Fiona Lyall

Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms.

The abnormal umbilical artery Doppler waveform represented by absent end-diastolic flow velocity (AEDFV) identifies a group of preterm small-for-gestational age fetuses that are at high risk of perinatal death due to chronic fetal hypoxia. The placental ischaemia that results from inadequate trophoblast invasion of spiral arterioles leads to an assumption of placental villous hypoxia, though an alternative explanation is that the placenta fails to adequately transfer oxygen to the fetus from the intervillous space. Because oxygen transport takes place within the terminal villi, we undertook the first detailed studies of villous ultrastructure structure and immunohistochemistry in order to determine the likely origin of fetal hypoxia in this condition. Terminal villi were examined ultrastructurally using transmission electron microscopy and by immunohistochemical localization of matrix molecules (laminin and collagens I, III and IV) and a marker of cell proliferation (MIB-1), in 16 small-for-gestational age pregnancies with AEDFV in the umbilical artery [deemed to have intrauterine growth restriction (IUGR)] and in 16 gestation age-matched controls. Terminal villi from the IUGR cases were smaller in diameter (P < 0.02) and had several abnormal features in comparison with the controls; increased syncytial nuclei (P < 0.01), reduced cytotrophoblast nuclei (P < 0.01), thickened basal lamina (P < 0.01), and increased stromal deposition of collagens and laminin. The amount of proliferating cytotrophoblast was reduced in the IUGR group (P < 0.014) and the degree of capillary erythrocyte congestion within terminal villous capillaries was increased (P < 0.001). Several of the structural differences in the terminal villi of the IUGR group such as reduced cytotrophoblast proliferation and stromal fibrosis are incompatible with the prevailing view of placental hypoxia in IUGR. Rather thickening of the basal lamina and congestion of the capillaries by erythrocytes are predicted to limit oxygen transfer from the intervillous space to the fetus and may represent an equilibration of oxygen tension between intervillous space and the terminal villi. Despite the known reduction in uteroplacental blood flow in IUGR, fetoplacental blood flow is compromised to a far greater extent in the presence of AEDFV such that maternal blood leaving the placenta has a higher oxygen content than under normal circumstances.

Nitrotyrosine Residues in Placenta: Evidence of Peroxynitrite Formation and Action

The interaction of nitric oxide and superoxide produces peroxynitrite anion, a strong, long-lived oxidant with pronounced deleterious effects that may cause vascular damage. The formation and action of peroxynitrite can be detected by immunohistochemical localization of nitrotyrosine residues. We compared the presence and localization of nitrotyrosine and of the endothelial isoform of nitric oxide synthase in placental villous tissue from normotensive pregnancies (n = 5) with pregnancies complicated by preeclampsia (n = 5), intrauterine growth restriction (n = 5), and preeclampsia plus intrauterine growth restriction (n = 4), conditions characterized by increases in fetoplacental vascular resistance, fetal platelet consumption, and fetal morbidity and mortality. In all tissues, absent or faint nitrotyrosine immunostaining but prominent nitric oxide synthase immunostaining were found in syncytiotrophoblast. In tissues from normotensive pregnancies, faint nitrotyrosine immunostaining was found in vascular endothelium, and nitric oxide synthase was present in stem villous endothelium but not in the terminal villous capillary endothelium. In contrast, in preeclampsia and/or intrauterine growth restriction, moderate to intense nitrotyrosine immunostaining was seen in villous vascular endothelium, and immunostaining was also seen in surrounding vascular smooth muscle and villous stroma. The intensity of nitrotyrosine immunostaining in preeclampsia (with or without intrauterine growth restriction) was significantly greater than that of controls. Intense nitric oxide synthase staining was seen in endothelium of stem villous vessels and the small muscular arteries of the terminal villous region in these tissues and may be an adaptive response to the increased resistance. The presence of nitrotyrosine residues, particularly in the endothelium, may indicate the formation and action of peroxynitrite, resulting in vascular damage that contributes to the increased placental vascular resistance.

Circulating angiogenic factors in early pregnancy and the risk of preeclampsia, intrauterine growth restriction, spontaneous preterm birth, and stillbirth

OBJECTIVE: To estimate the relationship between maternal serum levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) in early pregnancy with the risk of subsequent adverse outcome. METHODS: A nested, case–control study was performed within a prospective cohort study of Down syndrome screening. Maternal serum levels of sFlt-1 and PlGF at 10–14 weeks of gestation were compared between 939 women with complicated pregnancies and 937 controls. Associations were quantified as the odds ratio for a one decile increase in the corrected level of the analyte. RESULTS: Higher levels of sFlt-1 were not associated with the risk of preeclampsia but were associated with a reduced risk of delivery of a small for gestational age infant (odds ratio [OR] 0.92, 95% confidence interval [CI] 0.88–0.96), extreme (24–32 weeks) spontaneous preterm birth (OR 0.90, 95% CI 0.83–0.99), moderate (33–36 weeks) spontaneous preterm birth (OR 0.93, 95% CI 0.88–0.98), and stillbirth associated with abruption or growth restriction (OR 0.77, 95% CI 0.61–0.95). Higher levels of PlGF were associated with a reduced risk of preeclampsia (OR 0.95, 95% CI 0.90–0.99) and delivery of a small for gestational age infant (OR 0.95, 95% CI 0.91–0.99). Associations were minimally affected by adjustment for maternal characteristics. CONCLUSION: Higher early pregnancy levels of sFlt-1 and PlGF were associated with a decreased risk of adverse perinatal outcome. LEVEL OF EVIDENCE: II

Suppression of serum vascular endothelial growth factor immunoreactivity in normal pregnancy and in pre‐eclampsia

Objective To determine whether vascular endothelial growth factor (VEGF) concentrations are altered in pre‐eclampsia.

The cell adhesion molecule, VCAM‐1, is selectively elevated in serum in pre‐eclampsia: does this indicate the mechanism of leucocyte activation?

Objective To determine whether circulating levels of cell adhesion molecules, markers of endothelial damage and leucocyte activation, were increased in pre‐eclampsia.

Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function

The purpose of this study was to examine the expression of hemeoxygenases HO‐1 and HO‐2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO‐1 and HO‐2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO‐2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO‐2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO‐2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO‐2. HO‐1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO‐1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose‐dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function. Lyall, F., Barber, A., Myatt, L., Bulmer, J. N., Robson, S. C. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function. FASEB J. 14, 208–219(2000)

Nitric oxide concentrations are increased in the fetoplacental circulation in preeclampsia

OBJECTIVE The aim of this study was to measure serum concentrations of total nitrites, as an index of nitric oxide synthesis, in the maternal and fetal circulations of normal pregnancies and in pregnancies complicated by preeclampsia. STUDY DESIGN We studied 32 women with preeclampsia and 36 with uncomplicated pregnancies. Maternal venous blood samples were collected from all of the patients, and umbilical venous blood was collected from 13 of the preeclamptic group and 17 of the control group. Serum nitric oxide concentrations were determined with the Greiss reaction by measuring combined oxidation products of nitric oxide, serum nitrite and nitrate after reduction with nitrate reductase. RESULTS There were no significant differences in maternal serum nitrite concentrations between the groups (control group 29.8 +/- 1.07 mumol/L, preeclamptic group 29.5 +/- 1.06 mumol/L). Significantly higher serum nitrite concentrations were found in umbilical venous serum in the preeclamptic group compared with the control group (34.59 +/- 1.12 mumol/L vs 23.90 +/- 1.05 mumol/L, p < 0.01). CONCLUSIONS Total nitrites are increased in the fetoplacental circulation in preeclampsia. These results support the hypothesis that increased nitric oxide production may be a compensatory response to improve blood flow or may play a role in limiting platelet adhesion and aggregation.

Spiral Artery Remodeling and Trophoblast Invasion in Preeclampsia and Fetal Growth Restriction Relationship to Clinical Outcome

Failure to transform uteroplacental spiral arteries is thought to underpin disorders of pregnancy, including preeclampsia and fetal growth restriction (FGR). In this study, spiral artery remodeling and extravillous-cytotrophoblast were examined in placental bed biopsies from normal pregnancy (n=25), preeclampsia (n=22), and severe FGR (n=10) and then compared with clinical parameters. Biopsies were immunostained to determine vessel wall integrity, extravillous-cytotrophoblast location/density, periarterial fibrinoid, and endothelium. Muscle disruption was reduced in myometrial spiral arteries in preeclampsia (P=0.0001) and FGR (P=0.0001) compared with controls. Myometrial vessels from cases with birth weight <5th percentile (P<0.001), abnormal uterine Doppler (P<0.01), abnormal umbilical artery Doppler (P<0.001), and preterm delivery (P<0.001) had less muscle destruction compared with >5th percentile. Fewer extravillous-cytotrophoblast surrounded both decidual and myometrial vessels in the normal group and preeclampsia group compared with the FGR group (P=0.001). For myometrial vessels, the normal group contained more intramural extravillous-cytotrophoblast than in preeclampsia (P=0.015). Decidual vessels in the FGR group had less fibrinoid deposition compared with controls (P=0.013). For myometrial vessels, less fibrinoid was deposited in both the preeclampsia group (P=0.0001) and the FGR group (P=0.01) when compared with controls, and less fibrinoid was deposited in the preeclampsia group when compared with FGR group (P<0.001). Myometrial vessels obtained from birth weights <5th percentile had less periarterial fibrinoid than those with >5th percentile (P<0.02). A major defect in myometrial spiral artery remodeling occurs in preeclampsia and FGR that is linked to clinical parameters. Interstitial extravillous-cytotrophoblast is not reduced in preeclampsia but is increased in FGR.

Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction.

During early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR.

Heme oxygenase expression in human placenta and placental bed: reduced expression of placenta endothelial HO-2 in preeclampsia and fetal growth restriction

In this study we tested the hypothesis that expression of heme oxygenases HO‐1 and HO‐2, which are responsible for the production of carbon monoxide, are reduced in the placenta and placental bed of pregnancies complicated by preeclampsia (PE) and fetal growth restriction (FGR) compared with control third‐trimester pregnancies. Placental protein expression was determined by Western blotting (n=10 in each group) and immunohistochemistry (controls n=18, PE n=19, FGR n=10). Extravillous trophoblast expression was determined by immunohistochemistry of placental bed biopsy samples (controls n=17, PE n=19, FGR n=10). Western blot analysis of placental homogenates showed no overall differences in HO‐2 among groups. However, immunohistochemical analysis showed a reduction in HO‐2 expression in endothelial cells in both abnormal groups (PE P<0.01;FGR P<0.0005 vs. control group) but no differences in villous trophoblast staining. HO‐1 was undetectable by Western blotting in control and abnormal pregnancies and immunoreactivity was very low, suggesting that there is little HO‐1 in the placenta. Within the placental bed, HO‐2 but not HO‐1 was detected on all populations of extravillous trophoblast, but expression of HO‐2 or HO‐1 did not change in PE or FGR. The reduced expression of HO‐2 on endothelial cells in PE and FGR may be responsible for reduced placental blood flow in these conditions. The data do not show changes in HO in the placental bed in PE or FGR.— Barber, A., Robson, S. C., Myatt, L., Bulmer, J. N., Lyall, F. Heme oxygenase expression in human placenta and placental bed: reduced expression of placenta endothelial HO‐2 in preeclampsia and fetal growth restriction.—Barber, A., Robson, S. C., Myatt, L., Bulmer, J. N., Lyall, F. Heme oxygenase expression in human placenta and placental bed: reduced expression of placenta endothelial HO‐2 in preeclampsia and fetal growth restriction. FASEB J. 15, 1158–1168 (2001)