Keywords
angiogenic factors - pre-eclampsia - PlGF - protein creatinine ratio - sFlt-1
Palavras-chave
fatores angiogênicos - pré-eclâmpsia - PlGF - relação proteína creatinina - sFlt-1
Introduction
The hypertensive disorders of pregnancy are a leading cause of maternal and perinatal
mortality and morbidity worldwide, especially in developing countries, affecting 10%
of pregnancies, and have been responsible for high costs to the health system.[1]
[2]
[3] Pre-eclampsia and gestational hypertension are characterized by the new onset of
hypertension (> 140 mm Hg systolic or > 90 mm Hg diastolic) after 20 weeks of gestation.
The next step is to define whether this represents pure gestational hypertension or
pre-eclampsia. Pre-eclampsia is diagnosed by hypertension and the coexistence of one
or more of the following conditions: proteinuria (urine protein/creatinine > 0.3 mg/mg
or > 300 mg/day); maternal organ dysfunction (renal insufficiency, liver involvement,
neurological complications, hematological complications); and uteroplacental dysfunction
(fetal growth restriction).[4]
[5] Although often accompanied by new onset proteinuria, hypertension and other signs
or symptoms of pre-eclampsia may present in some women in the absence of proteinuria.[6]
The pathogenesis of pre-eclampsia involves deficient trophoblast invasion that is
responsible for altered uterine blood flow and placental oxidative stress.[7] Recent observations support the hypothesis that altered expression of placental
antiangiogenic factors is responsible for the clinical manifestations of the disease.
The damaged placenta produces higher concentrations of Soluble fms-like tyrosine kinase-1
(sFlt-1), a soluble receptor for vascular endothelial growth factor (VEGF) and placental
growth factor (PlGF) that is released into the maternal circulation and is involved
in endothelial dysfunction.[8]
[9]
[10]
[11] Soluble fms-like tyrosine kinase-1 is an endogenous antiangiogenic protein that
is made by the placenta and acts by binding and neutralizing the proangiogenic proteins
VEGF and PlGF. Decreased concentrations of the circulating proangiogenic factor PlGF
and increased concentrations of the antiangiogenic factor sFlt-1 have been observed
in pre-eclamptic patients, suggesting that an imbalance between these factors has
a fundamental role in the pathogenesis of pre-eclampsia.[12]
[13]
[14] Thereby, both sFlt-1 and PlGF have been suggested to be useful for the diagnosis
of pre-eclampsia.
The goal of the present study was to evaluate the sensitivity, specificity, and the
best accuracy level of sFlt-1, PlGF, sFlt-1/PlGF ratio in maternal serum and protein/creatinine
ratio in urine sample to define the best cutoff point of these tests to discriminate
between the patients with gestational hypertension and the patients with pre-eclampsia,
to evaluate the possibility of using them as diagnostic methods. In addition, we evaluated
the degree of association of 24-hour proteinuria with sFlt-1, PlGF, sFlt-1/PlGF ratio
and protein/creatinine ratio.
Methods
A prospective longitudinal study evaluated 95 pregnant women with hypertension in
attendance at prenatal clinics and at the obstetric emergency of a tertiary university
hospital in the south of Brazil (Maternidade Mário Totta – Santa Casa de Misericórdia
de Porto Alegre, state of Rio Grande do Sul, Brazil) over a period of 12 months (October
2010 to October 2011). All included patients signed an informed consent form. The
present study was approved by the Institutional Review Board (CEP UFCSPA 10–628).
The present study included pregnant women after 20 weeks of gestation with systolic
blood pressure ≥ 140 mm Hg or diastolic blood pressure ≥ 90 mm Hg, measured according
to a standard protocol,[15]
[16] and with ≥ 1 occurrences of protein on a dipstick or a protein/creatinine ratio ≥ 40 mg/mmol.
These tests were considered screening methods. Thus, patients with positive screening
had 24-hour proteinuria collection to confirm or exclude pre-eclampsia, considering
this test as the evaluation parameter used in our institution for the diagnosis of
the disease. Thereby, the 24-hour proteinuria was used to stratify patients into two
groups, gestational hypertension and pre-eclampsia. The 24-hour collection was performed
during hospitalization of the patient, under supervision of the nursing staff, following
standard procedures established by institutional guidelines, which contributes to
more accurate results. We excluded patients with diabetes mellitus and vascular disease
prior to the pregnancy or preexisting kidney disease.
Blood samples were collected and serum concentrations of sFlt-1 and PlGF were measured
at the time of the diagnosis. The Elecsys (Roche Diagnostics Brazil São Paulo, SP,
Brazil) immunoassays for determination of sFlt-1 and PlGF and analysis of blood samples
were performed at the central laboratory of the Santa Casa. Clinical information was
verified through data collection during hospitalization, searching for maternal and
gestational data, risk factors, gestational prognosis (eclampsia and HELLP Syndrome),
and other relevant evaluation parameters (hyperuricemia, severe hypertension, proteinuria ≥ 5 g,
fetal growth restriction). We made a separate analysis with the primigravida group.
In addition, patients were stratified into two groups, early-onset pre-eclampsia (<
34 weeks of gestation) and late-onset pre-eclampsia (≥ 34 weeks of gestation).
The quantitative variables were described by mean and standard deviation (SD), or
median and interquartile range (IQR). To compare averages between groups, the t-test was applied. For asymmetric variables, we used the Mann Whitney test. To compare
proportions, the chi-squared test or the Fisher exact test was applied. Serum levels
of sFlt-1 and PlGF, as well as biochemical parameters, were evaluated for sensitivity,
specificity, and the optimal cutoff point by receiver operating characteristic (ROC)
curve. The Spearman correlation coefficient (r) was used to evaluate the degree of
association between the tests. Sample size was calculated considering an α of 0.05
and a β of 0.20 and setting the null hypothesis in an area under the ROC curve of
0.75. A total of 75 patients were necessary. Statistical analysis was performed using
PASW Statistics for Windows, Version 18 (SPSS Inc., Chicago, IL, USA) and the significance
level adopted was 0.05.
Results
[Table 1] shows maternal and gestational data, risk factors (chronic/preexisting hypertension,
pre-eclampsia in a previous pregnancy and family history of pre-eclampsia) and some
relevant evaluation parameters (uric acid > 6 mg/dL, systolic blood pressure ≥ 160 mm
Hg or diastolic blood pressure ≥ 110 mm Hg, proteinuria > 5 g in 24 hours and fetal
growth restriction). Besides, we can observe the screening methods, biomarkers sFlt-1
and PlGF, and 24-hour proteinuria levels. A total of 95 pregnant patients had blood
samples collected to measure serum concentrations of biomarkers sFlt-1 and PlGF. One
patient has been excluded from the analysis because there was no screening test performed,
and another patient has been excluded because there was no registered value of PlGF.
Thus, the statistical analysis included 93 pregnant patients with hypertension.
Table 1
Sample characterization of patients included in the study
|
Variables[*]
|
Total Sample (n = 93)
|
Pre-eclampsia (n = 73)
|
Gestational Hypertension (n = 20)
|
p-value
|
|
Demographic data
|
|
|
|
|
|
Maternal age (years old)
|
29.1 ± 7.7
|
29.5 ± 7.7
|
27.9 ± 7.8
|
0.422
|
|
Body mass index (kg/m2)
|
34.0 ± 6.7
|
34.1 ± 7.1
|
33.3 ± 4.4
|
0.666
|
|
Gestational data
|
|
|
|
|
|
Gestational age at diagnosis (weeks)
|
34.2 ± 4.0
|
34.2 ± 4.4
|
34.1 ± 2.5
|
0.915
|
|
Nulliparity
|
39 (41.9)
|
31 (42.5)
|
8 (40.0)
|
1.000
|
|
Risk factors
|
|
|
|
|
|
Chronic/preexisting hypertension
|
42 (45.2)
|
34 (46.6)
|
8 (40.0)
|
0.787
|
|
Preeclampsia in a previous pregnancy
|
6 (6.5)
|
5 (6.8)
|
1 (5.0)
|
1.000
|
|
Family history of preeclampsia
|
3 (3.2)
|
3 (4.1)
|
0 (0.0)
|
1.000
|
|
Evaluation parameters
|
|
|
|
|
|
Uric acid ≥ 6 mg/dL
|
22 (23.9)
|
18 (24.7)
|
4 (21.1)
|
1.000
|
|
Severe blood pressure elevation
|
47 (50.5)
|
38 (52.1)
|
9 (45.0)
|
0.759
|
|
Proteinuria ≥ 5 g in 24 hours
|
1 (1.5)
|
1 (2.0)
|
0 (0.0)
|
1.000
|
|
Fetal growth restriction
|
12 (12.9)
|
8 (11.0)
|
4 (20.0)
|
0.280
|
|
Laboratory tests
|
|
|
|
|
|
Protein/creatinine (mg/mmol)
|
0.47 (0.36–0.96)
|
0.54 (0.41–1.13)
|
0.33 (0.24–0.42)
|
< 0.001
|
|
Protein/creatinine ratio ≥ 0.4
|
62 (68.9)
|
56 (80.0)
|
6 (30.0)
|
< 0.001
|
|
Positive preeclampsia screening
|
70 (75.3)
|
60 (82.2)
|
10 (50.0)
|
0.007
|
|
24-hour proteinuria (g)
|
0.37 (0.29–0.57)
|
0.46 (0.34–0.64)
|
0.21 (0.15–0.27)
|
< 0.001
|
|
PlGF (pg/mL)
|
100 (62.3–235)
|
97.7 (62.3–235)
|
119 (56.7–283)
|
0.495
|
|
sFlt-1 (pg/mL)
|
5253 (2649–9071)
|
5469 (2703–9375)
|
3920 (1613–8574)
|
0.169
|
|
sFlt-1/PlGF ratio
|
55.3 (14.7–113)
|
58.7 (16.5–122)
|
34.3 (6.5–95.5)
|
0.258
|
|
Uric acid (mg/dL)
|
5.16 ± 1.30
|
5.21 ± 1.30
|
4.97 ± 1.31
|
0.475
|
Abbreviations: P1GF, placental growth factor; sFlt-1, Soluble fms-like tyrosine kinase-1.
* described by mean ± standard deviation, median (percentiles 25 - 75) or n (%).
Pre-eclampsia was confirmed in 73 patients (78.5%). A total of 29 (39.7%) patients
had early-onset pre-eclampsia and 44 (60.3%) had late-onset disease. When evaluating
the pre-eclampsia group and the gestational hypertension group, there was no association
with nulliparity and no significant difference between early or late-onset pre-eclampsia.
Only one patient developed eclampsia and HELLP syndrome (1.1%). Most of the pregnant
patients were in the 3rd trimester when they entered in the study, justifying higher values for the body mass
index (BMI), which was calculated at the moment the patient was included and not in
the beginning of pregnancy.
We analyzed laboratory tests individually and could observe a significant association
of protein/creatinine ratio and 24-hour proteinuria with the diagnosis of pre-eclampsia.
The biomarkers sFlt-1 and PlGF did not have a good accuracy for disease diagnosis.
Demographic and gestational data, risk factors and other evaluation parameters did
not have a significant association with pre-eclampsia in our study.
We determined the sensitivity and the specificity for different thresholds of a parameter,
defining the best cutoff point for each test. Calculating the area under the ROC curve,
we evaluated the performance of the studied tests ([Table 2]). The sFlt-1/PlGF ratio demonstrated a sensitivity of 57.5% and a specificity of
60% using 50.4 as a cutoff point. The protein/creatinine ratio showed the best sensitivity
(78.9%) and specificity (70%) using 0.4 as a cutoff point and was the test with the
best accuracy level in pre-eclampsia diagnosis, showing an area under the ROC curve
of 0.80 (p < 0.001). In [Figs. 1] and [2], the sensitivity, specificity and the area under the ROC curve for the sFlt-1, PlGF,
sFlt-1/PlGF ratio and protein/creatinine ratio can be observed.
Table 2
Evaluation of the best cutoff point and the area under the ROC curve of the studied
tests for the diagnosis of pre-eclampsia, with their sensitivity and specificity
|
Tests
|
Sensitivity
|
Specificity
|
Cutoff point
|
AUC ROC
|
95%CI
|
p-value
|
|
sFlt-1/PlGF
|
57.5
|
60.0
|
> 50.4
|
0.58
|
0.44–0.73
|
0.258
|
|
sFlt-1
|
61.6
|
60.0
|
> 4671
|
0.60
|
0.46–0.74
|
0.169
|
|
PlGF
|
57.5
|
60.0
|
< 104.1
|
0.55
|
0.41–0.69
|
0.495
|
|
Protein/creatinine
|
78.9
|
70.0
|
> 0.40
|
0.80
|
0.68–0.92
|
< 0.001
|
Abbreviations: AUC, area under the curve; CI, confidence interval; P1GF, placental
growth factor; ROC, receiver operating characteristic; sFlt-1, Soluble fms-like tyrosine
kinase-1.
Fig. 1 ROC curve of sFlt-1/PlGF ratio (A), sFlt-1 (B) and PlGF (C) in the diagnosis of preeclampsia.
We used Spearman correlation to evaluate the degree of association between 24-hour
proteinuria with sFlt-1, PlGF, sFlt-1/PlGF ratio and protein/creatinine ratio. For
sFlt-1 (rs= 0.175; p = 0.163), PlGF (rs= - 0.066; p = 0.599) and sFlt-1/PlGF (rs= 0.107; p = 0.396), there was no degree of association. The only test that showed significant
association with 24-hour proteinuria was protein/creatinine ratio, with a regular
association (rs= 0.403; p = 0.001).
Fig. 2 ROC curve of protein/creatinine ratio in preeclampsia diagnosis.
Discussion
Several studies have evaluated the role of biochemical markers or a combination of
biochemical and biophysical markers in the prediction of pre-eclampsia in the 1st and 2nd trimesters of pregnancy. Clinical, ultrasonographic, and laboratory parameters have
been explored during early pregnancy as tools for predicting who will later develop
pre-eclampsia. None of these, individually, have sufficient sensitivity and predictive
values to be useful clinically, even among women at increased risk.[17]
[18]
[19]
[20]
[21] Regardless of the parameters used, screening for pre-eclampsia in low-risk women
is associated with very low positive predictive values ranging from 8 to 33%.[22]
The most studied antiangiogenic and proangiogenic markers have been sFlt-1 (soluble
receptor for VEGF and PlGF) and PlGF. Studies show that lower concentrations of PlGF
and higher concentrations of sFlt-1 during pregnancy confer an increased risk for
the subsequent development of pre-eclampsia. Some have noted that maternal serum concentrations
of these factors significantly separated healthy pregnant women and women with pre-eclampsia,
showing the value of these markers in the prediction of pre-eclampsia and in the differential
diagnosis of patients with atypical presentations of the disease. In addition, in
high-risk women, the sFlt-1/PlGF ratio is altered prior to pre-eclampsia onset.[23]
[24]
[25]
[26]
Recent studies have evaluated the performance of a newly developed assay for biomarkers
PlGF and sFlt-1, which has been studied for the prediction, diagnosis and prognosis
of patients with pre-eclampsia. A study conducted by Hagmann et al[27] showed that in early onset pre-eclampsia, the sFlt-1/PlGF ratio changes 11 weeks
before delivery. Zeisler et al[28] observed that a sFlt-1/PlGF ratio < 38 rules out pre-eclampsia, irrespective of
gestational age, for at least 1 week. In women with an elevated sFlt-1/PlGF ratio > 85
(early-onset pre-eclampsia) or > 110 (late-onset pre-eclampsia), the diagnosis of
pre-eclampsia or placenta related disorders is highly likely. Severely elevated sFlt-1/PlGF
ratios (> 655 at < 34 weeks; > 201 at ≥ 34 weeks) are associated closely with the
need to deliver within 48 hours.[29]
Many studies evaluated biomarkers for the prediction of pre-eclampsia. Different from
those studies, we evaluated angiogenic factors for the purpose of diagnostic correlation
with pre-eclampsia. In a longitudinal prospective study, with adherence to methodological
criteria, reviewed in detail, excluding any pregnant woman who could be configured
to bias the results analysis, we studied the role of biomarkers PlGF and sFlt-1 in
the diagnostic of pre-eclampsia. The present study demonstrated the cutoff point for
PlGF (104.1) and for sFlt-1 (4671). Considering these biomarkers, the sFlt-1/PlGF
ratio revealed the best association with pre-eclampsia diagnosis using 50.4 as a cutoff
point. However, our results showed that sFlt-1, PlGF, and sFlt-1/PlGF ratio did not
have good diagnostic accuracy.
Although the literature presents favorable evidence, there are many controversies
on the benefits that these biomarkers may provide in the assessment of pre-eclampsia.
A study conducted at Cambridge University showed that higher levels of sFlt-1 were
not associated with the risk of pre-eclampsia but were associated with a reduced risk
of delivery of a small for gestational age infant, spontaneous preterm birth, and
stillbirth associated with abruption or growth restriction.[30] A case-control study showed that sFlt-1 levels had low capacity to discriminate
between healthy patients and pre-eclampsia patients.[31] A systematic review accomplished at Oxford University demonstrated that a 3rd trimester increase in sFlt-1 and decrease in PlGF levels were associated with pre-eclampsia,
specifically severe disease; however, the authors concluded that the evidence is insufficient
to recommend these biomarkers to be used for screening.[32]
The most reasonable conclusions seem to be that determination of sFlt-1/PlGF ratio
can serve as an aid in the diagnosis of hypertensive disorders in pregnancy. The performance
of maternal levels of these factors, especially on early onset pre-eclampsia, could
be further improved by combining several markers. Combining biomarkers with maternal
history, mean blood pressure and uterine artery Doppler achieves a detection rate
of ∼ 90% of cases to develop pre-eclampsia.[33]
Some important considerations of our study are presented below. When our study was
initiated, in October 2010, the protein/creatinine ratio was considered a screening
method. Thus, pregnant women with positive screening (≥ 1 on dipstick or protein/creatinine
ratio ≥ 40 mg/mmol) were considered inclusion criteria of the study, requiring the
24-hour proteinuria collection to define or exclude the diagnosis of pre-eclampsia.
We used a cutoff level of 0.4 mg/mg for protein/creatinine ratio according to previous
studies performed at our institution, because this cutoff point showed the best accuracy
level for pre-eclampsia screening in our pregnant women population. Midrange protein/creatinine
ratio (0.3 mg/mg) had poor sensitivity and specificity in our study. In another hospital,
located in the same city of our study, they found that hypertensive pregnant women
with a protein/creatinine ratio ≥ 0.3 mg/mg had worse maternal and perinatal outcomes
than those with a protein/creatinine ratio < 0.3 mg/mg.[34] The latest guidelines already consider the use of protein/creatinine ratio as part
of criteria for the diagnosis of pre-eclampsia, but many authors and institutions
continue to use and consider a full 24-hour urine test for accurate results.[35] The existing evidence is not, however, sufficient to determine how the protein/creatinine
ratio should be used in clinical practice, owing to the heterogeneity in test accuracy
and prevalence across studies.[36]
We made a separate analysis with the primigravida group to assess the correlation
with the diagnosis of pre-eclampsia; however, in our study, there was no association
with nulliparity. Besides, patients were stratified into 2 groups, early-onset pre-eclampsia
(< 34 weeks of gestation) and late-onset pre-eclampsia (≥ 34 weeks of gestation) and,
similarly, there was no significant difference between the groups. Maybe these associations
could be observed if we had a larger sample.
Although some criteria are currently no longer considered as severity, when analyzed
together, they may have clinical significance. Therefore, we evaluated hyperuricemia,
severe hypertension, proteinuria ≥ 5 g and fetal growth restriction as relevant parameters.
The evaluation of these parameters did not show a significant difference when comparing
pre-eclampsia and gestational hypertension. Twenty-four-hour proteinuria > 5 g has
not been considered as a maternal prognosis to indicate pregnancy resolution.[6]
The BMI was calculated at the entry of the study, not in the beginning of the pregnancy,
justifying higher values for this ratio. Furthermore, as the sample comprises hypertensive
pregnant women, we could observe higher values for the BMI, since obesity is a risk
factor for hypertension. Moreover, in recent decades, there has been an increase in
weight in the general population. The growing prevalence of obesity is increasingly
recognized as one of the most important risk factors for the development of hypertension.[37]
Concluding, we can observe that there is considerable heterogeneity among reports.
There are differences in the analyte and storage conditions, in the gestational periods
selected for blood sampling, and in inclusion and exclusion criteria. Some reports
include women with risk factors for pre-eclampsia, whereas others excluded this group;
some of the study population was exclusively nulliparous women, whereas all parities
were included in others. The evidence is neither strong enough nor sufficient to recommend
PlGF and sFlt-1 to be used for pre-eclampsia diagnosis or to screen women at risk
to develop the disease. Therefore, the use of proangiogenic and antiangiogenic factors
in the assessment of pre-eclampsia is a subject of controversy and is currently under
investigation. Prospective studies employing rigorous laboratory and study design
criteria are needed to determine the clinical usefulness of these tests.
Conclusion
In summary, in our research, no studied laboratory test proved to be fairly accurate
for the diagnosis of pre-eclampsia, except for the protein/creatinine ratio. The evidence
is insufficient to recommend sFlt-1 and PlGF to be used for the diagnosis of pre-eclampsia.
The identification of biomarkers that can contribute to early detection of pre-eclampsia
is essential to apply better surveillance and treatment protocols. Besides, demonstrating
the clinical utility of these angiogenic markers could affect the management decisions
of the obstetrician, improve health outcomes, and reduce costs to the healthcare system.
