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Decreased Maternal Serum Placenta Growth Factor in Early Second Trimester and Preeclampsia

From the Department of Obstetrics and Gynecology, and the Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

Address reprint requests to: Fon-Jou Hsieh, MD Department of Obstetrics and Gynecology National Taiwan University Hospital No. 7, Chung-Shan South Road Taipei Taiwan E-mail: fjhsieh{at}ha.mc.ntu.edu.tw

	Abstract

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Objective: To compare early second-trimester maternal serum placenta growth factor concentrations in patients with subsequent development of preeclampsia and those with normal pregnancies.

Methods: We conducted a case-control analysis of stored maternal serum of 27 women who subsequently developed preeclampsia and 227 randomly selected normal controls during the gestational period of 14–19 weeks. Using such a sample size, there was a greater than 95% power to test a difference in the primary study interest. A quantitative sandwich enzyme immunoassay was used to measure the maternal serum placenta growth factor concentration. For statistical analysis, Mann–Whitney U tests, multiple linear regression analysis, multivariable logistic regression model, and receiver-operating characteristic (ROC) curve were used. P < .05 was considered statistically significant.

Results: Maternal serum placenta growth factor concentration was associated with the occurrence of subsequent preeclampsia (P < .001) and gestational age (P < .001). The median (interquartile range) of multiples (MoM) of the gestational age stratified median for placenta growth factor in preeclampsia was 0.55 (0.33, 0.85). The ROC curve revealed that the specificity was 70% when the diagnostic sensitivity was 70%, and the optimal cutoff value of placenta growth factor MoM was 0.76. The risk of developing preeclampsia subsequently was increased 2.5-fold for maternal serum placenta growth factor concentration decrements of 0.1 MoM.

Conclusion: A decreased maternal serum placenta growth factor concentration in the early second trimester is highly associated with the subsequent development of preeclampsia, but a large prospective study is needed to explore its use as an early predictor for the condition.

Preeclampsia is one of the most important complications of pregnancy and is the leading cause of maternal and perinatal morbidity and mortality in the world.^1,2 It is a complex clinical syndrome potentially involving all of the organ systems. Despite intensive research, the etiology of preeclampsia remains unknown. Current hypotheses include vascular-mediated factors, placental ischemia, genetic predisposition, and immune maladaptation, all of which have been stated to contribute to the development of preeclampsia.^3–6 Angiogenesis and vascular transformation are important processes in the normal development of the placenta.⁷ Abnormal development of the fetal vessels within the placental villi has been associated with fetal growth restriction and preeclampsia.⁸

Placenta growth factor, a member of the vascular endothelial growth factor family, is a 132-amino acid residue, 50-kDa dimeric glycoprotein, and shares a number of biochemical and functional features with other members of the vascular endothelial growth factor family.^9–11 Placenta growth factor is an important local mediator of angiogenesis and prominent expression of placenta growth factor has been identified in placenta, human umbilical vein endothelial cells, and choriocarcinoma cell lines.^10–12 In the placenta, placenta growth factor is produced mainly by the cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast.¹³ Previously, decreased levels of placenta growth factor in preeclamptic patients after the clinical manifestations occurred had been reported, mostly in the third trimester.^14,15

We designed this case-control study retrospectively to evaluate the early second-trimester maternal serum levels of placenta growth factor in pregnancies with the subsequent development of preeclampsia compared with that of normal pregnancies.

	Materials and Methods

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Second-trimester screening for Down syndrome and neural tube defects at 14–19 weeks’ gestation by using the combination of maternal serum alpha-fetoprotein and free beta-human chorionic gonadotropin is part of routine obstetric care at National Taiwan University Hospital. From January 1997 through June 1999, 7386 serum samples taken from women receiving the screening examination in the obstetric care clinics were kept in a -70C frozen serum bank. All the women were followed prospectively for their pregnancy outcomes. A total of 27 preeclampsia patients diagnosed in the study period who received the second-trimester screening for Down syndrome and delivered at the National Taiwan University Hospital enrolled in this study. To compare the effect of placenta growth factor on the incidence of preeclampsia, another 227 subjects with normal pregnancy outcomes matched for the gestational age of blood sampling were chosen from the same population. The control subjects were selected randomly from our serum bank to ensure a sufficient number of cases in each week of gestation between 14 and 19 weeks’ gestation. A total of 254 serum samples were analyzed in this study. Patients with a singleton pregnancy resulting in the full-term delivery of a live-born healthy infant were eligible for inclusion in the normal control group. Women who did not deliver their infants with known outcomes at National Taiwan University Hospital, or with medical or obstetric complications other than preeclampsia such as preterm labor, premature rupture of membranes, or known infectious disease, and those with fetal complications such as multifetal gestation, hydatidiform mole, or chromosomal or major congenital anomalies were excluded.

Preeclampsia was defined as new hypertension (two measurements of diastolic blood pressure [BP] above 90 mmHg obtained more than 4 hours apart or a single diastolic BP above 110 mmHg) after 20 weeks’ gestation, accompanied by proteinuria (at least 0.3 g/L in two samples of urine collected more than 4 hours apart or at least 0.3 g in a 24-hour urine collection) with no known history of hypertension or renal disease before pregnancy.¹⁶

At the time of second-trimester serum screening for Down syndrome, blood samples were drawn into serum tubes and allowed to clot, then immediately separated by centrifugation and stored at -70C before use. For the assay of serum placenta growth factor, a quantitative sandwich enzyme immunoassay technique (R & D Systems Inc., Minneapolis, MN) was performed. This antibody had no significant cross-reactivity or interference with other human growth factors or cytokines except to cross-react to recombinant human placenta growth factor/vascular endothelial growth factor heterodimer up to 5%, and the minimal detectable dose of placenta growth factor is typically less than 7 pg/mL (R & D Systems Inc.). In this immunoassay kit, wells of a microplate were coated with a murine monoclonal antibody against placenta growth factor stored at 4C overnight. All reagents were brought to room temperature before use. Diluent buffer was added to each well to rinse the plates. Standard or serum sample (100 µL) was added to each well. Wells were incubated for 2 hours at room temperature. Each well was aspirated and washed with 400 µL of wash buffer, and the process was repeated three times for a total of four washes. After the last wash, the remaining wash buffer was removed by aspiration. The plate was then inverted and blotted against paper toweling. Next, 200 µL of polyclonal antibody against placenta growth factor conjugated to horseradish peroxidase were added to each well and incubated for 2 hours at room temperature. Washing of each well with 400 µL of wash buffer was repeated three times. Substrate solution (200 µL) was added to each well. The reaction was incubated for 30 minutes at room temperature and then terminated with 50 µL of stop solution. The optical density of each well was determined within 30 minutes, using a microplate reader set to 450 nm.

According to our previous experience, the mean difference of log-transformed placenta growth factor between preeclampsia and normal was estimated to be 0.24, and the overall standard deviation of log (placenta growth factor) was assumed to be 0.22. Under such conditions with a two-tailed significance level of .05, there is a higher than 95% power to test difference of the primary study interest using such a sample size in this study. Mann–Whitney U test was used to compare the characteristics between the normal and preeclamptic pregnant women. The relationship between placenta growth factor and several other factors was evaluated using multiple linear regression analysis. Placenta growth factor, after logarithm transformation, was explored using a scatter plot as a function of gestational age. To adjust the differences of placenta growth factor in different weeks of gestation, weekly specific median of placenta growth factor was calculated from normal pregnant group. Placenta growth factor in units of multiples of median (MoM) for each woman was computed according to gestational week at sampling. A receiver-operating characteristic (ROC) curve was used to analyze the predictive validity of placenta growth factor on the preeclamptic group.¹⁷ Multivariable logistic regression analysis was used to identify the risk factors of preeclampsia. The two-tailed P values < .05 were considered to be statistically significant. Statistical analysis was performed using SPSS 9.0 for Windows (SPSS, Inc., Chicago, IL).

	Results

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Table 1 shows the clinical characteristics of the normal pregnant and subsequent preeclamptic groups. Preeclampsia was associated with greater maternal age, higher maternal BP, lower parity, lower gestational age at delivery, and lower fetal birth weight. There was no significant difference between the groups in body mass index (BMI) or gestational age at sampling.

View larger version (12K): [in this window] [in a new window]   Figure 1. Scatter plot showing relationship of log (maternal serum placenta growth factor) with gestational age in normal and preeclamptic pregnancies.

View larger version (17K): [in this window] [in a new window]   Figure 2. Levels of maternal serum placenta growth factor, expressed as multiples of the control median in each gestational age (gestational age stratified multiples of the median [MoM]), in 227 normal pregnancies and 27 preeclamptic pregnancies. Dashed line indicates the optimal cutoff value at 0.76 for placenta growth factor multiples of the median.

View larger version (14K): [in this window] [in a new window]   Figure 3. The receiver-operating characteristic curves for assay of maternal serum placenta growth factor. The area under the curve (AUC) shows the ability of the test to differentiate preeclampsia from normal pregnancies, with the value 1.0 representing 100% sensitivity and specificity, and the value 0.50 no discriminatory power. The area under the curve is 0.79 for placenta growth factor.

	Discussion

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Preeclampsia, which complicates approximately 7% of all pregnancies, remains a major cause of maternal and perinatal morbidity and mortality.¹⁸ Early identification of an at-risk group of pregnant women is essential for preventing and reducing both the maternal and perinatal morbidity and mortality.

In this study, we found that maternal serum placenta growth factor concentration in the early second trimester, maternal age, and the parity were independent determinants for the risk of subsequent preeclampsia. To eliminate the impact of gestational age, we transformed the placenta growth factor level to multiples of the appropriate gestational median. We proved that the value of placenta growth factor MoM was as useful as placenta growth factor concentration itself statistically and much easier to handle. In our study, the risk of developing preeclampsia subsequently was increased 2.5-fold for maternal serum placenta growth factor concentration decrements of 0.1 MoM.

Angiogenesis, the sprouting of new capillaries from existing vessels, is an essential component of placenta development. In humans, vascular transformation occurs in the placental bed where extravillous trophoblast cells within the maternal decidual tissue transform the spiral arteries into high-flow, low-resistance vessels that are more efficient at supplying the placenta with maternal blood.¹⁹ The placenta is a relatively rich source of angiogenic growth factors, and the regulation of vascular development in the placenta is thought to be highly associated with such factors. In recent years, great emphasis has been focused on vascular endothelial growth factor, a key regulator of angiogenesis.^20–22 Placenta growth factor, based on its 50% overall amino acid residual similarity with vascular endothelial growth factor, has been classified as a member of the vascular endothelial growth factor family of growth factors.⁹ It also shares a number of biochemical and functional similarities with other members of the vascular endothelial growth factor family.^9–11 Moreover, it has the distinctive characteristic of being highly expressed only in the placenta, especially in the villous cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast.¹³ These features suggest that placenta growth factor could play a significant role in the regulation of the vascular development in the placenta.

Shore et al¹⁰ reported that placenta growth factor expression decreased in trophoblasts cultured under hypoxic conditions. This finding suggests that the hypoxic trophoblast will downregulate placenta growth factor expression. This finding was consistent with the observation of reduced maternal serum levels of placenta growth factor in patients with the clinical appearance of preeclampsia during the third trimester reported by Torry et al¹⁴ and Reuvekamp et al.¹⁵ In vitro, hypoxia had been found to induce significant morphologic changes in trophoblasts and reduce cytotrophoblast differentiation with less syncytial formation.^23–25 These morphologic changes including limited cytotrophoblast invasion to the superficial decidua and few breached arterioles were also observed in the placenta of preeclamptic patients.^26,27 The phenomenon of poor vascular formation is possibly the response to hypoxia mediated through the activity of a hypoxia-regulated factor such as placenta growth factor. It seems that hypoxia will lead to poor vascular development of the placenta, and the poor vascular formation will in turn cause more severe hypoxia. This cascade of events will finally present the clinical manifestations of preeclampsia.

In the early second trimester before 20 weeks’ gestation, there is still not enough evidence to prove that hypoxia exists before the clinical manifestations of preeclampsia become evident. However, because decreased placenta growth factor was found in the early second trimester in women who subsequently developed preeclampsia, similar to that seen in the third trimester, it may be postulated that hypoxia also exists in the early second trimester in women with subsequent preeclampsia, downregulating the expression of placenta growth factor. Bower et al^28,29 reported that reduced uteroplacental blood flow and increased vascular resistance as revealed by Doppler flow velocity waveform study in the early second trimester were associated with later development of preeclampsia. This finding indirectly supports the contention that hypoxia did exist in the early second trimester in women with subsequent preeclampsia.

There is still not enough evidence to support the notion that decreased serum levels of placenta growth factor in preeclamptic patients are the consequence or the cause of a placentation deficit. Moreover, the proper function of placenta growth factor in the physiologic and pathologic development of placentation needs further elucidation. But we did prove that lowered maternal serum placenta growth factor in the early second trimester was strongly associated with the subsequent occurrence of preeclampsia. Accordingly, we concluded that maternal serum placenta growth factor might be useful in screening the high-risk group of preeclampsia before the clinical appearance of maternal disease (hypertension, proteinuria, and edema). Because of the lack of a proven prophylaxis for preeclampsia, prediction of risk to identify patients for more intensive care and possible intervention is attractive. Although placenta growth factor has been proved to be associated with preeclampsia and could be a potential marker for the early prediction of preeclampsia before the clinical manifestations are identified, further prospective, large-scale, longitudinal studies are essential to determine the usefulness of placenta growth factor in predicting preeclampsia.

	Footnotes

 
PII S0029-7844(01)01341-2

Received September 25, 2000. Received in revised form January 2, 2001. Accepted January 18, 2001.

	References

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