DOI: 10.1002/pd.

4370

REVIEW

Screening for abnormal placentation and adverse pregnancy

outcomes with maternal serum biomarkers in the second trimester
Katherine R. Goetzinger and Anthony O. Odibo*
Department of Obstetrics and Gynecology, Washington University, St. Louis, MO, USA
*Correspondence to: Anthony O. Odibo. E-mail: odiboa@wudosis.wustl.edu

ABSTRACT
Second trimester biomarkers were initially introduced with the intent of screening for neural tube defects and then
subsequently for Down syndrome. It was soon realized that these markers can be indirect evidence of abnormal
placentation and, therefore, can be used for screening for adverse pregnancy outcomes. Several new biomarkers have
subsequently been described with conicting ndings regarding their efciency for screening for adverse pregnancy
outcomes. Although a biologically feasible mechanism has been proposed for the role of these biomarkers, they still
fall short of an ideal screening test to be clinically useful. 2014 John Wiley & Sons, Ltd.

Funding sources: None

Conicts of interest: None declared

INTRODUCTION
Second trimester maternal serum biomarker screening was rst
introduced into the eld of obstetrics in the 1970s with the
discovery of elevated levels of alpha-fetoprotein (AFP) in the
maternal serum of pregnancies affected by fetal open neural
tube defects (NTDs). Approximately one decade later, low
maternal serum levels of AFP were identied in pregnancies with
fetal trisomy 21. Since that time, AFP has been combined with
other maternal serum biomarkers, including human chorionic
gonadotrophin (hCG), unconjugated estriol, and inhibin A, in
order to improve the detection rate of fetal aneuploidy in the
second trimester of pregnancy.
These biomarkers are secreted by the placenta and enter the
maternal bloodstream in small amounts throughout gestation.
The detection of abnormal biomarker levels in fetal trisomies
suggest that aneuploid fetuses demonstrate a degree of fetal
placental immaturity that results in both unregulated
hypersecretion and undersecretion of both fetal and placental
products.1 This suggests that abnormal levels of this biomarker
may be representative of abnormal placentation and, thus,
associated with adverse pregnancy outcomes such as
preeclampsia, fetal growth restriction (FGR), preterm delivery,
and fetal loss. Abnormal maternal serum markers obtained during
the rst trimester of pregnancy are also risk factors for subsequent
adverse pregnancy outcomes; however, these rst trimester
markers will not be addressed in this review. The objective of this
review is to critically evaluate the current literature regarding
abnormal levels of maternal serum biomarker in the second
trimester of pregnancy and their association with and screening
efciency for adverse pregnancy outcomes.

Prenatal Diagnosis 2014, 34, 635641

Routine second trimester serum biomarkers

Alpha-fetoprotein
Alpha-fetoprotein was the rst maternal serum biomarker
identied as a screening tool for fetal structural malformations
and chromosomal anomalies. Subsequently, it is the most
studied biomarker to be associated with adverse pregnancy
outcome. AFP is an oncofetal protein that is synthesized by
the yolk sac early in gestation and later by the fetal liver. The
placenta transports small amounts of AFP into the maternal
circulation during early gestation, where it reaches peak
concentrations between 28 and 32 weeks.2 Elevated levels of
maternal serum AFP >2.5 multiples of the median (MoMs)
between 16 and 18 weeks gestation are associated with an
~80% detection rate for open NTDs.3 In the absence of an
NTD, the differential diagnosis for elevated levels of AFP in
maternal serum in the second trimester of pregnancy includes
false positive test reading, inaccurate pregnancy dating,
multiple gestations, intrauterine fetal demise, maternalfetal
hemorrhage, aneuploidy, congenital infection such as
Parvovirus, and other fetal structural malformations, most
commonly abdominal wall defects. However, approximately
1% of women persistently will have an abnormally elevated
serum level of AFP that cannot be accounted for by these
diagnoses.4 It has been demonstrated that these women
remain at increased risk for adverse pregnancy outcomes, with
an incidence as high as 20% to 38% in some studies.5 In 1988,
Barbara Burton demonstrated that patients with an
unexplained elevated maternal serum AFP level 2.5 MoMs
were at a signicantly increased risk for fetal loss beyond
20 weeks gestation, low birth weight, and neonatal death as

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K. R. Goetzinger et al.

636

compared with controls with a normal maternal serum AFP

level.6 Milunsky and colleagues further quantied these risks
demonstrating that patients with unexplained elevated
maternal serum AFP levels 2.0 MoMs were at a twofold
increased risk for preeclampsia, a threefold increased risk for
placental abruption, a fourfold increased risk for low birth
weight, and an eightfold increased risk for fetal death.7 In
1991, Waller and colleagues published a case-control study of
612 women whose pregnancies ended in fetal death and
compared them with 2501 live births. After controlling for
confounders, this study demonstrated a 10.4-fold increased risk
for fetal death in women with a serum AFP level greater than
3.0 MoMs and a 2.4-fold increased risk for fetal death in women
with a serum AFP level between 2.0 and 2.9 MoMs. Most
interestingly, this study also demonstrated that the increased risk
for fetal death persisted throughout gestation, months after the
second trimester AFP level was obtained.8 Most recently, Smith
and colleagues published data suggesting an increased risk for
sudden infant death syndrome [odds ratio (OR) 2.8, 95%
condence interval (CI) 1.45.4] in children born to women with
a maternal serum AFP level in the highest quintile as compared
with those in the lowest quintile; however, this nding may be
partially mediated by FGR and preterm birth.9
The mechanism for these associations remains unclear.
Elevated AFP levels have been found in women who have
experienced early vaginal bleeding that may occur in the setting
of recent or impending fetal death. However, the fact that the risk
for fetal death persists far into the third trimester of pregnancy
suggests an alternative mechanism. It has been hypothesized that
a disruption in the maternalfetal interface allows increased
transfer of AFP into the maternal circulation.10 Others suggest that
elevated AFP levels are a surrogate marker for abnormal
implantation and placental malfunction that are the underlying
pathologic mechanisms for many adverse obstetric outcomes.11,12
This is further substantiated by research demonstrating a large
range of both sonographic and pathologic abnormalities in both
the placenta and umbilical cord in patients with an elevated
maternal serum AFP level.13 Salaa and colleagues identied
distinct placental lesions including chronic villitis, thrombosis,
and infarction in women with elevated maternal serum AFP
levels.14 Furthermore, an increased risk for abnormal placental
adherence (i.e. placenta accreta/increta/percreta) also has been
demonstrated in women with an elevated maternal serum AFP
level, especially in the presence of a placenta previa.15,16
Risk stratication has been evaluated to identify those
women at highest risk for adverse pregnancy outcome
among those with an elevated AFP level. Using maternal
characteristics and medical comorbidities, Chandra and
colleagues assigned women into high-risk and low-risk groups
based on their a priori risk of having an obstetric complication.
They found that women with an unexplained elevated
maternal serum AFP level were at increased risk for obstetric
complications regardless of their prepregnancy risk status.12
Biophysical measurements such as uterine artery Doppler also
have been combined with serum levels of AFP in order to
improve screening efciency. Multiple small studies have
reported an increased risk for adverse pregnancy outcomes in
patients with abnormal second trimester uterine artery
Prenatal Diagnosis 2014, 34, 635641

Doppler studies in the setting of an unexplained elevated

maternal serum AFP level.1721 Bromley and colleagues
demonstrated that severe grade II uterine artery notching was
associated with a greater than threefold increased risk for a
composite perinatal morbidity including preterm delivery <37
weeks, birth weight <10th percentile for gestational age, and
fetal/neonatal death.19 Similarly, Konchak and colleagues
demonstrated an increased risk for preeclampsia, preterm
birth, and low birth weight in patients with uterine artery
notching and an elevated maternal serum AFP level.20 Despite
these associations, the positive predictive value (PPV) of these
combined tests remains relatively low. In one of the largest
published series, the combination of an elevated serum level
of AFP and bilateral uterine artery notching demonstrated a
PPV of 21% and 43% for preeclampsia and FGR, respectively.21
Obstetric surveillance and pregnancy management in these
patients remains controversial. Although many practitioners will
opt to follow these patients with increased frequency of antenatal
visits, uterine artery Doppler studies, serial ultrasounds for fetal
growth, and antenatal surveillance, there is no existing evidence
to support any specic obstetric surveillance protocol.22 In 2001,
Huerta-Enochian and colleagues published a retrospective cohort
study of 136 pregnancies with maternal serum AFP levels >2.0
MoMs. This study demonstrated that intensive antenatal
surveillance consisting of twice-weekly nonstress tests and
amniotic uid index determinations did not achieve earlier or
improved detection of adverse pregnancy outcome. In fact, all
adverse outcomes were either detected with routine pregnancy
care or remained undetected despite intensive antenatal
surveillance.23 Given the retrospective design of this study, timing
of initiation of antenatal surveillance was left to the discretion of
the provider. It remains unclear whether increased obstetric
surveillance can improve outcomes in patients with an
unexplained elevated maternal serum AFP level.
Far less data exists on the association between unexplained
low levels of maternal serum AFP and adverse pregnancy
outcomes. There are a few small cohort studies suggesting a
paradoxically increased risk for low birth weight and fetal
demise in patients with maternal serum AFP levels <0.25
MoMs. Regrettably, chromosomal analysis in many of these
demise cases was not obtained; therefore, fetal trisomy, a
well-established cause of low AFP levels, cannot be denitely
ruled out in all cases of demise.6,8,24,25 This limitation could
introduce signicant ascertainment bias to the results of such
studies, thereby, decreasing the robustness of their ndings.
Other studies have suggested an increased incidence of high
birth weight infants and associated delivery trauma in women
with low maternal serum AFP levels.26 Finally, still other
studies suggest a much more favorable pregnancy outcome
in the setting of low maternal serum AFP levels, reecting an
intact maternalfetalplacental unit.12,27,28

Human chorionic gonadotrophin

Soon after the discovery that low levels of maternal serum AFP
were associated with an increased risk for trisomy 21, it also
was discovered that high levels of maternal serum hCG
(typically 2.5 MoMs) also could be found in aneuploid
pregnancies.29 hCG is a glycoprotein produced by the placental
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Second trimester biomarkers screening

syncytiotrophoblast. It has been hypothesized that decreased

placental perfusion, with resultant hypoxic injury, may lead to
increased hCG production.30 Placental pathologic evaluation from
pregnancies with unexplained elevated maternal serum hCG
levels shows an increased incidence of retroplacental hematomas,
large for gestational age placentas, increased decidual plasma
cell inltrates, and low mean fetoplacental weight ratios.
Morphometric placental analysis also demonstrates an increased
volume of hCG-positive trophoblast per unit surface area.31
Together, these ndings suggest that elevated levels of hCG may
be reective of placental dysfunction and, therefore, also may
have an association with adverse pregnancy outcomes.
In a cohort study of over 25 000 pregnancies, Benn and
colleagues demonstrated a signicantly increased risk for
fetal/neonatal death, preterm birth, low birth weight, and
preeclampsia in patients with a maternal serum hCG >3.0
MoMs.32 Subsequent studies have produced similar
results.12,3335 In fact, Chandra and colleagues demonstrated
that the risk for adverse outcome was increased even when
maternal hCG levels exceeded 2.0 MoMs, with the most
pronounced risk being for intrauterine fetal demise [relative
risk (RR) 4.91, 95% CI 2.688.98].12 Although Lepage and
colleagues were underpowered to study intrauterine fetal
demise, they did demonstrate an association between elevated
maternal serum hCG levels and spontaneous miscarriage,
small for gestational age, pregnancy-associated hypertensive
disorders, and preterm delivery. Interestingly, they also were
able to demonstrate a doseresponse relationship, as 87%
(13/15) of pregnancies with extremely elevated maternal serum
hCG levels (10 MoMs) in their cohort experienced at least one
adverse pregnancy outcome.33 Alternatively, a large casecontrol study by Spencer demonstrated that an isolated
elevated maternal serum hCG level (>2.0 MoMs) was not
associated with any adverse pregnancy outcome.36
Furthermore, Walton and colleagues showed only a slight
association between maternal serum hCG levels >2.0 MoMs
and the adverse outcomes of pregnancy-induced hypertension
and preterm delivery and no association with premature
rupture of membranes nor FGR. However, that study did show
that higher levels of hCG were associated with stillbirth with an
OR of 1.4 (95% CI 1.11.9) for every increase of one MoM.37
In rare case of elevated levels of both maternal serum AFP
and hCG, it is more apparent that the risk for adverse
pregnancy outcome is signicant.12,36,38 Spencer and
colleagues observed a sixfold increased risk for preterm
delivery and a sevenfold increased risk for stillbirth when both
AFP and free hCG were >2.0 MoMs.36 Chandra and colleagues
demonstrated a risk increase for FGR from a RR of 1.28 in the
setting of an isolated maternal hCG level to a RR of 4.11 in
the setting of elevated levels of both AFP and hCG. Similarly,
the risk of intrauterine fetal demise was reported to increase
to 13.8 when both AFP and hCG levels are elevated.12

Unconjugated estriol and inhibin A

Unconjugated estriol and Inhibin A are the other two serum
analytes that comprise the quadruple second trimester
aneuploidy screen. Unconjugated estriol levels are typically
<0.7 MoMs in pregnancies affected by trisomy 21, whereas
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637

Inhibin A levels are typically elevated >1.6 to 1.8 MoMs in

trisomy 21.3941 Low unconjugated estriol levels have been
associated with a number of adverse pregnancy outcomes.4246
Yaron and colleagues observed that unconjugated estriol
levels <0.5 MoM were associated with pregnancy-induced
hypertension, miscarriage, FGR, and fetal death.42 Dugoff and
colleagues later demonstrated that low unconjugated estriol
levels 0.5 MoMs were associated with an increased risk for birth
weight less than the fth percentile and less than the tenth
percentile for gestational age and fetal loss 24 weeks. In that
study, there was no association found between low estriol levels
and preterm birth, preeclampsia, or fetal demise >24 weeks.43 In
another study restricted only to cases of FGR, patients with a low
estriol level <0.5 MoMs were at a greater than vefold increase
for composite adverse perinatal outcome, with a sensitivity of
36% and a specicity of 87%. This study suggested that serum
biomarker screening in the second trimester may be able to
differentiate a subset of growth-restricted fetuses at highest
risk for adverse outcome.44 The pathophysiologic mechanism
behind these associations with low estriol level remains
unclear. In the 1970s, serial urine estriol measurements were
routinely obtained during the third trimester of pregnancy in
order to evaluate for fetal well-being, with normal levels
being highly predictive a healthy fetus.45 Successful biosynthesis
of unconjugated estriol requires contributions both from the
fetus and placenta; therefore, abnormally low estriol levels
may be representative of a dysfunctional maternalfetal
placental unit. This is further illustrated by the association of
extremely low estriol levels in both fetal genetic and metabolic
placental disorders such as placental sulfatase deciency and
SmithLemliOpitz syndrome.47,48
Inhibin A is a member of the transforming growth factor
superfamily. Outside of pregnancy, the ovary is primarily
responsible for its production; however, during pregnancy,
there is a shift towards increased placental production of this
glycoprotein.49,50 Inhibin A is primarily localized to the surface
layer of the syncytiotrophoblast. Abnormal trophoblastic
invasion into the myometrium with subsequent hypoxic
placental injury, as can be seen in preeclampsia, causes
damage to the syncytiotrophoblast and leakage of inhibin A
into the maternal circulation. Additionally, hypoxic placental
injury can also result in proliferation of the cytotrophoblast
layer, resulting in increased production of inhibin A.51,52 Either
or both of these mechanisms could explain the biologic
plausibility of these ndings.
Elevated inhibin A levels in chromosomally normal fetuses
have been most extensively studied for their association with
preeclampsia.43,51,5355 Aquilina and colleagues demonstrated
that women with inhibin A levels >2.0 MoMs are at an
increased risk for all preeclampsia (OR 9.4, 95% CI 4.619.3)
and preeclampsia necessitating delivery <37 weeks (OR 18.2,
95% CI 6.054.8).51 These ndings were conrmed in a follow
up study that demonstrated a sensitivity of 48.6% and
specicity of 90% for the prediction of preeclampsia.54 Other
studies have demonstrated an even higher screening efciency
for elevated inhibin A levels and preeclampsia, with a
sensitivity of 71.4% and specicity of 96.3%.55 Other studies
have demonstrated an association between elevated inhibin
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K. R. Goetzinger et al.

638

A levels and FGR; however, these associations appear to be less

robust.43,51 The addition of uterine artery Doppler studies to
enhance the screening efciency of inhibin A has been
proposed; however, studies have produced conicting
results.5558 Ultimately, it appears that while abnormal uterine
artery Doppler studies in the setting of elevated inhibin A levels
may increase the detection rate of adverse pregnancy
outcomes, the nding of normal uterine artery Doppler
studies does not negate the increased risk for adverse
pregnancy outcome in the setting of abnormal second
trimester serum screening.57

The quadruple screen: combinations of serum markers

Although it is clear that individual second trimester maternal
serum biomarkers can be predictive of multiple adverse
pregnancy outcomes, these biomarkers are rarely measured
individually. Instead, they are interpreted as part of the second
trimester quad screen for fetal aneuploidy. Currently, there
are no published studies that prospectively evaluate the use
of the quad screen for the primary intent of quantifying the risk
of adverse pregnancy outcomes; however, multiple studies
have evaluated these outcomes in patients pursuing second
trimester serum screening for the purpose of aneuploidy
detection. In a secondary analysis of the FASTER trial, Dugoff
and colleagues evaluated both the individual components of
the quad screen and their various combinations to determine
their screening efciency for adverse pregnancy outcome. That
study found that combinations of at least two abnormal
biomarkers were more strongly predictive for any adverse
pregnancy outcome compared with only one abnormal
marker. That association became stronger with increasing
numbers of abnormal biomarkers. For example, the adjusted
OR for fetal loss 24 weeks increased from 3.63 (95% CI 1.81
7.27) in the setting of elevated AFP levels 2.0 MoMs to 8.81
(95% CI 5.213.12) in the setting of two abnormal biomarkers.
This risk association increased even further to 23.15 (95% CI
12.6142.49) in the setting of three or more abnormal
biomarkers.43 Huang and colleagues observed similar results
when evaluating combinations of AFP, hCG, and unconjugated
estriol in addition to pregnancy-associated plasma protein A
(PAPP-A) levels obtained in the rst trimester of pregnancy.46
Despite the strong associations observed with multiple
abnormal serum markers, the sensitivity and PPV for adverse
pregnancy outcomes remain relatively low, and the false
positive rates can be high. As the rst trimester aneuploidy
screening and cell-free fetal DNA gain more and more
popularity, the frequency at which second trimester
aneuploidy screening will continue to be obtained remains
questionable. Given the modest predictive efciency of these
second trimester serum biomarkers and the lack of
management strategies that are proven to improve pregnancy
outcome, it is unlikely that second trimester serum biomarker
testing can be recommended as a population-based screening
tool for adverse pregnancy outcome; however, when screening
is performed for the clinical indication of aneuploidy
detection, abnormal serum screening results may be used to
identify high-risk patients and to guide clinical management
at the discretion of the physician.22
Prenatal Diagnosis 2014, 34, 635641

Beyond the quadruple screen

Pregnancy-associated plasma protein A
Pregnancy-associated plasma protein A is an insulin-like
growth factor binding protein (IGFBP) protease with specicity
for IGFBP 2 and 4.58 Reduced levels of PAPP-A may result in
increased amounts of IGF being bound to its carrier proteins
and, hence, not available at the cell receptor level to stimulate
fetal growth and trophoblast invasion of the decidua.59
Many studies have reported reduced maternal serum PAPP-A
concentration at 11 to 14 weeks and increased risk for
subsequent development of preeclampsia, small for gestational
age, and preterm delivery; however,58,6063 only few studies have
evaluated the association between PAPP-A and adverse
outcomes in the second trimester. For example, in a case-control
study, DAnna et al. compared PAPP-A levels drawn between 14
and 17 weeks in 40 women who developed preeclampsia with
562 that had uncomplicated pregnancies.64 The area under the
receiver operating characteristic curve for prediction of
preeclampsia was 0.88 with a 95% CI of 0.80 to 0.96. For a xed
false positive rate of 5%, the detection rate for preeclampsia
was 66.7%. The ndings from this study are yet to be validated
by other studies. In fact, another case-control study found no
difference in early second trimester PAPP-A levels between
77 cases with preeclampsia and 224 unaffected pregnancies.65

A disintegrin and metalloprotease

A disintegrin and metalloprotease 12 (ADAM12) is a placentaderived multidomain glycoprotein involved in controlling fetal
and placental growth and development. Human ADAM12
exists in two forms: ADAM12-L (long) and ADAM12-S (short).
ADAM12-S is the secreted form of ADAM12 and is also an
IGFBP protease but with specicity for IGFBP 3 and 5.66,67
ADAM12 can also bind to adhesion receptors and mediate
shedding of oxytocinase, which may be associated with
progressive growth of placenta.68,69
Reduced ADAM12 has been shown to be a potential marker of
a preeclampsia and intrauterine growth restriction (IUGR) in the
rst trimester.7072 Similar to PAPP-A, there are only a handful of
studies estimating the value of ADAM12 as a biomarker for
preeclampsia in the second trimester. DAnna et al., using the
same population mentioned earlier, evaluated ADAM12 in
40 cases with preeclampsia compared with 562 unaffected
pregnancies.64 The area under the curve for predicting
preeclampsia was 0.95, with a 95% CI of 0.91 to 0.98. For a xed
false positive rate of 6%, the detection rate for preeclampsia
using ADAM12 was 77.8%. These ndings were not conrmed
in an early second trimester study by Bestwick et al.65

Angiogenic factors
Circulating angiogenic factors include vascular endothelial
growth factor and placental growth factor. They are thought
to contribute to normal trophoblastic proliferation and
implantation.73 Soluble fms-like tyrosine kinase-1 (sFlt-1) is
able to block the effects of vascular endothelial growth factor
and PIGF by inhibiting interaction with their receptors.
Ischemic trophoblasts have been shown to synthesize
antiangiogenic factors, notably sFlt-1.74 This deprives the
maternal vascular endothelium of these essential angiogenic
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Second trimester biomarkers screening

factors and causes systemic endothelial dysfunction that may

culminate in the preeclamptic syndrome.75,76
Numerous studies have documented that placental growth
factor concentration in the second trimester is reduced in
women who go on to develop preeclampsia/IUGR73,7779 and
inversely correlates with the severity of the diseases.73,75,8082
On the other hand, sFlt-1 levels in the second trimester are
increased in women destined to develop preeclampsia/
IUGR.75 In the prospective multicenter cohort study Screening
for Pregnancy Endpoints, clinical risk factors, serum PIGF, sFlt1, and soluble endoglin were measured at 14 to 16 weeks in
3529 nulliparous women, including 47 that later developed
preterm preeclampsia needing early delivery. The combination
of clinical risk factors and PIGF resulted in the best area under
the curve of 0.84 (95% CI of 0.770.91). With a xed false
positive rate of 5%, the sensitivity for predicting preeclampsia
was only 45% .83 The addition of sFlt-1 or soluble endoglin
did not improve the prediction model any further. Repeating
the analysis with samples drawn between 19 and 21 weeks
and adding uterine artery Doppler did not signicantly
improve the screening efciency for preeclampsia. The authors
concluded that while these angiogenic markers can identify
nulliparous women at risk for developing preeclampsia, their
performance was not sufciently efcient to be introduced
into routine clinical practice.83

CONCLUSION
In recent years, much interest has been shown in identifying
biomarkers of placental dysfunction in the rst and second

639

trimesters of pregnancy that can predict women at risk for

preeclampsia and fetal growth restriction. The published
literature clearly supports a positive association between
abnormal levels of these serum markers, which is
strengthened in the presence of more than one abnormal
marker. Despite these associations, the predictive accuracy
of these biomarkers, both in isolation or combined with other
clinical risk factors, still falls short of the ideal for clinically
useful screening tests. Given the clinical importance of
recognizing those patients at high risk for developing adverse
pregnancy outcomes such as preeclampsia, continued
effort at identifying markers with better discrimination
should be encouraged.

WHATS ALREADY KNOWN ABOUT THIS TOPIC?

Several studies have been published on the role of placental
biomarkers in screening for adverse outcomes.
Although it is clear that strong associations between second
trimester serum biomarkers and adverse pregnancy outcomes
do exist, the predictive accuracy of these markers remain
limited for use as a population-based screening tool in
clinical practice.

WHAT DOES THIS STUDY ADD?

This review summarizes the studies on these biomarkers in a
systematic fashion and critically evaluates the current role of second
trimester biomarkers in screening for preeclampsia and other
adverse outcomes.

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