Changes in placental angiogenesis and their correlation with foetal intrauterine restriction

Summary

Type of study:
Summary review.

Setting:
Department of Gynaecology and Obstetrics, 1st Faculty of Medicine, Charles University and Hospital Na Bulovce, Prague; Department of Physiology, Faculty of Science, Charles University, Prague; Department of Children and Adolescent Medicine, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague.

Introduction:
Intrauterine growth restriction (IUGR) is one of the most common problems in obstetrics. Its incidence is ranging between 3–10%, according to the type of study population and chosen criteria. The cutoff value mainly used for defining the IUGR is weight below the 10^th percentile for gestational age. The minority of authors defines the cutoff value under the 5^th or 3^rd percentile. Any pathological interference with normal vascular development of placenta may have a critical impact on foetal growth and development. Ischaemia is the most common cause of IUGR in normally well-supplied placenta. IUGR is then a consequence of insufficient extension, branching, and dilatation of capillary loops during the formation of terminal villi.

Methods:
This paper is a review focused on up-to-date-known data concerning changes in placental angiogenesis and their impact on IUGR development.

Conclusion:
The aim of this review is to summarize the knowledge concerning the mechanisms of development of the vascular supply to the placenta under physiological conditions and in conditions that result in IUGR.

Keywords:
intrauterine growth restriction, angiogenic factors, placenta, foetal weight, angiogenesis

INTRODUCTION

The placenta is a unique organ that develops and is functioning only during pregnancy. It forms a physiological barrier between mother and foetus, serves to allow the diffusion of monosacharides, amino acids, hormones, vitamins, oxygen, carbon dioxide, water and other waste materials, provides the exchange of respiratory gases between the mother and the developing foetus, works as an excretory organ of foetus while releasing the nitrogenous waste materials into mother´s blood, manufactures fructose from glucose, and performs endocrine and immunological function [18]. Malfunction of placenta is responsible for a broad range of pregnancy complications. Foetal demands for diffusional exchange of blood gases and metabolites rise exponentially with progress of pregnancy, and disruption of development of maternal and foetoplacentar blood flow can result in severe pathological conditions or complicated placental vascular diseases resulting in low birth weight, preterm delivery, and increased perinatal morbidity and mortality [30].

INTRAUTERINE GROWTH RESTRICTION

In the early 1960s, the birthweight nomograms according to gestational week were developed, and the concept of appropriate weight for gestational age was introduced [35]. The foetus with an inherent growth potential is usually born as a healthy newborn of appropriate size. If the maternal-placental-foetal unit is not able to provide the needs of the foetus, intrauterine growth restriction (IUGR) can occur. It is a common diagnosis in obstetrics with an incidence of approximately 5% in the general obstetric population [39]. The IUGR incidence range is estimated to 3–10%, with respect to e.g. the examined newborn population, geographic loca-tion, and the standard reference growth curves [9].

Perinatal outcome of IUGR newborns is highly related to their weight. Infants of weight less than 2500 g at term are in 30 times higher risk of a perinatal death than infants whose birth weights are at the 50th percentile. In infants of a weight below 1500 g, the mortality rate is 70 to 100 times higher [10]. One of the most severe problems in IUGR newborns is also perinatal asphyxia involving multiple organ systems growth-restricted infants [39].

The physiologic counterpart of IUGR is small-for-gestational-age (SGA). SGA foetuses exhibit no maternal pathology and normal umbilical artery and middle cerebral artery Doppler results. Being SGA includes constitutional smallness, given by e.g. female sex of the foetus, maternal ethnicity, parity and mother‘s BMI [16]. The SGA newborns are well proportioned and developmentally normal and their risk of morbidity and mortality is not as high as in the IUGR infants. Within the foetuses population with a birth weight below the 10^th percentile, approximately 70 percent of foetuses are SGA. The remaining 30 percent represents IUGR [41]. In routine antenatal care, less than a quarter of all SGA babies are identified before birth [48]. Approximately 50% of non-anomalous stillborn infants are small for gestational age and survivors are at increased risk of neurodevelopmental delay, cerebral palsy [5] and later cardiovasular complications and diabetes [4]. SGA infants who are recogized and well managed before birth have been reported to have a four-fold reduction in perinatal death a severe fetal distress [34].

IUGR CLASSIFICATION AND ETHIOLOGY

The most common definition of IUGR is a foetus with estimated weight is below the 10th percentile for its gestational age and with abdominal circumference is below the 2.5th percentile [42]. However, the international consensus on the definition of IUGR is still lacking, with call for the need of standardisation of terminology, implementation of foetal weight customisation and national guidance [47].

According to biometric measurements, IUGR is usually classified as proportional (symmetric) and disproportional (asymmetric). Proportional IUGR is characterized by a reduction in all growth parameters, especially the biparietal diameter and abdominal circumference. In other words, it implies a foetus whose entire body is proportionally small. This growth restriction type is usually seen in the first half of pregnancy. A disproportional (asymmetric) IUGR foetus reveals a normal head dimension, but its abdominal circumference is small (caused by decreased liver size). The foetus usually has scrawny limbs (because of low muscle mass) and thinned skin with decreased amount of fat. Asymmetric IUGR is usually the result of placental insufficiency. A foetus is chronically undernourished and tries to maintain growth of vital organs (brain and heart). If the insult causing undernourishment is severe or long lasting, the foetus can be unable to compensate it at the expense of other non-vital organs and will become symmetrically growth-restricted [7].

Disproportional IUGR is more common and occurs in 70-80 %. It is usually seen in the second half of pregnancy [3]. Some authors mentiona third type of IUGR, mixed. It appears the last 2–3 weeks before birth. The cause is foetal-placental reduction in blood flow, reduced supply of oxygen, glucose and essential amino acids [11].

IUGR DIAGNOSIS BASEDON ULTRASOUND IMAGING

For estimating the diagnosis of IUGR, accurate dating in early pregnancy is essential. However, an accuracy of ultrasound assessment of gestational age is best up to 20th gestational week, with the margin of error of 7 to 10 days. The most precise assessment is achieved in an early ultrasound examination at 8 to 13 weeks of gestation. Ultrasound dating performed from 20 to 36 weeks of gestation can render an accuracy of about three weeks, and about three week at term [42].

The gold standard for assessment of foetal growth is represented by ultrasound biometry. The most common measured parameters include the biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL). The most sensitive indicator of IUGR is the abdominal circumference. Its sensitivity can reach over 95%, if the measurement is below the 2.5th percentile [8]. Estimated foetal weight (EFW) can be calculated from the measured parameters, using combination of them. In practice, the equation by Shepard and Hadlock is used most often [19].

Another useful parameter is the ratio of the head circumference to the abdominal circumference (HC/AC). In foetus of physiological development, the HC/AC ratio drops almost linearly from 1.2 to 1.0 between 20 and 36 weeks of gestation. In the foetus with symmetric IUGR, the HC/AC ratio remains normal, and in the foetus with asymmetric IUGR is elevated [42].

An important role in the diagnosis and classification of IUGR plays Doppler flowmetry, i.e. measuring blood flow in the placenta, uterine or foetal arteries and veins. Doppler flowmetry in IUGR foetuses suffering chronic intrauterine hypoxemia usually reveals downstream vascular resistance (umbilical artery, UA) and preferential organ blood flow (middle cerebral artery, MCA). In other words, the foetal adaptation to hypoxia is usually expressed as a decrease in cardiac output, slower heart rate, an increase in arterial pressure and the associated afterload, and major redistribution of the blood flow because of selective peripheral vasoconstriction [26]. Redistribution of blood to vital organs (brain and heart) occurs. Such brain-sparing flow (BSF) is for the IUGR foetuses typical [2, 17].

A classical signs of foetal hypoxemia accompanied by acidemia and an adverse perinatal outcome are pulsation in the umbilical vein associated with late umbilical artery abnormalities (absent or reversed diastolic flow) [27]. In the initial phase of chronic foetus hypoxemia, an elevation of pulsatile index value (PI) occurs. This reduction in end-diastolic flow serves as a first sign of feto-placental vascular insufficiency. The insufficiency can progress to absent end-diastolic flow, with reversed end-diastolic flow as the most severe stage of feto-placental vascular impairment [36]. Umbilical artery Doppler examination can be accompanied with uterine artery Doppler study. It is documented that one third of high-risk pregnancies with bilateral abnormal uterine artery Doppler results in a mid-gestation development of severe IUGR with absent/reversed end-diastolic flow in the umbilical arteries [46, 32].

With respect to Doppler flowmetry examina-tion of umbilical artery we distinguish

• IUGR with persistent end-diastolic flow in the umbilical artery (IUGR + PED)
• IUGR with unilateral or bilateral absent or even reverse end-diastolic flow (IUGR + ARED) [33].

Umbilical artery Doppler waveforms should be obtained with respect to avoid false-positive waveforms with low end-diastolic velocities, obtained especially at the foetal cord root or within the foetal abdomen surrounding the bladder. The most suitable flowmetry site is either a free loop of cord or near the placental end [29].

The middle cerebral artery (MCA) and ductus venosus Doppler flowmetry can also identify blood flow redistribution in the IUGR foetus. For explain, a decrease in vascular resistance in the middle cerebral artery (MCA) characterizes foetal cerebral hyperperfusion [14].

Changes in arterial blood flow are followed by changes in foetal venous flow. Abnormal early UA and MCA Doppler flowmetry measurements do not necessarily predict the outcome in IUGR foetuses. They are a good and valuable indicator of the brain-sparing effect; however, the foetus can still have adequate reverses to cope with the stress of labour and vaginal delivery [21]. For better prediction of the foetal outcome and the optimal time for delivery, the venous side of the foetal circulation (umbilical vein, ductus venosus etc.) should be also examined by the means of Doppler flowmentry in IUGR foetuses [14].

High numbers of IUGR placentas at delivery are small [13, 46] and visible lesions correlating with ultrasound examination are evident [44]. Thus, placental ultrasound examination can be a valid assessment tool in IUGR. There is an evidence suggesting that placental ultrasound imaging may be more relevant than Doppler flowmetry of uterine artery in the assessment the likelihood of placental insufficiency as the cause of IUGR [43].

FORMATION OF PLACENTAL CIRCULATION

The placentas of all placental (eutherian) mammals can be classified into four groups. The classification is based on divergent characteristics of (I) the distribution of contact sites between foetal membranes and endometrium, with respect to the gross shape of the placenta, and (II) the number of layers of tissue between maternal and foetal vascular systems. Humans have the hemochorial placenta, in which foetal chorionic epithelium is directly washed in maternal blood, because endometrial epithelium, connective tissue and uterine endothelium of maternal layers are not retained [15].

The establishment of a functional placenta requires formation of new blood vessels. Two cooperative and tightly regulated processes are involved in that: vasculogenesis and angiogenesis. With the term of vasculogenesis we mean the de novo development of the primitive vascular network from endothelial progenitor cells. Vasculogenesis is followed by angiogenesis, during which the vasculogenesis-formed vascular network is used as a template for new vessels [28].

Vascular adaptation to pregnancy occurs via different processes including vasodilatation, increased vascular permeability, trophoblast invasion of maternal uterine vessels, and uterine angiogenesis. Placental vascular development is mainly based on proliferation and differentiation of several cell types. Major steps are associated with the differentiation of placental villi. In humans, primary villi are formed around Day 13 post conception (p.c.). Secondary villi are characterized by intravillous extraembryonic mesoderm around Day 15 p.c. At this point of time, the endothelial cell surface marker CD34 is detected within the villi. Hemangiogenic progenitor cells are found dispersed in early villi and quickly form string-like aggregates of polygonal cells that are called „hemangiogenic cords“. Foetal mesoderm growing into the primary placental villi precedes villous vasculogenesis. Villous trophoblast cells also seem to affect the differentiation of intravillous mesoderm. Between the 21st and 32nd day p.c., endothelial tubes are formed resulting in establishment of tertiary villi. Around Day 28 p.c., the first hematopoietic stem cells in the placenta develop by delamination from the primitive vessel walls into the early lumen. At this time, endothelial tubes are still isolated, as no circulation exists. Vasculogenesis can be detected up to the 10th to 12th week of gestation. Between 32nd day p.c. and the 24th week p.c., angiogenesis is responsible for expanding the placental blood vessel system. By midgestation, the placental vascular tree is remodeled through the process of angiogenesis. At first, the primitive capillary network is created by elongation of newly formed tubes. This so-called non-branching angiogenesis is followed by ramification of these tubes by lateral sprouting. Immature villi are defined by capillary networks originating from branching angiogenesis while further elongation of poorly branched capillary loops characterize mature and terminal chorionic villi. A continuous transformation and ripening of the villous vessel system takes place until term.

Major players during placental vascular development include cellular components (e.g., trophoblast, chorionic stromal cells, hemangiogenic progenitor cells), growth factors and cytokines, as well as components of extracellular matrix.

GROWTH FACTORS

Placental vascular development can be divided into three molecular and morphologic steps. In the first step, villous cytotrophoblasts produce a variety of factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [20]. The induction of differentiation, proliferation, and migration of pluripotent mesenchymal cells takes place via a paracrine manner. Hemangiogenic progenitor cells differentiate locally in the villi from the embryonic derived mesenchymal cells. Those hemangiogenic progenitor cells will further form both angioblastic and hematopoietic lineages. During the second step, VEGF and placental growth factor (PlGF) activate angiogenic cell cords causing the further differentiation of endothelial precursor cells. The third step depends on extracellular matrix components and perivascular cells, which induce remodeling of primary blood vessels. Further vessel stabilization is mainly supported by angiopoietin (Ang), its respective receptors Tie-1 and Tie-2, VEGF, PlGF, nitric oxide (NO), vascular endothelial-cadherin, and low tissue oxygen level.

Angiogenic growth factors (VEGF, PlGF) play an important role in remodelling and function of maternal vasculature during gestation [37]. Their function is modulated by sFlt-1, a splice variant of the VEGF receptor Flt-1 lacking the transmembrane and cytoplasmic domains, which acts as a potent VEGF and PlGF antagonist.

The major role of sFlt-1 is to bind VEGF and suppress the physiological levels of VEGF via binding. VEGF activity is also modulated by binding of free VEGF to circulating binding proteins including the soluble extracellular portions of VEGF receptors (sVEGF-R1 and sVEGF-R2. sVEGFR-2 placental expression seems to be more limited to the fetoplacental endothelium and the syncytiotrophoblast; it has also been found in human umbilical vein endothelial cells. In contrast, sVEGFR-1 is expressed and secreted by the placenta. By binding free VEGF, sVEGF-R1 serves as a negative functional regulator of VEGF activity. By day 30 p.c., the decline in circulating free VEGF was associated with a corresponding rise in sVEGF-R1 leaking from early trophoblasts into the maternal circulation.

IUGR AND PATHOLOGY OF PLACENTAL TRANSPORT

Disordered placentation is in the background of many pregnancy pathologies, including intrauterine growth restriction (IUGR) [45]. A high number of pathologic findings is characteristic for placentas of IUGR infants, such as reduced syncytiotrophoblast surface area, increased thickness of the exchange barrier formed by the trophoblast and fetal capillary endothelium, and an increase in placental apoptosis [1, 12, 22, 24]. In the early 1980s, Nylund et al. demonstrated a 30 ± 50% reduction in uteroplacental blood flow compared with normal pregnancies [40]. A significant decrease in villi vascular density together with a decrease in branching angiogenesis is associated with IUGR [23]. A decrease in surface area, volume, and number of terminal villi as well as a reduced number of capillaries in the stroma of IUGR placentas is also well described [25, 31]. Alltogether, the data suggest that reduced uteroplacental blood flow with reduced oxygen delivery to the placental intervillous space and aberrant vascular formation can take part in increasing of umbilical blood flow resistance observed during IUGR.

IUGR + PED (UTERO-PLACENTAL HYPOXIA)

Late-onset IUGR + PED belongs to the uteroplacental type of hypoxia. This condition is a result of early pregnancy deficient trophoblast invasion of endometrial arteries. The delivery of blood to the intervillous space is compromized and ischaemic hypoxia occurs. Placental villi suffer uneven quality of perfusion and oxygenation [37].

Literary data considering molecular regulation of vaculogenesis and angiogenesis under condition of IUGR + PED are inconsistent. This can be ascribed as well to the fact that many authors describe IUGR + PED together with preeclampsia, which is very often, but not unconditional co-morbidity of mothers with IUGR foetuses. Many authors also do not take in consideration the storage and release of VEGF-A from platelets, which can significantly affect the dynamics of its serum level. We try to summarize the often-controversial literature data. Summarized, behaviour of the selected angiogenic factors may be following in IUGR + PED pregnancies in comparison with normal pregnancies (for more comprehensive information see Mayhew et al., 2004):

• Levels of hypoxia inducible factor (HIF) and VEGF-A would be normal or increased before mid-gestation and at term, levels of VEGFR-1 can be reduced.
• There may be a present decrease in serum PlGF levels, especially after mid-gestation. Expression in placental tissues may be increased.
• The level of Ang-2 mRNA is reduced, the level of Ang-1 mRNA unchanged.
• The level of fibroblast growth factor (FGF-2) in the maternal serum remained unchanged.
• The level of angiogenin in serum and placenta is unchanged.

For IUGR + PED, the increase of branching angiogenesis is typical. There is a decrease in the volume and surface of the capillaries of the villi and decrease in capillary length. Despite the decrease of capillary volume and surface, capillarisation can be maintained and even increased [38]. The vascularisation itself is not affected. Changes in the number of endothelial cells are not known.

An increase of branching angiogenesis can be a breeding ground for the characteristic malformation of terminal villi with in knob-like, multiply indented villous surfaces [6].

IUGR + ARED (POST-PLACENTAL HYPOXIA)

Early onset IUGR + ARED is usually attended by early preeclampsia, although it can manifest alone as well. It is also known as placental hyperoxia, because of changes in the fetoplacental unit circulation greatly limit uptake of oxygen from the intervillous space. Oxygenation of the foetus is then significantly reduced, whereas the intervillous space partial pressure of oxygen is higher than in normal pregnancies (hence the term postplacental hypoxia) [37].

Molecular regulation of vasculogenesis and angiogenesis slightly differ from that of IUGR + PED:

• Expression of VEGF-A in the syncytiotrophoblast is reduced.
• Expression of VEGFR-1 is decreased and level of VEGFR-2 is maintained.
• PlGF mRNA and protein levels are up-regulated, especially in the second half of pregnancy.
• The levels of Ang-2 mRNA in placental tissues do not different from levels detected in healthy controls; however, immunoreactivity of Ang-2 protein is significantly reduced. These changes probably reflect different translational regulation and/or premature maturation of fetal vessels in terminal villi.
• Plasma levels of angiogenin with ARED + IUGR pregnancies are reduced compared to controls, in placental tissue is the level of angiogenin comparable or increased.

Morphometric analyses reveal relatively few capillary loops, which are long but slender and substantially longer and more poorly branched than in controls. The long and unbranched capillary loops increase the diffusion distance of the mother-child. Artery wall in villi is stronger and vessels have a smaller diameter. The umbilical cord enters into placenta often at the edge of the placenta, not in the middle. A number of the cotyledons is often reduced.

CONCLUSION

Impaired vasculogenesis and angiogenesis are associated with placental and foetal mal-development and can result in complicated pregnancies, such as IUGR. Although these processes have been extensively studied, their role in placental development is not still clarified in full. Rational therapeutic strategies that target these processes should be considered to provide safe antenatal surveillance of the IUGR foetus, climaxing in a planned delivery. General management includes good maternal nutrient therapy, cessation of substance abuse (cigarette smoking), and institution of bed rest, which may maximize uterine blood flow. Antenatal testing can include the non-stress test, repeated biometric profile examination, and an oxytocin challenge test. In subsequent pregnancies, low-doses of aspirin may help reduce the presence of IUGR in selected high-risk women. Hovewer, routine use of aspirin in pregnancy is not recommended.

More effective use of current evidence as well as further studies is crucial for improvement of the perinatal prognosis of IUGR due to placental insufficiency.

This study was supported by grants GAUK 12642/2014 and RVO-VFN 64165/2012.

MUDr. Petra Bolehovská

Gynekologicko-porodnická klinika

Nemocnice Na Bulovce

Budínova 67/2

180 81 Praha 8

e-mail: petrabolehovska@seznam.cz