.3.3. Blood levels of angiogenesis‐related factors in patients with missed abortion at 11–14 weeks* 

Missed abortion detected at of 11–14 weeks was characterized by a significant decrease in sVEGF‐R1, PLGF, MMP‐2 and MMP‐9 compared with the control group and main sub‐ group A. Level of TIMP‐2 was significantly lower than in the main subgroup A, but close to

The end of the first trimester of pregnancy is marked by the start of the fetal period of intra‐ uterine human development, fading of the first wave of trophoblast invasion and prepara‐ tion for a second wave at 16‐18 weeks of pregnancy. Significant decrease in soluble forms of VEGF‐R1 and PLGF in the main subgroup B could evidence the role of these factors in the pathologic processes leading to pregnancy loss. PLGF is the main regulatory factor in the first trimester of physiological pregnancy [2, 24] that acts as paracrine regulator of decidual angiogenesis and autocrine regulator of trophoblast function [24]. The synergy of effects of PLGF and VEGF on angiogenesis manifests with the morphogenesis of more mature and stable vasculature [24, 27]. Its effect is more significant in angiogenesis than in vasculogenesis [2]. Besides inhibition of angiogenesis, soluble form of the receptor VEGF‐R1 also provides "support," and thus, the effect depends on the factor blood level. Probably in this situation, Dynamic balance in the system ligand/receptor provides a state of the system which empow‐ ers the implementation of its function, if this system tends towards the harmonic balance. The equilibrium point in such systems is always moving, because ligand/receptor pairs, being complex systems, are influenced by a variety of factors. In terms of the dynamic bal‐ ance theory, ligand's positions in this study were occupied by the factors which specifically and nonspecifically affect angiogenesis, while the receptor's positions were occupied by its inhibitors. Studied ligands and receptors have multiple substrate specificity, but because their activity is affected by other factors that are present in the bloodstream and were not included in this study, investigation of ligand/receptor pairs in terms of classical concepts is difficult. Moreover, current models describing interactions of VEGF family members with the recep‐ tors [26] as well as of the matrix metalloproteinase family members with the inhibitors [31] do not provide quantitative binding characteristics of the system receptor/ligand for these molecules. The situation is complicated by the different levels of expression of these factors by various cell types. Therefore, the use of any index or ratio characterizing the dynamic situa‐ tion in the selected time interval as a pro‐angiogenic or nonproangiogenic state does not seem possible. To describe such situations, "surrogate" indexes characterizing the ligand/receptor ratio may be most appropriate to define any process, particularly angiogenesis. Such ratios are used to calculate the risk of development of pregnancy complications, such as preeclamp‐ sia [41–44].

Changes of the ligand/receptor pair ratio (VEGF/VEGF‐R1, VEGF/VEGF‐R2, PLGF/VEGF‐R1, MMP‐9/TIMP‐1, MMP‐2/TIMP‐2) in the studied groups are presented in **Figure 1**.

#### *4.4.1. VEGF/VEGF‐R1 and VEGF/VEGF‐R2 ratios*

VEGF/VEGF‐R1 ratio (**Figure 1A**) in the control group before 6 weeks was significantly higher than in the main subgroups A and B (0.562; 0.178 and 0.0312, respectively). In the control group, we observed significant differences between the terms before 6 weeks of gestation and other terms. Interestingly, the pattern of the VEGF/VEGF‐R1 ratio change in the main sub‐ group A had tendencies similar to the control group. In the main subgroup B, the values of ligand/receptor pairs were significantly low, except for the last study term (0.0086) compared with the control group (0.006) and the main subgroup A (0.0024).

VEGF/VEGF‐R2 ratio changed in a similar way (**Figure 1B**). Before 6 weeks of pregnancy, the significant changes were noted only in the main subgroup B (0.0023), compared with the control group (0.020) and the main subgroup A (0.011).

Tendencies of changes of VEGF/VEGF‐R2 were similar in the control and the main subgroup A, demonstrating at the same time significant differences at 7‐8 weeks. The main subgroup B was characterized by minimal values of the ratio which significantly differed in terms before 6 and 7–8 weeks compared with the other groups, but do not change within group in all terms of pregnancy.

It is known that realization of different mechanisms of angiogenesis depends on the type of receptor interacting with VEGF. Activation of VEGF‐R2 leads to stimulation of angiogenesis

**Figure 1.** Factor/receptor (A, B, C) and factor/inhibitor (D, E) ratio in the blood of women at early terms of pregnancy. **\***Significant difference from the control group; **\*\***significant difference from the main subgroup A; **•**significant differences inside the control group in comparison with terms before 6 weeks; **••**significant difference inside the control group in comparison with terms 7‐8 weeks; **#** significant difference inside the main subgroup A in comparison with terms before 6 weeks; **##**significant difference inside the main subgroup A in comparison with terms 7–8 weeks; significant difference inside the main subgroup B in comparison with terms before 6 weeks; significant difference inside the main subgroup B in comparison with terms 7–8 weeks (*p* < 0.05).

by triggering proliferation, migration, differentiation and inhibition of apoptosis of endo‐ thelial cells. Activated VEGF‐R1 receptor stimulates intercellular interactions, branching of vascular network and regulates the trophoblast invasion into the spiral arteries [27]. Use of blocking antibodies for VEGF‐R2 causes reduction in decidual angiogenesis and pregnancy loss in mice, while use of antibodies of similar effect for VEGF‐R1 does not cause such effects [37]. In other studies, the lack of VEGF‐R1 in experimental animals led to overdevelopment of disorganized vessels and clusters of endothelial cells, and the absence of VEGF‐R2—to reduc‐ tion in development of vasculature [2]. It has been demonstrated that interaction of VEGF/ VEGF‐R2 regulates the development of fetoplacental complex [27] and acts as a paracrine system in the processes of formation of primitive embryo vascular network [24, 29]. On the contrary, the formation of an active complex VEGF/VEGF‐R1 mostly affects the processes of differentiation and migration of trophoblast and also regulates invasion [27].

## *4.4.2. PLGF/VEGF‐R1 ratio*

PLGF factor affects endothelium through specific binding with the receptor VEGF‐R1 (**Figure 1C**). According to the published data, PLGF more impacts the processes of angio‐ genesis, than vasculogenesis; however, PLGF and VEGF‐R1 also affect the mobilization of mesenchymal progenitors of endothelial cells, which are involved in vasculogenesis [2]. PLGF enhances angiogenesis acting synergistically with VEGF, and it is also able to replace VEGF in the complex with VEGF/VEGF‐R1 releasing it for VEGF‐R2 activation. It is also known that PLGF is a paracrine regulator of decidual angiogenesis and autocrine regulator of tropho‐ blast's functions in differentiation and invasion [24] and also the main regulating factor in normal pregnancy in the first trimester [2, 24].

The ratio of PLGF/VEGF‐R1 for each group of pregnant women had its own tendencies. There were significant differences between the main subgroup A and control group before 6 and at 7–8 weeks. However, there was notable misbalance of factors in ligand/receptor pair PLGF/ VEGF‐R1 manifesting with significant deviations from the average value of the factors ratio at 7‐8 weeks of pregnancy in the main subgroup B. Moreover, low ratio values were noted in the ligand/receptor pair of the main subgroup A at all studied points. The reduced value of this ratio in the main subgroup A may be due to the changes in the dynamic system PLGF/ VEGF‐R1 (both toward the increase in sVEGF‐R1 and toward the decrease in PLGF). Obtained ratio values for the given ligand/receptor pair for all groups at early terms of pregnancy may evidence the acceptable fluctuations of the values of the factors in this pair, which are nonsig‐ nificant for the development of pregnancy.

## *4.4.3. MMP‐9/TIMP‐1 and MMP‐2/TIMP‐2 ratio*

**Figure 1.** Factor/receptor (A, B, C) and factor/inhibitor (D, E) ratio in the blood of women at early terms of pregnancy. **\***Significant difference from the control group; **\*\***significant difference from the main subgroup A; **•**significant differences inside the control group in comparison with terms before 6 weeks; **••**significant difference inside the control group

before 6 weeks; **##**significant difference inside the main subgroup A in comparison with terms 7–8 weeks; significant difference inside the main subgroup B in comparison with terms before 6 weeks; significant difference inside the

significant difference inside the main subgroup A in comparison with terms

in comparison with terms 7‐8 weeks; **#**

main subgroup B in comparison with terms 7–8 weeks (*p* < 0.05).

86 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Analysis of the obtained results showed no significant differences of ratios of free forms of MMPs and tissue inhibitors TIMPs (MMPs/TIMPs) between the groups at the most studied terms of pregnancy (**Figure 1D, E**). Significant differences were shown for the MMP‐9/TIMP‐1 ratio at 11‐14 weeks for the main subgroup B (0.91) and the control group (1.48). The signifi‐ cant decrease in this ratio in the main subgroup B may evidence degradation processes and autolysis in missed abortion. The character of changes of the ligand/receptor ratio within studied groups confirms some stable dynamic equilibrium of the factors concentrations. Probably, nonspecific effects of the factors in the studied ligand/receptor pairs on angiogen‐ esis processes are of somewhat conservative nature comprising prevention of excessive pro‐ tease activity, and sufficient for an adequate angiogenesis at the studied terms of pregnancy. However, use of these ratios is not informative to characterize the pathologic processes at studied terms in patients of these groups, excluding the ratio MMP‐9/TIMP‐1 at 11‐14 weeks of pregnancy. The observed reduction in MMP‐9/TIMP‐1 at 11‐14 weeks may serve as an alert of the development of critical events.

## **5. Conclusions**

This study revealed the features of humoral systems regulating angiogenesis during physi‐ ological pregnancy and in patients with successful and unsuccessful perinatal outcomes. We found that deviations in the peripheral blood contents of angiogenesis‐related factors: VEGF‐ R1, VEGF‐R2, MMP‐9 and TIMP‐1 in patients with the history of missed abortion do not reflect critical for angiogenesis events in the first trimester of pregnancy. However, a significant dis‐ balance of soluble factors, regulating angiogenesis, detected in patients with missed abor‐ tion shows that matrix metalloproteinases and their tissue inhibitors play the leading role in pregnancy losses before 6 and 7–8 weeks. Analysis of ligand/receptor ratios complements the obtained results, as we have found a significant decrease in the VEGF/VEGF‐R1 and VEGF/ VEGF‐R2 ratios before 6 weeks of pregnancy despite the fact that there were no significant differences between individual molecules forming these pairs. The nature of VEGF/VEGF‐R1 and VEGF/VEGF‐R2 ratios alterations within the groups at the studied terms suggests the presence of a single mechanism that regulates interactions between VEGF and its receptors VEGF‐R1 and VEGF‐R2. In patients with pregnancy losses at 11‐14 weeks, we found low con‐ centrations of PLGF and sVEGF‐R1 and also of MMP‐2 and MMP‐9, and reduction in MMP‐2/ TIMP‐2 ratio, which are probably insufficient for an adequate angiogenesis at this term. Since an adequate angiogenesis is the determining factor for the development of pregnancy, early identification of criteria alerting about a trouble in fetoplacental system will also have diagnos‐ tic and prognostic value. Detection of the markers is especially important in cases of habitual pregnancy loss of unknown origin, because the disturbance of angiogenesis may be one of the causes of missed abortion. Taking into account difficulties with obtaining placental tissue at the studied terms of pregnancy, the angiogenesis‐related factors may serve as unbiased indi‐ cators of placental angiogenesis. The obtained results allow to presume various mechanisms of pregnancy pathology at early terms and to demonstrate the possibility of using the analysis of ligand/receptor pairs to characterize the angiogenesis processes in early pregnancy.

## **Acknowledgements**

The authors express their deep appreciation and gratitude to employee of Department of Perinatal Pathology, Kulikova G.V and the Department of Library and Information Resources and Telemedicine, A.L. Komarovsky, V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology of the Russian Ministry of Health, for his help in preparing this manuscript.
