A, Mean urinary placental growth factor (PlGF) concentrations in normotensive
women (controls) and in women (cases) before onset and after onset (active
preeclampsia [PE]) of clinical PE, according to gestational age. Also shown
for women who subsequently developed PE (cases) are the mean urinary concentrations
of PlGF after excluding specimens obtained within 5 weeks before onset of
PE (open circles). Error bars represent SEs. P values
for the comparisons between specimens from cases before the onset of PE and
specimens from controls obtained during the same gestational age interval,
after logarithmic transformation and accounting for patients with varying
numbers of specimens, were significant at 25 to 28 weeks (P<.001), 29 to 32 weeks (P = .002),
and 33 to 36 weeks (P = .005). Comparisons
between controls and cases more than 5 weeks before onset of PE were also
significant at 25 to 28 weeks (P = .005)
and 29 to 32 weeks (P = .02). The comparisons
between specimens obtained from women with active PE and from controls were
significant at 29 to 32 weeks (P <.001), 33 to 36 weeks (P <.001), and 37 to 42 weeks (P <.001). The comparisons
between specimens obtained from women with active PE and from women in whom
PE later developed were also significant at 29 to 32 weeks (P<.001), 33 to 36 weeks (P<.001), and
37 to 42 weeks (P = .003). Note that PlGF
concentrations before onset of PE do not include specimens obtained after
appearance of hypertension or proteinuria (active PE). Mean urinary creatinine
concentrations between cases and controls were not significantly different
for the various gestational windows (171 vs 147 mg/dL at 8-12 weeks, 136 vs
139 mg/dL at 13-16 weeks, 118 vs 112 mg/dL at 17-20 weeks, 109 vs 102 mg/dL
at 21-24 weeks, 104 vs 117 mg/dL at 25-28 weeks, 110 vs 129 mg/dL at 29-32
weeks, 95 vs 98 mg/dL at 33-36 weeks, and 108 vs 109 mg/dL at 37-42 weeks).
B, Mean urinary PlGF concentrations in normotensive women (controls) and in
women (cases) before onset and after onset (active PE) of clinical PE according
to gestational age, using only first morning urine specimens. Error bars represent
SEs. P values for the comparisons between specimens
from cases before the onset of PE and specimens from controls obtained during
the same gestational age interval were significant at 25 to 28 weeks (P = .002) and at 33 to 36 weeks (P = .02). The comparisons between specimens obtained from
women with active PE and specimens obtained from controls were significant
at 29 to 32 weeks (P<.001) and at 33 to 36 weeks
(P = .006). The comparisons between specimens
obtained from women with active PE and from those in whom PE later developed
were also significant at 29 to 32 weeks (P = .003).
C, Mean PlGF concentrations before and after onset of clinical PE, using only
random urine specimens. Error bars represent SEs. P values
for the comparisons between specimens from cases before the onset of PE and
specimens from controls obtained during the same gestational age interval
were significant at 29 to 32 weeks (P = .01)
and at 33 to 36 weeks (P = .02). The comparisons
between specimens obtained from women with active PE and specimens obtained
from controls were significant at 29 to 32 weeks (P<.001),
33 to 36 weeks (P<.001), and 37 to 42 weeks (P = .05). The comparisons between specimens obtained
from women with active PE and specimens obtained from women in whom PE later
developed were also significant at 29 to 32 weeks (P <.001)
and at 33 to 36 weeks (P = .002).
Urinary placental growth factor (PlGF) concentrations in picograms per
milliliter and in picograms of PlGF per milligram of creatinine are shown
at 21 to 32 weeks of gestation in controls and in women who subsequently developed
clinical preeclampsia (PE) according to whether they had mild PE, severe PE,
PE with an onset at less than 37 weeks of gestation, PE and a small-for-gestational-age
(SGA) infant, or PE with an onset at less than 34 weeks of gestation. All
specimens from women in whom PE later developed were obtained before the onset
of clinical symptoms. The P values given are for
comparisons with specimens from controls. Error bars represent SEs.
Measurements were obtained from paired urine and serum specimens obtained
from 20 women before development of preeclampsia at less than 37 weeks of
gestation and from 69 normotensive controls.
Urinary placental growth factor (PlGF) concentrations in picograms per
milliliter and in picograms of PlGF per milligram of creatinine are shown
at 21 to 32 weeks of gestation in normotensive (NT) women whose infants were
not born small for gestational age (SGA), NT women with SGA infants, women
who subsequently developed gestational hypertension (GH), and women who subsequently
developed preeclampsia (PE) before 37 weeks of gestation. Specimens from women
in whom GH or PE developed were obtained before onset of clinical disease.
The mean gestational age at specimen collection was similar in all groups.
The P values given are for comparisons with specimens
from controls (NT without SGA). Error bars represent SEs.
Levine RJ, Thadhani R, Qian C, Lam C, Lim K, Yu KF, Blink AL, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. Urinary Placental Growth Factor and Risk of Preeclampsia. JAMA. 2005;293(1):77-85. doi:10.1001/jama.293.1.77
Author Affiliations: Division of Epidemiology,
Statistics, and Prevention Research, National Institute of Child Health and
Human Development, Department of Health and Human Services, Bethesda, Md (Drs
Levine and Yu and Ms Blink); Departments of Medicine and Obstetrics, Massachusetts
General Hospital, and Harvard Medical School (Dr Thadhani), Renal Division,
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical
School (Drs Lam, Epstein, Sukhatme, and Karumanchi), and Department of Obstetrics
and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School,
and Harvard School of Public Health (Drs Lim, Sachs, and Karumanchi), Boston,
Mass; Allied Technology Group, Rockville, Md (Mr Qian); and Department of
Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati,
Ohio (Dr Sibai).
Context Preeclampsia may be caused by an imbalance of angiogenic factors. We
previously demonstrated that high serum levels of soluble fms-like tyrosine
kinase 1 (sFlt1), an antiangiogenic protein, and low levels of placental growth
factor (PlGF), a proangiogenic protein, predict subsequent development of
preeclampsia. In the absence of glomerular disease leading to proteinuria,
sFlt1 is too large a molecule to be filtered into the urine, while PlGF is
Objective To test the hypothesis that urinary PlGF is reduced prior to onset of
hypertension and proteinuria and that this reduction predicts preeclampsia.
Design, Setting, and Patients Nested case-control study within the Calcium for Preeclampsia Prevention
trial of healthy nulliparous women enrolled at 5 US university medical centers
during 1992-1995. Each woman with preeclampsia was matched to 1 normotensive
control by enrollment site, gestational age at collection of the first serum
specimen, and sample storage time at −70°C. One hundred twenty pairs
of women were randomly chosen for analysis of serum and urine specimens obtained
Main Outcome Measure Cross-sectional urinary PlGF concentrations, before and after normalization
for urinary creatinine.
Results Among normotensive controls, urinary PlGF increased during the first
2 trimesters, peaked at 29 to 32 weeks, and decreased thereafter. Among cases,
before onset of preeclampsia the pattern of urinary PlGF was similar, but
levels were significantly reduced beginning at 25 to 28 weeks. There were
particularly large differences between controls and cases of preeclampsia
with subsequent early onset of the disease or small-for-gestational-age infants.
After onset of clinical disease, mean urinary PlGF in women with preeclampsia
was 32 pg/mL, compared with 234 pg/mL in controls with fetuses of similar
gestational age (P<.001). The adjusted odds ratio
for the risk of preeclampsia to begin before 37 weeks of gestation for specimens
obtained at 21 to 32 weeks, which were in the lowest quartile of control PlGF
concentrations (<118 pg/mL), compared with all other quartiles, was 22.5
(95% confidence interval, 7.4-67.8).
Conclusion Decreased urinary PlGF at mid gestation is strongly associated with
subsequent early development of preeclampsia.
Preeclampsia is a common hypertensive disorder of pregnancy characterized
by systemic endothelial dysfunction and diagnosed by the appearance of hypertension
and proteinuria.1,2 For this reason,
it is recommended that women undergo blood pressure and urinary protein screening
at each prenatal visit throughout gestation.3 Potentially
life-threatening complications of preeclampsia include seizures, cerebral
hemorrhage, disseminated intravascular coagulation, and renal failure; and
the time between the first detection of hypertension and proteinuria and the
subsequent development of these complications can be extremely short.3,4 The only known cure for preeclampsia
is delivery of the placenta. If maternal signs develop before the fetus is
mature, the risk of neonatal morbidity and mortality due to premature delivery
is markedly increased.
Evidence from our group and others suggests that preeclampsia may be
caused by an imbalance of angiogenic factors.5- 12 Circulating
soluble fms-like tyrosine kinase 1 (sFlt1, also referred to as sVEGFR1), an
antiangiogenic protein, binds the proangiogenic proteins, vascular endothelial
growth factor (VEGF) and placental growth factor (PlGF), preventing their
interaction with endothelial cell receptors and thereby inducing endothelial
dysfunction.13 Administration of sFlt1 to rats
results in hypertension, glomerular endotheliosis, and proteinuria, the hallmarks
of preeclampsia.6 Among participants in the
Calcium for Preeclampsia Prevention (CPEP) trial,14 we
recently demonstrated that elevated serum concentrations of sFlt1 are evident
approximately 5 weeks before the onset of clinical preeclampsia. Low serum
concentrations of free PlGF, beginning at 13 to 16 weeks of gestation, and
reduced free VEGF also antedated the clinical signs of preeclampsia.5
Although longitudinal measures of these angiogenic mediators in serum
might be ideal for ascertaining the risk of preeclampsia, obtaining such measurements
during routine prenatal care could be challenging. An alternative and less
invasive screening method may be to measure these proteins in urine. Although
sFlt1 is too large a molecule (≈100 kDa) to be filtered into urine in the
absence of renal damage, PlGF and VEGF, much smaller proteins (≈30 kDa
and ≈45 kDa, respectively), are readily filtered. Unlike urinary PlGF,
which is derived entirely from circulating blood, the major sources of urinary
VEGF are cells of the kidney itself (glomerular podocytes and tubular cells15,16); thus, urinary VEGF is unlikely
to reflect the circulating angiogenic state. Therefore, we used archived urine
samples to test the hypothesis that urinary PlGF is reduced well before the
onset of hypertension and proteinuria and might predict preeclampsia.
The CPEP trial was a randomized, double-blind clinical trial conducted
in 1992-1995 to evaluate the effects of daily supplementation with calcium
or placebo on the incidence and severity of preeclampsia.14,17 A
total of 4589 healthy nulliparous women with singleton pregnancies were enrolled
between 13 and 21 weeks of gestation at 5 participating US medical centers
and were followed up until 24 hours after delivery. Written informed consent
was obtained from all participants. Subsequently, 326 women developed preeclampsia.
Serum and urine specimens were requested from participants before enrollment
in the trial, at 26 to 29 weeks of gestation, at 36 weeks if they were still
pregnant, and when hypertension or proteinuria was noted. Both first morning
and 24-hour urine specimens were requested; if neither was available, a random
or “spot” urine specimen was collected. Twenty-four-hour urine
specimens were requested from patients in whom preeclampsia was suspected.
Because the studies reported here used data and specimens that could not be
linked to identifiable women, the office of Human Subjects Research of the
National Institutes of Health granted them exemptions from the requirement
for review and approval by the institutional review board.
Main Study. For the present study, we selected
women with complete outcome information, serum samples obtained at less than
22 weeks of gestation, and a live-born male infant. This group had previously
been selected for a study of fetal DNA and preeclampsia, in which fetal and
maternal DNA were differentiated through the amplification of a gene on the
Y chromosome.18 Furthermore, we have demonstrated
that alterations in circulating sFlt1 and PlGF antedate clinical preeclampsia
in these patients.5 Analysis of previous work
revealed no significant differences in maternal serum sFlt1 or PlGF concentrations
according to infant sex.5,6
Of the 4589 women enrolled in the CPEP trial, we excluded 253 who were
lost to follow-up, 21 whose pregnancy ended before 20 weeks, 13 who had missing
data on maternal or perinatal outcomes, 4 who had no data on smoking history,
9 in whom the presence of hypertension had not been verified by the team that
reviewed each chart, and 32 others who had a stillbirth, leaving 4257 women.
Of these women, 2156 had a male infant. After exclusion of 1 woman whose infant
had a chromosomal abnormality, 381 women with gestational hypertension, and
43 without a baseline serum specimen, 1731 women remained. Preeclampsia developed
in 175 of these women, whereas 1556 remained normotensive during pregnancy.
Calcium supplementation did not affect urinary levels of PlGF. Specimens
collected at 8 to 20 weeks of gestation were considered the baseline specimens
and were obtained before the administration of calcium or placebo. At 21 to
32 weeks, mean concentrations of PlGF were 223 vs 228 pg/mL (P = .63) in women receiving placebo vs calcium, respectively;
at 33 to 42 weeks, these concentrations were 187 vs 166 pg/mL (P = .53). Similarly, at 21 to 32 weeks, mean levels of PlGF
per milligram of creatinine were 226 vs 219 pg/mg (P = .66)
and at 33 to 42 weeks were 222 vs 178 pg/mg (P = .62).
Since calcium supplementation had no effect on the risk or severity
of preeclampsia14 or on the concentrations
of angiogenic factors in serum5 or urine, women
were chosen without regard to whether they had received calcium supplementation
or placebo. For each woman with preeclampsia, 1 normotensive control was selected,
matched according to enrollment site, gestational age at the collection of
the first serum specimen (within 1 week), and storage time of the samples
at –70°C (within 12 months). A total of 120 of 159 matched pairs
were randomly chosen for analysis of all serum and urine specimens obtained
before labor or delivery. If a woman had more than 1 urine specimen obtained
on the same day, we selected 1 specimen, preferring first morning to random
and random to 24-hour specimens. We identified 348 urine specimens from 120
preeclampsia cases and 318 urine specimens from 118 normotensive controls.
Two normotensive controls from the serum study had no eligible urine specimens
and were excluded from further analyses. Of the 238 women in the urine specimen
study, 26 (10.9%) contributed 1 urine specimen, 55 (23.1%) contributed 2 specimens,
111 (46.6%) contributed 3, 35 (14.7%) contributed 4, 10 (4.2 %) contributed
5, and 1 (0.4%) contributed 7.
For all controls and cases with onset of preeclampsia before term (<37
weeks), we examined separately samples of urine obtained at 21 to 32 weeks
of gestation for which a serum specimen from the same woman had been collected
within 3 days of the urine specimen (mean difference, 0.5 days). Among the
90 resulting pairs of urine-serum specimens, 2 were from the same woman; for
this woman, we included only the pair closest to the mid point of the gestational
age interval. A total of 89 urine-serum specimen pairs remained from 20 cases
of preterm preeclampsia and 69 normotensive controls.
Ancillary Study. We performed an ancillary
study to ascertain whether urinary PlGF at 21 to 32 weeks of gestation might
differ between women with male or female infants and to determine if concentrations
of urinary PlGF might be lower than normal in women with gestational hypertension
and in women who remained normotensive during pregnancy but who delivered
a small-for-gestational-age (SGA) infant. Among the 4256 women in the CPEP
trial with adequate data who delivered a live-born infant not known to have
a chromosomal abnormality, we excluded 239 with term preeclampsia (≥37
weeks). Of the 4017 women remaining, 3303 had at least 1 urine specimen obtained
at 21 to 32 weeks of gestation before onset of labor or delivery and before
onset of preeclampsia or gestational hypertension. Among these women, we randomly
selected 120 whose pregnancy was normotensive and whose infant was not SGA,
60 with normotensive pregnancy who delivered an SGA infant, 60 with gestational
hypertension, and 59 with preterm (<37 weeks) preeclampsia. In each group,
we chose half the women who delivered male infants and half who delivered
female infants, except for the group with preterm preeclampsia. In this group,
we selected 30 with male infants but could find only 29 with female infants.
Placental growth factor was analyzed in all urine specimens obtained at 21
to 32 weeks of gestation.
Preeclampsia was defined as a newly elevated diastolic blood pressure
of at least 90 mm Hg and proteinuria of at least 1+ (30 mg/dL) on dipstick
testing, each on 2 occasions 4 to 168 hours apart. Severe preeclampsia was
defined as the HELLP syndrome (hemolysis, elevated liver enzyme levels, and
a low platelet count), eclampsia, or preeclampsia with either severe hypertension
(diastolic blood pressure ≥110 mm Hg) or severe proteinuria (urinary protein
excretion ≥3.5 g per 24 hours or findings of ≥3+ [300 mg/dL] on dipstick
testing). Gestational hypertension was hypertension as defined herein in the
absence of proteinuria. Detailed definitions have been published.14,17 The time of onset of preeclampsia
was defined as the time of the first elevated blood pressure or urine protein
measurement leading to diagnosis of preeclampsia. Similarly, onset of gestational
hypertension was the time of the first elevated blood pressure measurement
that led to diagnosis. An SGA infant was defined as an infant whose birth
weight was below the 10th percentile according to US tables of birth weight
for gestational age that accounted for race, parity, and infant sex.19
Assays were performed by personnel who were unaware of pregnancy outcomes.
Specimens were randomly ordered for analysis. Enzyme-linked immunosorbent
assays for sFlt1, free PlGF, and free VEGF were performed in duplicate, as
previously described, with the use of commercial kits (R&D Systems, Minneapolis,
Minn).6 The minimum detectable doses in the
assays for sFlt1, PlGF, and VEGF were 5, 7, and 5 pg/mL, respectively, with
interassay and intra-assay coefficients of variation of 7.6% and 3.3%, respectively,
for sFlt1; 10.9% and 5.6% for PlGF; and 7.3% and 5.4% for VEGF. The enzyme-linked
immunosorbent assay kits for sFlt1, VEGF, and PlGF were validated for use
in urine specimens with 96%, 98%, and 99% recovery from spiked urine samples,
respectively. Urinary creatinine was measured using a commercially available
picric acid colorimetric assay (Metra creatinine assay kit, Quidel Corp, San
The χ2 test was used for comparison of categorical variables
and the t test for comparison of continuous variables.
Although arithmetic mean concentrations are reported in the text and figures,
statistical testing was conducted within each time interval individually after
logarithmic transformation, using the generalized estimating equations method
(SAS/PROC GENMOD procedure; SAS, version 8.0, SAS Institute Inc, Cary, NC)
in crude and adjusted analyses to account for patients with varying numbers
of specimens. Odds ratios (ORs) were adjusted with the use of logistic regression
analysis. Since matching was complete only for analyses of the earliest serum
specimen in the entire study population, matching was not accounted for in
the statistical analyses. For all analyses, P <.05 was considered
Characteristics of the Women. Of the 120 women
with preeclampsia, 80 had mild and 40 had severe disease. Compared with controls,
women with preeclampsia had greater body mass index (P =
.007), higher systolic and diastolic blood pressure at enrollment in the CPEP
trial (P = .001 and.006, respectively), and larger
proportions of their current pregnancies complicated by preterm delivery (P = .002) or resulting in SGA infants (P = .002).5 Patient and infant characteristics
have been described previously5 and are briefly
summarized in Table 1.
Differences in Urinary PlGF After Onset of Preeclampsia. We first ascertained that urinary levels of PlGF were altered in women
after development of clinical preeclampsia. Among 22 pairs of women with preeclampsia
and gestational age–matched controls, specimens of urine obtained after
onset of clinical disease had lower levels of PlGF than specimens from controls
(mean PlGF level, 32 vs 234 pg/mL; P<.001 and
50 vs 227 pg/mg of creatinine; P<.001).
Gestational Changes in Urinary PlGF. To evaluate
gestational patterns, we performed cross-sectional analyses of urine obtained
within gestational age intervals of 4 to 5 weeks, with PlGF levels expressed
as concentrations (Figure 1A) or as
picograms per milligram of creatinine. Patterns expressed as concentrations
and picograms per milligram of creatinine were similar because mean urinary
creatinine concentrations within gestational age intervals did not differ
significantly between cases and controls. The PlGF levels in controls increased
during the first 2 trimesters, with a more rapid increase after 21 to 24 weeks,
reaching a peak at 29 to 32 weeks and decreasing thereafter. The levels in
women who subsequently developed preeclampsia followed a similar pattern but
were significantly lower at 25 to 28, 29 to 32, and 33 to 36 weeks.When specimens
obtained within 5 weeks before the onset of preeclampsia were excluded, the
differences in the preceding gestational age intervals between controls and
women who later had preeclampsia were less pronounced. Among women with specimens
obtained in the same gestational age interval, those who already had clinical
preeclampsia had significantly lower concentrations at 29 to 32, 33 to 36,
and 37 to 42 weeks than those who developed preeclampsia later. Similar gestational
age patterns among controls and cases before and after onset of clinical preeclampsia
were observed when restricting the analysis of specimens to either first morning
(Figure 1B) or random (Figure 1C) urine specimens.
Relationship of Urinary PlGF and Severity of Preeclampsia. Before the onset of preeclampsia, there were particularly large differences
between the levels of urinary PlGF in controls and those in women who later
had preeclampsia with onset before 37 weeks or who had preeclampsia and an
SGA infant. Figure 2 shows PlGF concentrations
and PlGF expressed as picograms per milligram of creatinine in urine obtained
at 21 to 32 weeks of gestation.
Alterations in urinary PlGF levels were also more pronounced in women
who subsequently developed preeclampsia before term (<37 weeks of gestation)
than in women who had an onset of preeclampsia at term (≥37 weeks) (at
21-32 weeks, 87 pg/mL in women with preeclampsia before term vs 223 pg/mL
in women with preeclampsia at term; P<.001; at
33-42 weeks, 22 pg/mL in women with preeclampsia before term vs 118 pg/mL
in women with preeclampsia at term; P<.001). Results
were similar when using PlGF expressed as picograms per milligram of creatinine
or after adjusting PlGF concentrations for creatinine, gestational age at
specimen collection, storage time, body mass index, and maternal age. Furthermore,
PlGF levels in specimens of urine obtained before onset of preeclampsia from
women who later had preeclampsia and an SGA infant were lower than in women
who later had preeclampsia but whose infants were not SGA (at 21-32 weeks,
62 vs 205 pg/mL; P = .002; at 33-42 weeks,
42 vs 123 pg/mL; P = .06).
Odds Ratios for Preeclampsia Associated With Urinary
PlGF. To determine the risk of preeclampsia according to urinary PlGF
in specimens obtained before the onset of clinical signs, we divided PlGF
values into quartiles based on the distribution in controls and calculated
adjusted ORs for preeclampsia in each quartile, compared with the highest
quartile (Table 2) or with all other
quartiles. Among specimens obtained at 21 to 32 weeks of gestation, the lowest
quartile of PlGF was associated with a greatly increased risk of preterm preeclampsia
and a small increased risk of preeclampsia at term. For preterm preeclampsia,
after adjustment for gestational age at specimen collection, storage time,
body mass index, and age, using PlGF concentration, the OR for the lowest
quartile vs all others was 22.5 (95% confidence interval [CI], 7.4-67.8);
using picograms of PlGF per milligram of creatinine, the OR was 16.4 (95%
CI, 5.9-45.5). After restricting specimens to first morning urine, adjusted
ORs were 39.5 (95% CI, 6.5-240.8) and 20.4 (95% CI, 4.5-92.3) for PlGF concentration
and picograms of PlGF per milligram of creatinine, respectively. Using random
urine specimens, adjusted ORs were 13.5 (95% CI, 2.3-79.8) and 11.1 (95% CI,
2.0-61.3), respectively. On average, urine specimens obtained at 21 to 32
weeks of gestation from women who developed preeclampsia before 37 weeks were
collected 46 days prior to the onset of clinical disease.
For term preeclampsia, after adjustment for the factors noted herein
and using all urine specimens obtained at 21 to 32 weeks, ORs were 2.2 (95%
CI, 1.2-4.3) and 2.1 (95% CI, 1.1-4.1), respectively, for the lowest quartile
vs all other quartiles. The lowest quartile of PlGF was also associated with
an increased risk of term preeclampsia vs all other quartiles in specimens
obtained at 33 to 42 weeks of gestation (adjusted OR, 2.3; 95% CI, 1.2-4.5
for picograms of PlGF per milligram of creatinine).
When we performed the same analyses in specimens obtained at 21 to 32
weeks of gestation for women who developed preeclampsia during a pregnancy
complicated by an SGA infant, we found that the estimates were unstable (adjusted
OR, 405; 95% CI, 27-5983 for picograms of PlGF per milligram of creatinine).
This was because there were only 20 such women, all of whom were in the lowest
(n = 19) or next lowest (n = 1) quartiles of urinary PlGF.
Nevertheless, the data indicate that low urinary PlGF is associated with a
substantial increase in risk for preeclampsia with an SGA infant.
Relationship of Urinary PlGF to Proximity to Preeclampsia. Urinary concentrations of PlGF in specimens obtained at 21 to 32 weeks
of gestation and within 5 weeks before the onset of preeclampsia were lower
(43 pg/mL) than in specimens obtained more than 5 weeks before clinical disease
(196 pg/mL; P<.001). In specimens obtained at
33 to 42 weeks of gestation, concentrations were 110 pg/mL vs 187 pg/mL, respectively
(P = .05). There was little difference
when PlGF was normalized for creatinine.
Figure 3A is a scatter plot of
urinary PlGF concentrations at 21 to 32 weeks from all 69 controls and all
20 cases who subsequently developed preeclampsia before term (<37 weeks)
and who had a serum specimen obtained within 3 days of the urine specimen
(mean difference, 0.5 days). Women who developed preeclampsia before term
had lower urinary PlGF concentrations than normotensive controls. Concentrations
were lowest (all <150 pg/mL) in specimens obtained within 5 weeks before
the onset of clinical disease. However, a number of control specimens also
had low urinary PlGF. To distinguish these specimens from specimens obtained
within 5 weeks prior to preeclampsia, we examined serum measurements of the
ratio of sFlt1 to PlGF. The ratio accounts for both the increased sFlt1 and
decreased PlGF observed before onset of preeclampsia. A scatter plot of the
ratios of sFlt1 to PlGF concentrations in paired sera is shown in Figure 3B. Ratios are elevated (>5) in all specimens
obtained within 5 weeks before the onset of preeclampsia and exceed almost
all control values.
Urinary sFlt1 and Urinary VEGF in Preeclampsia.
We randomly selected 22 cases and 22 controls for analysis of urinary
sFlt1 and VEGF in samples obtained at 21 to 32 weeks of gestation before onset
of clinical preeclampsia. In 16 of 22 case specimens (73%) and 19 of 22 control
specimens (86%), urinary sFlt1 was undetectable. In contrast, urinary VEGF
was detected in all specimens but was not significantly altered in cases before
or after the onset of hypertension and proteinuria (before onset, 272 vs 248
pg/mL in the groups of 22 randomly selected cases and controls, respectively; P = .56 and after onset, 167 vs 103 pg/mL in
22 gestational age–matched cases and controls, respectively; P = .61).
To further test the hypothesis that decreased urinary PlGF is specific
for early-onset preeclampsia, we performed a second study in which we analyzed
urine specimens obtained at 21 to 32 weeks from women with other obstetrical
conditions that may share similarities of pathogenesis. We compared women
with gestational hypertensionand women who remained normotensive during pregnancy
but delivered an SGA infant with normotensive women whose infant was not SGA
(controls) and with women with preeclampsia before 37 weeks. The clinical
characteristics of the women in this study and of their infants are summarized
in Table 3. The characteristics of women
with preeclampsia and their infants were similar to those reported for such
women in the main study. Compared with normotensive women whose infants were
not SGA, women with gestational hypertension had greater body mass index and
infants of greater birth weight and normotensive women with an SGA infant
had lower body mass index and infants of lower birth weight. Normotensive
women with SGA infants were most likely and women with hypertensive disorders
of pregnancy were least likely to have smoked during pregnancy.
Figure 4 depicts urinary PlGF
at 21 to 32 weeks of gestation, expressed as concentrations and as picograms
per milligram of creatinine. Placental growth factor levels in women who remained
normotensive during pregnancy but delivered an SGA infant did not differ from
those of normotensive controls whose infant was not born SGA. Similarly, levels
in patients with gestational hypertension did not differ from those of normotensive
controls. However, levels of urinary PlGF in patients who developed preeclampsia
before 37 weeks of gestation (collected on average 42 days prior to clinical
disease onset) were much lower than controls (77 vs 206 pg/mL; P<.001). Within each group, PlGF concentrations among women who
delivered male or female infants did not differ significantly.
In this study of 120 women with preeclampsia and 118 normotensive controls,
urinary concentrations of PlGF were significantly lower beginning at 25 to
28 weeks of gestation among the women who subsequently developed preeclampsia.
Differences between the 2 groups became more pronounced at 29 to 36 weeks.
In our previous study of serum concentrations of angiogenic proteins, serum
free PlGF was lower in cases than in controls beginning at 13 to 16 weeks
of gestation, becoming even lower after 25 weeks of gestation.5 As
predicted from serum measurements, in the current study, urinary PlGF at 21
to 32 weeks of gestation was especially decreased in women who developed preeclampsia
before 37 weeks or during a pregnancy complicated by an SGA infant and in
those within 5 weeks of the onset of clinical signs. Furthermore, among women
in the lowest quartile of urinary PlGF concentrations (<118 pg/mL) at 21
to 32 weeks of gestation, the risk of developing preeclampsia before 37 weeks
of gestation or during a pregnancy complicated by an SGA infant was markedly
elevated. Risk was high irrespective of adjustment for urinary creatinine
concentrations and evident even in random urine specimens—although the
association was stronger with first morning specimens, which are likely to
be more concentrated. Thus, urinary PlGF was especially useful for identifying
patients who would benefit most from early diagnosis. We have also demonstrated
that a strategy of following urine measurement of PlGF with serum measurements
of sFlt1 and PlGF in selected patients may minimize false-positive results
from urine testing.
Urinary PlGF was much lower at 21 to 32 weeks of gestation in women
who developed preeclampsia before 37 weeks than in women who developed gestational
hypertension or delivered an SGA infant, 2 obstetrical conditions with similarities
to preeclampsia. Thus, it appears that a low urinary PlGF concentration at
this stage of pregnancy may distinguish preeclampsia from gestational hypertension
and intrauterine growth retardation.
Urinary VEGF concentrations were reported recently to be modestly elevated
in 37 women with severe preeclampsia compared with 32 with uncomplicated pregnancy.20 We found nonsignificant elevations of urinary VEGF
before and after the onset of preeclampsia, consistent with the hypothesis
that urinary VEGF reflects primarily local renal VEGF production. Since urinary
VEGF originates almost entirely from renal podocyte and tubular cells,15,16 it has not been exposed to circulating
sFlt1, which is too large a molecule to filter freely through an intact glomerulus.
Therefore, while reduced urinary PlGF in women with preeclampsia likely reflects
reduced circulating free PlGF (the result of binding to excess circulating
sFlt1), urinary VEGF does not reflect the angiogenic imbalance in the blood.
Limitations of this study must be acknowledged. We used specimens obtained
almost 10 years ago, and although we did find differences between cases and
controls, specimen deterioration may have affected reported values. Moreover,
since there was an average of only 3 urine specimens per woman throughout
pregnancy, we could not follow up suspicious results with repeated measurements
of urine and serum to search for trends, as could be done in clinical practice.
Finally, we did not determine whether there are alterations in urinary PlGF
throughout gestation in obstetric conditions with similarities to preeclampsia,
such as gestational hypertension or pregancy complicated by an SGA infant.
Nevertheless, our data suggest that at a time when alterations in urinary
PlGF are dramatic in women who will develop early-onset preeclampsia, normotensive
women who subsequently develop gestational hypertension or deliver an SGA
infant have none.
The identification of angiogenic proteins that appear to mediate the
maternal syndrome of preeclampsia may present specific targets for therapeutic
intervention to restore angiogenic balance.6 Prevention
and treatment are especially needed for women with preeclampsia of early onset
or complicated by an SGA infant. However, such women must first be identified
before the onset of clinical disease. If a reliable and valid urinary dipstick
assay can be developed, one scenario might be to screen all women for low
urinary PlGF concentrations. Among those with low levels, serial serum measurements
of sFlt1 and PlGF could then be used to identify more precisely individuals
at high risk. Prospective longitudinal studies with measurements throughout
pregnancy are needed to assess the validity of these observations.
Corresponding Author: S. Ananth Karumanchi,
MD, Beth Israel Deaconess Medical Center, Renal Division, Dana 517, 330 Brookline
Ave, Boston, MA 02215 (firstname.lastname@example.org).
Financial Disclosures: Drs Sukhatme and Karumanchi
are named as coinventors on a pending patent filed by Beth Israel Deaconess
Medical Center for the use of angiogenesis-related proteins for diagnosis
and treatment of preeclampsia.
Author Contributions: Drs Levine and Karumanchi
had full access to all of the data in the study and take responsibility for
the integrity of the data and the accuracy of the data analysis.
Study concept and design: Levine, Thadhani,
Acquisition of data: Levine, Lam, Sibai, Karumanchi.
Analysis and interpretation of data: Levine,
Thadhani, Qian, Lam, Lim, Yu, Blink, Sachs, Epstein, Sukhatme, Karumanchi.
Drafting of the manuscript: Levine, Thadhani,
Critical revision of the manuscript for important
intellectual content: Levine, Thadhani, Qian, Lam, Lim, Yu, Blink,
Sachs, Epstein, Sibai, Sukhatme, Karumanchi.
Statistical analysis: Qian, Yu.
Obtained funding: Levine, Karumanchi.
Administrative, technical, or material support:
Levine, Qian, Blink.
Study supervision: Levine, Karumanchi.
Calcium for Preeclampsia Prevention Study Group: University
of Alabama at Birmingham: J. C. Hauth, R. Goldenberg, B. S. Stofan; University
of New Mexico at Albuquerque: L. B. Curet, G. M. Joffe, V. Dorato; University
of Tennessee at Memphis: B. M. Sibai, S. A. Friedman, B. M. Mercer, T. Carr;
Case Western Reserve University at MetroHealth Medical Center, Cleveland,
Ohio: P. M. Catalano, A. S. Petrulis, L. Barabach; Oregon Health Sciences
University, Portland: C. Morris, S. L. Jacobson, K. McCracken; EMMES Corp,
Rockville, Md: J. R. Esterlitz, M. G. Ewell, D. M. Brown; National Institute
of Child Health and Human Development (NICHD): R. J. Levine, R. DerSimonian,
J. D. Clemens, M. A. Klebanoff, E. G. Raymond, J. G. Rigau-Perez, H. Shifrin;
National Heart, Lung, and Blood Institute (NHLBI): J. A. Cutler, D. E. Bild.
Data Safety and Monitoring Board: M Lindheimer, C. Begg, T. Chalmers, M. Druzin,
Funding/Support: This study was funded by the
American Society of Nephrology Carl W. Gottschalk Research Scholar Award,
Beth Israel Deaconess Medical Center Department of Medicine, Obstetrics &
Gynecology seed funds to Dr Karumanchi, and by intramural funding of Dr Levine
from the NICHD. Salary support for Dr Karumanchi was provided through a National
Institutes of Health physician-scientist award (KO8-DK-002825). The CPEP trial
was supported by the NICHD under contracts N01-HD-1-3121 through 3126, N01-HD-3154,
and N01-HD-5-3246, with cofunding from the NHLBI. Dr Thadhani is funded by
grant R01-HD-3-9223 from the NICHD.
Role of the Sponsors: The funding organizations
had no role in the design and conduct of the study; collection, management,
analysis, and interpretation of the data; or the preparation and review of
the manuscript for submission. The manuscript was approved for submission
by the Division of Epidemiology, Statistics, and Prevention Research of the
Acknowledgment: We are indebted to the CPEP
Study Group, who assembled the database and specimen repository used here,
and to the patients who took part in the study. We thank Patricia Moyer for
assistance with the figures.