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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1042
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 1 285-292
Copyright © 2008 by The Endocrine Society

Placental Expression of Soluble fms-Like Tyrosine Kinase 1 is Increased in Singletons and Twin Pregnancies with Intrauterine Growth Restriction

Ori Nevo, Ariel Many, Jing Xu, John Kingdom, Ettore Piccoli, Stacy Zamudio, Martin Post, Alan Bocking, Tullia Todros and Isabella Caniggia

Department of Obstetrics and Gynecology (O.N., J.K., A.B., I.C.) and Samuel Lunenfeld Research Institute (O.N., J.X., A.M., J.K., A.B., I.C.), Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; Hospital for Sick Children (M.P.), Toronto, Ontario, Canada M5G 1X8; Department of Obstetrics and Gynecology (O.N.), Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5; Department of Physiology (M.P., A.B., I.C.), University of Toronto (O.N., J.K., M.P., A.B., I.C.), Toronto, Ontario, Canada M5S 1A8; Department of Obstetrics, Gynecology, and Women’s Health (S.Z.), New Jersey Medical School, Newark, New Jersey 07101; and Department of Obstetrics and Gynecology (T.T., E.P.), University of Turin, 10124 Turin, Italy

Address all correspondence and requests for reprints to: Isabella Caniggia, M.D., Ph.D., Mount Sinai Hospital, Samuel Lunenfeld Research Institute, 600 University Avenue, Room 871C, Toronto, Ontario, Canada M5G 1X5. E-mail: caniggia{at}mshri.on.ca.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Objective: Intrauterine growth restriction (IUGR) is characterized by decreased placental perfusion. Low oxygen has been shown to increase soluble fms-like tyrosine kinase 1 (sFlt-1) expression in the human placenta. The objective of this study was to examine sFlt-1 expression in different types of IUGR pregnancies, including early-onset severe cases characterized by abnormal umbilical and uterine artery Doppler and discordant IUGR twins in which the normal cotwin represents the optimal control because both placentas share the same uterine environment.

Patients: Placentas from four subgroups were collected: early severe IUGR with umbilical artery absent end diastolic flow (n = 19), small for gestational age with normal uterine and umbilical artery Doppler (n = 11), severely growth-restricted dichorionic and monochorionic twins with abnormal umbilical artery Doppler (n = 9), preeclamptic twins (n = 3), and age-matched normal singletons (n = 19) and twin controls (n = 8).

Results: Expression of sFlt-1 mRNA and protein was significantly increased in IUGR placentas compared with small for gestational age and normal control placentas. sFlt-1 expression levels were also significantly greater in the small IUGR twin placentas from discordant twin pregnancies compared with the normal cotwin. In preeclamptic twins, sFlt-1 expression was increased in only one of the two placentas.

Conclusions: Our results demonstrate that sFlt-1 expression is increased in severe IUGR placentas with abnormal umbilical artery Doppler of singletons and also in discordant IUGR twins. Reduced placental perfusion may contribute to the increased expression of sFlt-1 in IUGR pregnancies. Our data are compatible with differential sFlt-1 expression in placentas from discordant twins.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Severe intrauterine growth restriction (IUGR) is one of the major complications of pregnancy and accounts for significant neonatal mortality and morbidity (1). Approximately 50% of women with growth-restricted fetuses will develop preeclampsia (2). Severe growth restriction and preeclampsia often necessitate preterm delivery, further impacting neonatal morbidity. The intrauterine environment of low-birth-weight neonates not only contributes to increased morbidity early in life but is also associated with increased risk for developing cardiovascular and metabolic disorders later in life (3, 4).

The definition of fetal growth restriction is important, because not all small fetuses are growth restricted. True growth restriction is present when the fetus fails to achieve its growth potential (5). Although IUGR may be related to several causes (6, 7), most often its development is attributable to a defect in proper placental development (8). Histomorphological studies indicate that IUGR is associated with structural alterations of the placenta such as abnormally developed terminal capillary loops and sparse arrangement of functional terminal villi (9). These morphological alterations are responsible for increased fetoplacental vascular resistance and consequently decreased blood flow and availability of nutrients and oxygen to the fetus.

The soluble vascular endothelial growth factor receptor 1 (VEGFR-1), also known as soluble fms-like tyrosine kinase 1 (sFlt-1), is a truncated form of the VEGFR-1 gene that is physiologically secreted by the human placenta (10). Circulating sFlt-1 binds VEGF and placental growth factor (PlGF) with high affinity, thereby decreasing their availability for binding to the transmembrane receptors VEGFR-1 and VEGFR-2 (10), events required for physiological endothelial cell function (11). During normal pregnancy, sFlt-1 serum levels increase with advancing gestation. Recent studies have shown that both circulating and placental sFlt-1 expression levels are markedly increased in preeclamptic pregnancies (12, 13, 14). We have shown recently that sFlt-1 is regulated by oxygen via hypoxia-inducible transcription factor 1, supporting a key role for low oxygen in regulating sFlt-1 expression in the human placenta (15). Doppler studies indicate that pregnancies complicated by IUGR exhibit decreased uterine blood flow and higher resistance indices. This is attributable to both failure in spiral artery remodeling found in IUGR pregnancies (16) and abnormal development of the tertiary villous vasculature (17, 18), which then limits the amount of oxygen to the placenta. We therefore hypothesized that lowered oxygen availability in IUGR placentas may lead to increased sFlt-1expression. However, the data regarding sFlt-1 expression in placentas from IUGR pregnancies are controversial. One report (19) using a heterogeneous group of IUGR pregnancies showed a 2-fold increase in placental sFlt-1 expression compared with controls, whereas another study reported no changes in sFlt-1 expression in IUGR placentas (20).

In the present study, we examined the expression of sFlt-1 in placental samples from severe IUGR pregnancies using singletons and discordant twin samples in which one twin was severely growth restricted. The singleton IUGR pregnancies were rigorously classified according to onset and severity as documented by Doppler analysis. These singleton IUGR placentas were compared with normal control tissue and with placentas from small for gestational age (SGA) pregnancies. Our second example, of discordant twins, is advantageous in having two placentas developing in the same maternal uterine environment, thereby creating an optimal intrauterine control twin for the growth-restricted twin. Understanding sFlt-1 expression in IUGR placenta is critical, because alteration in its expression may be related to the abnormal angioarchitecture found in placental vasculature and may in part contribute to the increased incidence of preeclampsia found in severe IUGR pregnancies.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and tissue collection

Local Ethics Committees approval was obtained for the study, and all women gave written informed consent. All women were healthy nonsmokers, with no signs of preeclampsia, infections, or other causes of IUGR. The clinical characteristics of women with singletons are shown in Table 1Go. Gestational age was determined by the date of the last menstrual period and first trimester ultrasound measurement of crown rump length.


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  		TABLE 1. Clinical characteristics of participating singleton pregnancies

 
IUGR was defined as birth weight less than the fifth centile accompanied by abnormal umbilical artery Doppler defined as absent or reverse end diastolic velocity (AREDV) and in most of cases with increased resistance to flow in uterine arteries defined as early diastolic notch or pulsatility index more than 1.45. We used these strict criteria to distinguish severe placental dysfunction as the cause of the growth restriction vs. constitutionally small fetuses, a significant confounder in previous studies (19, 20). The neonates were examined after delivery, and none showed signs of infection or chromosomal abnormalities. Newborns with birth weight less than or equal to the 10 centile but with normal umbilical and uterine artery Doppler were defined as SGA. Controls were selected as age-matched healthy pregnancies with normally grown fetuses that did not have signs of placental dysfunction. Birth weight, gestational age, laboratory values, and clinical observations relevant to the health of the mother were taken from the clinical records. Relevant ultrasound and Doppler data that were collected during pregnancy were recorded. Subjects suffering from diabetes, essential hypertension, or renal disease were excluded. Preterm deliveries were attributable to incompetent cervix, preterm rupture of membrane, and spontaneous preterm deliveries without identifiable cause. All preterm (PTC) and term (TC) control groups did not show clinical or pathological signs of preeclampsia, infections, or other maternal or placental disease.

Discordant twins were selected as a separate group in which a normal twin and a growth-restricted twin developed in the same maternal and uterine environment. Discordant growth was defined as discordancy of more than 25% in birth weight in conjunction with IUGR in one fetus with AREDV and normal growth and Doppler study in the cotwin. Dichorionic and monochorionic twins were considered as two separate groups. The clinical parameters of the twin pregnancies are shown in Table 2Go. Additionally, dichorionic twin pregnancies with signs of preeclampsia, but without IUGR, were included as a separate group and were compared with control twins without preeclampsia. Preeclampsia was diagnosed according to the American College of Obstetrics and Gynecology criteria (21). The clinical characteristics of twins with preeclampsia are shown in Table 2Go.


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  		TABLE 2. Clinical characteristics of participating twin pregnancies

 
All specimens were randomly collected immediately after delivery from normal-looking cotyledons. Areas with calcified, necrotic, or visually ischemic tissue were omitted from sampling. Pathological analysis of IUGR placentas showed the presence of multiple infarcts, intervillous thrombosis, fibrin deposition, and the existence of numerous immature villi. Because the human placenta is known to be a heterogeneous tissue, we further collected multiple samples from the same placenta in a subgroup of patients with severe IUGR and controls using a preformed grid and random sampling method (22). Five samples were collected from each placenta.

In all intrauterine growth-restricted cases, cord blood was obtained at birth from the double-clamped umbilical vein and processed immediately for fetal blood gases analysis. IUGR fetuses exhibited pO2 and pH values below the fifth centile in 70 and 89% of the cases, respectively.

RNA isolation and real-time quantitative RT-PCR

Total RNA was isolated using a TRIzol-based approach according to the protocol of the manufacturer (Invitrogen, Carlsbad, CA). DNA contamination was enzymatically removed by deoxyribonuclease-I digestion before RNA RT. Quantitative PCR (qPCR) (sFlt-1) was performed on the Opticon II Light Cycler system (MJ Research, Waltham, MA) as described previously (23). TaqMan Universal MasterMix and specific TaqMan primers and probe for soluble VEGFR-1 (sFlt-1) and 18S were used (Applied Biosystems, Foster City, CA) based on the protocol of the manufacturer (Applied Biosystems). Sequences for sFlt-1 primers and probe were as follows: forward, 5'-GGGAAGAAATCCTCCAGAAGAAAGA-3'; reverse, 5'-GAGATCCGAGAGAAAACAGCCTTT-3'; and probe, 5'-CAGTGCTCACCTCTGATTG-3'. For the relative quantitation, PCR signals were compared among groups after normalization using 18S as an internal reference. Relative expression was calculated according to Livak and Schmittgen (24).

Western blot analysis

Western blot analysis for sFlt-1 was performed using 50 µg of total placental protein lysates that were subjected to 8% (wt/vol) SDS-PAGE gel. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. Nonspecific binding was blocked by incubation in 5% (wt/vol) BSA in Tris-buffered saline containing 0.1% (vol/vol) Tween 20 (TBST) for 60 min. Membranes were then incubated with 1:200 diluted rabbit anti-soluble VEGFR-1 (sFlt-1) antibody (Zymed Laboratories, San Francisco, CA) in 5% (wt/vol) BSA in TBST at 4 C or with mouse monoclonal anti-Flt-1 antibody (1:1000; Abcam, Cambridge, MA) in 5% (wt/vol) milk in TBST. After overnight incubation, membranes were washed with TBST and incubated for 60 min at room temperature with 1:10,000 diluted horseradish peroxidase-conjugated antirabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) in 5% (wt/vol) BSA in TBST or with 1:10,000 horseradish peroxidase-conjugated antimouse IgG (Santa Cruz Biotechnology) in 5% milk in TBST. After washing with TBST, blots were exposed to chemiluminescent reagent (ECL; GE Healthcare, Little Chalfont, UK). All Western blots were checked for equal protein loading at all times using Ponceau staining.

Immunohistochemistry

Paraffin sections were mounted on glass slides, dewaxed in xylene, and rehydrated in a descending ethanol gradient. Antigen retrieval was performed by heating in 10 mM sodium citrate solution. Endogenous peroxidase was quenched with 3% (vol/vol) hydrogen peroxide in PBS for 30 min. After blocking [5% (wt/vol) normal goat serum for 1 h], the slides were incubated overnight with primary antibody (rabbit antihuman sFlt-1, 1:150 dilution; Zymed Laboratories). Slides were washed in 1x PBS and exposed to biotinylated secondary goat antirabbit antibody (1:300; Vector Laboratories, Burlingame, CA) for 45 min at room temperature. Finally, avidin biotin complex (Vector Laboratories) was applied for 1 h, and positive immunoreactivity was visualized with the diaminobenzidine chromogen after 5 min. Slides were counterstained with hematoxylin. Primary antibody was omitted and replaced by blocking solution in negative controls.

Statistics

Statistical analyses were performed using Prism software (GraphPad Software, San Diego, CA). For comparison of data between multiple groups with normal distribution, we used one-way ANOVA with post hoc Dunnett’s test. For comparison between two groups, we used the Mann-Whitney U test and paired or unpaired t test when applicable. Significance was defined as P < 0.05. Results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
sFlt-1 expression in placentas of singletons with severe IUGR

Early-onset severe IUGR with documented pathological umbilical artery Dopplers exhibit substantial clinical differences compared with SGA pregnancies with normal Doppler (Table 1Go). In a first set of experiments, we examined the expression of sFlt-1 in two different pathological groups, namely placentas from severe IUGR without any sign of preeclampsia and placentas from SGA. Age-matched PTC and TC placentas were used as controls. Real-time qPCR analysis showed a significant increase (>2.5-fold) in sFlt-1 mRNA expression in severe singleton IUGR placentas compared with the SGA, PTC, and TC placentas (Fig. 1AGo). No significant changes in sFlt-1 mRNA expression were found between SGA and control groups.


Figure 1
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  		FIG. 1. sFlt-1 expression in IUGR, SGA, and control placental tissue. A, Expression of sFlt-1 mRNA, assessed by real-time qPCR, in severe IUGR (n = 19), SGA (n = 11), PTC (n = 19), and TC (n = 16) placentas. *, P < 0.05, IUGR vs. SGA, PTC, and TC. B and C, Representative sFlt-1 immunoblots of IUGR, SGA, PTC, and TC placental samples. AMC, Age-matched control; PE, preeclampsia. D, sFlt-1 protein densitometric analysis of Western blots of IUGR, SGA, PTC, and TC. *, P < 0.05, IUGR vs. SGA, PTC, and TC. All values are represented as the mean ± SE.

 
Because the human placenta is a heterogeneous tissue, we next examined sFlt-1 expression in multiple samples randomly collected from a subgroup of severe IUGR and PTC (22). Combining the transcript levels obtained from all samples within the same group, we found that sFlt-1 mRNA expression was increased in the severe IUGR group compared with the controls (IUGR, 2.15 ± 0.4 vs. PTC, 0.89 ± 0.21 arbitrary sFlt-1 mRNA levels; n = 3, P < 0.05). These data support that increased sFlt-1 expression in IUGR is a global placental event and not confined to a limited area of the placenta.

Western blot analysis demonstrated greater sFlt-1 protein levels in IUGR placentas compared with SGA, PTC, and TC, in agreement with increased mRNA expression (Fig. 1Go, B and C). Although variability in sFlt-1 protein levels was noted in both IUGR and control placentas, sFlt-1 was consistently elevated in the pathological group. No changes in sFlt-1 protein levels were noted between SGA, PTC, and TC placentas (Fig. 1CGo). Densitometric analysis revealed a 2-fold increase in sFlt-1 protein content in severe IUGR placentas compared with control placentas (Fig. 1DGo). The spatial localization of sFlt-1 in severe IUGR and control placentas was examined by immunohistochemistry. Strong positive immunoreactivity for sFlt-1, mostly localized to trophoblast layers, was observed in IUGR placentas (Fig. 2Go). Negative or weak staining for sFlt-1 was found in PTC and SGA placentas, respectively. Low to absent stromal or perivascular staining was noted in both IUGR and control placentas.


Figure 2
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  		FIG. 2. Immunolocalization of sFlt-1 in IUGR, SGA, and control placental tissue. Top left, PTC. Top right, Negative control (no primary antibody). Bottom left, IUGR. Bottom right, SGA. Brownish stainingrepresents positive sFlt-1 immunostaining. ST, Syncytiotrophoblast; ET, endothelium.

 
sFlt-1 expression in IUGR discordant twin placentas

Twin pregnancies share the same maternal environment, and, therefore, hypothetically placentas are exposed to the same intrauterine milieu. Thus, discordant twins consisting of a severe IUGR and normally grown twin provides a natural example in which the normally grown twin serves as an internal control for the IUGR twin. We have used this example to compare sFlt-1 expression in placentas of growth-restricted twin vs. normally grown cotwin from dichorionic and monochorionic twin pregnancies.

In dichorionic twins, the sFlt-1 transcript level was significantly greater in the IUGR twin placenta compared with its normally grown cotwin placenta (IUGR twin vs. cotwin, 3.52 ± 0.5 vs. 1.08 ± 0.13 arbitrary sFlt-1 mRNA levels; P < 0.05) and placenta of normal control twins (Fig. 3AGo). In line with the dichorionic twins, sFlt-1 mRNA expression in monochorionic twins was also increased in the growth-restricted twin placenta compared with the cotwin placenta (IUGR twin vs. cotwin, 1.7 ± 0.19 vs. 0.79 ± 0.2 arbitrary sFlt-1 mRNA levels; P < 0.05) and placentas of normal control twins without growth restriction (Fig. 3BGo). Interestingly, sFlt-1 transcript levels were greater in the dichorionic than the monochorionic growth-restricted twin placentas (dichorionic vs. monochorionic IUGR, 3.52 ± 0.5 vs. 1.7 ± 0.19 arbitrary sFlt-1 mRNA levels).


Figure 3
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  		FIG. 3. sFlt-1 expression in discordant twin placentas. A, Expression of sFlt-1 mRNA, assessed by real-time qPCR, in placentas of discordant dichorionic twins (DCDA; n = 4) compared with normal control twins (Twin A and Twin B; n = 8). B, Expression of sFlt-1 mRNA in discordant monochorionic twins (MCDA; n = 5) compared with normal control twins (Twin A and Twin B; n = 8). C, Left, Representative sFlt-1 immunoblot of placental tissues from discordant (DC) and normal (MC) control twins (L, large twin; S, small twin). Right, sFlt-1 protein densitometric analysis. Data are normalized vs. controls. *, P < 0.05 small twin vs. large twin. All values are represented as the mean ± SE.

 
Western blot and densitometric analysis of placental lysates from the discordant twins showed increased sFlt-1 protein content in the placentas from monochorionic and dichorionic IUGR twins when compared with their cotwins and control twins without growth restriction (Fig. 3CGo).

sFlt-1 expression in placentas from preeclamptic twins

Because placentas from control twins did not show changes in sFlt-1 expression, we examined an additional group of concordant twin pregnancies associated with preeclampsia attributable to elevated sFlt-1 expression in singleton preeclamptic pregnancies (14). Placental tissue from dichorionic twin pregnancies complicated by clinical signs of preeclampsia that did not show discordancy between fetuses was used for the sFlt-1 analysis. Unlike measurements of circulating sFlt-1 that do not differentiate between the different sources of sFlt-1, sampling of each placenta separately enabled us to quantify the relative sFlt-1 expression in each placenta. Real-time qPCR analysis of preeclamptic twin placentas consistently showed increased sFlt-1 transcripts in one placenta relative to the other, indicating discordance in sFlt-1 expression. Therefore, the preeclamptic and control twin pairs were divided into high-expression (HE) and low-expression (LE) subgroups according to the level of sFlt-1 RNA expression detected. We found significantly greater sFlt-1 mRNA expression in one of the preeclamptic twin placentas compared with the other and with that of twin controls (Fig. 4Go). sFlt-1 protein content of the same groups of preeclamptic concordant twins placentas was analyzed by Western blotting. As shown in Fig. 4BGo, the protein content of sFlt-1 in HE preeclamptic twin placenta was greater than that of the LE preeclamptic twin placenta and control twin placentas. The spatial localization of sFlt-1 in preeclamptic concordant twin placentas was next examined by immunohistochemistry (Fig. 4Go). Strong positive immunoreactivity for sFlt-1, which localized predominantly to trophoblast layers, was consistently observed in sections of one placenta but not in those of the other cotwin placenta. This staining pattern agrees with the data obtained by qPCR and Western blotting.


Figure 4
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  		FIG. 4. sFlt-1 expression in preeclamptic twin placentas. A, Placental expression of sFlt-1 mRNA, as assessed by real-time qPCR, in twins with preeclampsia (n = 3) compared with control twins. B, Representative Flt-1 immunoblot of placental lysate from preeclamptic and control twins. *, P < 0.05, HE vs. LE. All values are represented as the mean ± SE. C, Immunohistochemical localization of sFlt-1 in placental tissue from preeclamptic twin pregnancy. Left, Representative section of preeclamptic twin placenta with low expression (PE Twin-LE). Right, Representative section of preeclamptic twin placenta with high expression (PE Twin-HE).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The characterization of specific placental subpathologies is of utmost importance when studying the basis of molecular events underlying different pregnancy-related disorders. In the present study, we show that placental sFlt-1 expression is increased in placentas from severe early-onset IUGR pregnancies with documented absent/reverse end diastolic velocity but not in SGA pregnancies with normal Doppler. Second, we demonstrate that placental sFlt-1 expression is increased in singleton IUGR pregnancies and in the growth-restricted twin from discordant twins pregnancies compared with control twins. Additionally, we show that elevated sFlt-1 expression in preeclamptic twin pregnancies with no sign of growth discordancy originates in one but not both placentas.

The elevated placental sFlt-1 expression observed in singleton IUGR pregnancies in the present study is in agreement with a previous report of increased sFlt-1 transcript levels in placentas from IUGR pregnancies compared with control placentas (19). The previous study used a smaller number (n = 10) of heterogeneous (less stringently classified) IUGR pregnancies. Herein, we used a larger (n = 19) and more homogeneous group of severe IUGR samples classified by documented AREDV, delivery at mean age of 31.5 wk, and birth centile of 2.1. The finding of elevated sFlt-1 expression in placental tissues from pregnancies associated with AREDV is supported by recent reports. One study showed that increased circulating sFlt-1 levels in patients with documented abnormal uterine artery Doppler is a useful predictor of the development of preeclampsia (25). Two other studies reported increased serum sFlt-1 levels in IUGR pregnancies during the third trimester of the pregnancy (26, 27). Whether the placenta was the origin of the increase in circulating sFlt-1 was not addressed in these studies. Our observation of similar increases of placental sFlt-1 expression in the IUGR twins and singletons with severe IUGR further corroborates that IUGR placentas express higher levels of sFlt-1. This increased expression of sFlt-1 in IUGR cannot be attributed to the process of labor because we have shown recently that there is no difference in placental sFlt-1 expression between spontaneous delivery and cesarean section (15).

Our result partly disagree with another report showing no changes in sFlt-1 expression in IUGR placentas (20). This discrepancy could be attributable to the use of a different and more heterogeneous patients’ population, which included milder, late-onset IUGR patients delivering at mean gestational age of 39.5 wk, documented abnormal umbilical artery Doppler in only 3 of 24 patients, and a birth weight centile of 3.8, a group that would have been classified as SGA in our study. Interestingly, 58% of the SGA group in the above-mentioned study were smokers (20). Smoking is associated with decreased fetal weight and IUGR (28, 29) and was shown previously to cause a decrease in sFlt-1 expression in nonpregnant patients (30, 31) as well as in pregnant women (32). Because of this known effect of smoking on sFlt-1 expression, in our study we excluded women who smoked.

In the present study, we used discordant twin pregnancies as a natural example in which the IUGR and normal fetus share the same maternal environment. Hypothetically, the maternal environment should affect the two placentas and the fetuses in a similar manner. Our data demonstrate that, in discordant twin pregnancies, sFlt-1 expression is increased in the placental tissue from the growth-restricted twin, confirming that the fetal discordancy leads to molecular placental differences (33). As we reported previously (15), low oxygen via hypoxia-inducible transcription factor 1 is one of the main triggers for sFlt-1 expression in the placenta. Thus, the increased sFlt-1 expression in IUGR placentas is likely attributable to reduced placental perfusion experienced by the growth-restricted placenta, resulting in low oxygenation. Additional support for placental hypoxia being causally associated with IUGR, and not hyperoxia as was postulated (34), stems from an IUGR murine model induced by maternal hypoxia (35) and from a recent human study using gene array analysis in IUGR placentas showing increased expression of hypoxia-regulated genes (36). The idea that the fetal circulation is hypoxic and thus creates placental hypoxia via diffusion is quite old. In 1953, Clemetson and Churchman (37) argued that ischemia in the fetus (diminution/slowing of cord blood flow) results in fetal hypoxia, and this in turn causes placental hypoxia. The more recently postulated hyperoxia argument is based on limited clinical data showing that the blood exiting the uteroplacental circulation through the uterine vein has a higher pO2 in IUGR than in control pregnancies (38). Because the gas is equilibrating on a constant and ongoing basis, the placenta is being exposed to variable pO2 levels (as little as 5–10 mm Hg from the blood exiting the fetus through the cord artery to as high as 80 mm Hg from the blood entering through the maternal arteries into the intervillous space). The pO2 measurements likely reflect a moment in time when equilibration between maternal and fetal circulation is ongoing. The fact that the blood exiting the maternal uterine vein may have a slight higher pO2 is irrelevant to what is seen in the placenta, which is highly variable and encompasses the value of the pO2 exiting through the uterine vein (39). Moreover, it is established that a high percentage of the uterine blood flow is normally shunted to the uterine vein and as such does not contribute to the intervillous gas exchange (40). However, we cannot exclude that severe IUGR is associated with decreased oxygen extraction by the placenta as was postulated previously (41).

Considering that one twin develops normally in the same uterine environment whereas the IUGR twin exhibits AREDV in the umbilical artery, it is likely that the process of decreased placental perfusion found in singleton IUGR leading to placental hypoxia holds true in discordant twin pregnancies. Only a few studies on molecular differences in discordant twin placentas are available. One study reported increased apoptosis in placentas from IUGR twins (42) compared with their normally grown cotwin, indicative of abnormal trophoblast turnover. Thus, in discordant twins, despite of the same maternal environment, the two placentas present different morphology, which is accompanied by differences in sFlt-1 expression. Our findings indicate that sFlt-1 expression was higher in dichorionic growth-restricted twins compared with the monochorionic (3-fold increase in IUGR dichorionic vs. 1.7-fold increase in monochorionic). The cause for increased sFlt-1 levels in dichorionic placenta can be related to differences in the mechanism underlying the pathogenesis of the chorionicity in twin pregnancies. Whereas the alteration found in dichorionic placenta is likely attributable to impaired placental development similar to that found in singletons IUGR, the placental pathology in monochorionic IUGR is further complicated by the unequal sharing of the placenta in which the IUGR twin has smaller area of the placenta and the presence of vascular anastomoses between the two twins (43).

Studies on growth-restricted fetuses with abnormal blood flow in the umbilical artery have revealed that the increased resistance found in these vessels is attributable to the presence of nonbranching intermediate villi and decreased number of terminal villi capillaries that are responsible for most of the increased resistance in the placental circulation (9, 18). The precise mechanisms involved in the pathogenesis of the abnormal fetoplacental vasculature in IUGR remain elusive. The present data may in part explain the abnormal angioarchitecture found in IUGR placentas. Previous studies have shown that IUGR placentas are characterized by decreased VEGF expression and increased PlGF expression (44). As such, elevated placental sFlt-1 production, leading to increased sFlt-1 circulating levels in the fetoplacental circulation (45), can bind locally produced VEGF and PlGF, impairing their binding to the VEGF receptors. This would result in an anti-angiogenic balance in IUGR placentas, thereby altering the terminal differentiation of placental capillary loops. This may contribute to the abnormal angiogenesis found in IUGR in the intermediate and terminal IUGR villi.

Although it is established that preeclampsia is more prevalent in twin pregnancies, it remains to be established whether both placentas contribute equally to the development of preeclampsia. Our finding of high sFlt-1 expression in one placenta, but not in the other, suggests that only one placenta can trigger preeclampsia in this subgroup of nondiscordant twin pregnancies. The notion that one placenta in twins can cause preeclampsia is known from cases in which selective fetocide reverses preeclampsia in discordant twins (46) or resolution of mirror syndrome after demise of the diseased fetus (47). The reason for this selective increase in sFtl-1 expression in one of the twins placenta is as yet unknown.

In conclusion, our data demonstrate that sFlt-1 expression is increased in severe IUGR with documented abnormal umbilical blood flow (AREDV) in both singleton and discordant dichorionic and monochorionic twin pregnancies. This is true despite a shared intrauterine environment in twins and is consistent with placental hypoxia.


   Acknowledgments
 
We thank Dr. Ljiljiana Petkovic and Dragica Curovic for placental collection.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (CIHR) Grant MT-1406 (to I.C.) and a Physicians Services Incorporated grant (to O.N., A.B., and I.C.). I.C. is the recipient of an Ontario Women’s Health CIHR/Institute of Gender and Health Mid-Career Award.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 23, 2007

Abbreviations: AREDV, Absent or reverse end diastolic velocity; HE, high-expression subgroup; IUGR, intrauterine growth restriction; LE, low-expression subgroup; PlGF, placental growth factor; PTC, preterm control; qPCR, quantitative PCR; sFlt-1, soluble fms-like tyrosine kinase 1; SGA, small for gestational age; TBST, Tris-buffered saline containing 0.1% Tween 20; TC, term control; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

Received May 10, 2007.

Accepted October 16, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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