This document discusses preeclampsia (PE), a serious pregnancy complication characterized by high blood pressure and protein in the urine. It summarizes several potential biochemical markers for predicting and diagnosing PE, including PAPP-A, PP13, sFlt-1/PlGF ratio, HbF/A1M, and cystatin C. New algorithms combining multiple factors can achieve prediction rates of 60-100% for early onset PE, but no single marker currently fulfills all the ideal characteristics of being pathogenically central, early-appearing, easy to measure, and highly sensitive and specific.

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2.  Preeclampsia (PE) is one of the most serious
pregnancy complications. The worldwide
prevalence of PE ranges from 3 to 8% of
pregnancies, affecting a total of 8.5 million
women worldwide.
 PE is responsible for about 18% of maternal
deaths and up to 40% of fetal mortality.
 At this time, PE still lacks a safe and effective
therapy, as well as a reliable, early means of
diagnosis or prediction

3.  The disease evolves in two stages. The first
stage is characterized by an altered formation of
the placenta .
 During placentation, a defective invasion of the
extra villous trophoblast cells into the muscle
layers of the spiral arteries has been shown .
 This contributes to a reduced uteroplacental
blood flow that can result in fetal intrauterine
growth restriction (IUGR), seen in one of four
women with PE.
 A growing body of evidence suggests that
oxidative stress further aggravates vascular
function in the placenta , which in turn gives rise
to insufficient blood perfusion , inflammation,
apoptosis and structural damage

4.  The second stage, the clinical manifestations
,i.e. hypertension and proteinuria, appears
from 20 weeks of gestation onwards.
 As the disease progresses, angiospasms in
the brain and brain edema may cause severe
epileptic seizures - eclampsia .

5.  According to the International Society for the
Study of Hypertension in Pregnancy (ISSHP),
PE can be defined as de novo hypertension
occurring after 20 weeks of pregnancy
together with proteinuria.
 Hypertension is defined as a systolic blood
pressure 140 and/or a diastolic blood
pressure 90 mmHg measured at two
occasions with at least 4 h in between.

6.  Proteinuria is defined as 300 mg per day
Proteinuria is questionable as a marker for PE
since it lacks predictive value and does not
correlate with severity of the disease.
 A severe form of PE is the Hemolysis, Elevated
Liver enzymes and Low Platelets syndrome
(the HELLP-syndrome).
 It is defined by the laboratory findings of
hemolysis, elevated liver enzymes and low
platelet count .

7.  . Altogether, the wide range of clinical
manifestations makes PE more syndrome-like
than a defined disease, which complicates the
clinical diagnosis .
 Lately, the time of onset of the clinical
manifestations, early onset PE (<34weeks of
gestation) and late onset PE (>34 weeks of
gestation), have been used to further
characterize PE, but the overall classification
still lacks stringency

8.  Primi gravida
 Family history
 Placental abnormalities
 Obesity
 Pre existing vascular disease
 thrombophilias

9.  Failure of trophoblast invasion
 Vascular endothelial damage
 Inflammatory mediators (cytokines)
 Immunological intolerance between maternal
and fetal tissues
 Coagulation abnormalities
 Increased oxygen free radicals
 Genetic predisposition(polygenic disorder)
 Dietary deficiency or excess

15.  Few biochemical markers have been proven
specific and sensitive as single markers to
predict and/or diagnose PE.
 Algorithms also include clinical
measurements such as Doppler ultrasound
and clinical risk factors, to further enhance
the prediction rate at a low false positive rate.
 In this review the most promising individual
biochemical markers are described for both
prediction and diagnosis of PE.
 The biochemical markers are presented in
the order they are shown to appear in
pregnancy, i.e. first, second or thirdtrimester

16.  PAPP-A
 HbF/A1M all show potential as predictive biochemical markers in the
first trimester
 PP 13
 Sflt-1 & s endoglin
 PIGF
 Cystatin C

17.  PAPP-A is a glycoprotein synthesized in the
placenta and the study of it as a biochemical
marker in pregnancy has been pursued for
almost 30 years .
 The maternal plasma concentration increases
through out pregnancy.
 PAPP-A has been used in combination with b-
human chorionic gonadotropin (b-hCG) and
nuchal translucency thickness, to screen for
trisomy 21, 13 and 18 at 11 to 13 weeks of
gestation

18.  In fetuses with normal chromosomes,
decreased levels of PAPP-A in the first
trimester have been associated with increased
risk for PE, IUGR, fetuses small for gestational
age (SGA) and preterm delivery

19.  PAPP-A has been evaluated as a predictive and
diagnostic biochemical marker for PE, but the
screening performance, when used as a single
biochemical marker, is only about 10 to 20 %
 Combined with Doppler ultrasound, PAPP-A is
a powerful predictive biochemical marker of PE
with prediction rates of 70% at false positive
rates of 5%.
 At term, plasma PAPP-A concentrations have
been shown to increase in pregnancies
complicated by PE and HELLP, but its
concentration is still not predictive .

20.  Recent reports suggest that free, extracellular fetal
hemoglobin(HbF) is involved in the pathogenesis of
PE.
 Furthermore the heme and radical scavenger a1-
microglobulin (A1M) is involved in the physiological
defence against HbF.
 Their concentrations in maternal serum or plasma
can be used as early predictive biochemical markers.
 Increased mRNA levels of HbF in the placental tissue
and free HbF protein in the placental vascular lumen
were described in women with PE .

21.  Hemoglobin is a highly reactive molecule that
is capable of damaging and disrupting cell
membranes , and binds and inactivates nitric
oxide (NO) with vasoconstriction as a
consequence .
 Its metabolites, heme and iron, damage lipids,
protein and DNA through direct oxidation
and/or generation of reactive oxygen species
(ROS).

22.  Heme is also a pro-inflammatory molecule that
activates neutrophils .
 Several Hb- and hemedetoxification systems
have been described in humans.
 Recently, the plasma and tissue protein A1M
was shown to bind and degrade heme , have
radical-scavenger properties , and protect cells
and tissues against extracellular Hb, heme and
ROS .

23.  A1Mexpression in liver and placental cells has
been shown to be upregulated by Hb, heme
and ROS . A pathogenic role of Hb and
protective role of A1M in PE is supported by ex
vivo placenta perfusion experiments .
 Studies evaluating maternal serum/plasma
concentrations of HbF and A1M, as predictive
and diagnostic markers for PE, have shown
promising results .

24.  In a cohort of 96 patients subsequently
developed PE the serum concentrations of HbF
and A1M were significantly increased at 10 to
16 weeks’ gestation in women who
subsequently developed PE.
 The plasma concentrations of HbF and adult
hemoglobin (HbA) were also significantly
correlated to maternal blood pressure in
patients with established PE . These markers
still need to be validated in larger cohorts

25.  PP13 is a member of the galectin family and is
produced by the placental trophoblast cells .
 The function(s) of PP13 is stillnot clearly understood,
but it is involved in normal placentation
 In normal pregnancies, serum levels of PP13 slowly
rise with gestational age.
 Several studies have shown lowered serum levels in
the first trimester in pregnancies that subsequently
developed PE.
 As a first trimester screening marker for PE, PP13
shows different prediction rates in different studies.

26.  In two different cohort studies, PP13 levels
were determined at 11 to 13 weeks of
gestation .
 Both studies showed significantly lower first
trimester levels of PP13 in women who later
developed PE.
 When combining serum screening with
Doppler ultrasound pulsatilityindex (PI), the
prediction rate increased to 71% at a false
positive rate of 10% .

27.  Romero et al. [40] studied a cohort of 300
patients 50 of whichdeveloped PE.
 At a false positive rate of 20% the detection rate
was 36% for all types of PE. For early onset PE it
was 100% (n ¼ 6) and for preterm PE 85% (n ¼
44).
 Preterm was defined as onset before 37 weeks.
 The prediction rate for severe PE at term was
24%
Based on t hese findings, PP13 was concluded to
be a reasonable biochemical marker for early
onset and preterm PE but a weak marker for PE at
term

28.  Two angiogenesis-related factors are particularly well
studied: soluble fms-like tyrosine kinase (sFlt-1), a soluble
VEGF receptor, and soluble endoglin (s-Eng), a co-receptor
for TGF-beta.
 Both are elevated in maternal plasma in patients with PE
compared to normal pregnancies .
 Elevated levels of sFlt-1 occur before the clinical
symptoms. The levels correlate with the time of onset of
clinically manifest PE and partly with disease severity.
 Early-onset PE exhibits higher levels of sFlt-1 .
 Moreover, in animal experiments, proteinuria and
hypertension, as well as a HELLP-like syndrome, were
induced by infusion of high levels of sFlt and endoglin .

29.  As a first trimester screening marker, s-Eng
shows conflicting results .
 Used in combination with Doppler
ultrasound (PI) and PlGF, the prediction rate
for early onset PE was 77.8% at a false
positive rate of 5% .

31.  The ratio of the PlGF/sFlt-1 is well described
and they are a promising set of biochemical
markers for prediction of PE .
 Automated fast analysis methods have been
developed for these proteins , but their role
as first trimester markers is not clear .
 Several studies have shown the predictive
power of PlGF/sFlt-1 ratio from the second
trimester.
 The prediction rate is about 89% .

32.  In a recent multicenter study by Verlohren et al. ,
including 351 patients (71 with PE), the sFlt-1/PlGF
ratio was measured longitudinally throughout
pregnancy.
 At a false positive rate of 5% the detection rate was
82% for all PE.
 For early onset PE, at a false positive rate of 3%, the
detection rate was 89%
 Hence, the sFlt-1/PlGF ratio has no predictive value
in the first trimester.
 As a single biochemical marker, PlGF has been
shown to predict 53.5% of early onset PE at a false
positive rate of 5% and 65% at a false positive rate of
10% in late first trimester.

33.  Metabolic profiling is a powerful strategy to
investigate the metabolites that a specific cellular
event leaves behind.
 Metabolic profiling can be used to reveal the
patho physiological mechanisms in a disease
such as PE .
 Recently, in a study of 60 patients who
subsequently developed PE and 60 normal
pregnancies, 45 metabolites were shown to be
significantly altered in the first trimester in
pregnancies that later developed PE.

34.  For early and late onset PE, the prediction
rate was between 73 and 77% at a 10% false
positive rate .
 The findings were validated in a cohort of 39
patients with subsequent PE matched with 40
normal pregnancies. Interestingly, 3 out of
the 40 up-regulated were shown to be
hemoglobin metabolites.

35.  Cystatin C is a protease inhibitor widely used
by clinicians as a sensitive marker for renal
function and for estimation of glomerular
filtration rate.
 The maternal plasma level of cystatin C is
increased in women with PE and studies have
demonstrated that the level of cystatin C is a
reliable diagnostic marker for PE.

36.  Increased levels of cystatin C are suggested
to be caused by either impaired renal
function and/or by increased placental
synthesis
 Cystatin C has recently been suggested as a
predictive first trimester marker for PE .
 However, given the low screening
performance of the study, cystatin C is
probably not clinically useful as a single
marker but could be useful in combination
with other biochemicalmarkers.

37.  As genomics, proteomics and metabolomics are being
developed and made more available, the number of
potential biochemical markers will increase.
 Ideally, the biochemical markers will give us new hints as
to the pathogenesis behind PE.
 These new techniques have revealed many of the above
mentioned biochemical markers, and worth mentioning
are free mRNAs and miRNAs in maternal blood.
 Both types of RNAs are expressed in the placenta and can
be found in the maternal circulation.
 Further investigation is needed but profiling of these
RNAs might show potential in predicting pregnancy
outcomes

38.  2.9. New algorithms
 The lack of a specific and sensitive biochemical
marker has led to the development of
mathematical models that combine several
factors in order to predict PE .
 Akolekar et al. combined maternal
characteristics, PI and mean arterial pressure
(MAP) with serum levels of PAPP-A, PlGF, PP13,
inhibin-A, activin-A, sEng,pentraxin-3 and p-
selectin in a large study (n ¼ 33,602) at 11 +0 to
13 + 6 weeks of gestation.

39.  . The prediction rates, at a false positive and
60.9% for late onset PE (intermediate onset PE
was defined as PE that led to delivery between 34
and 36 weeks o f gestation).
 Wortel boer et al. developed a model based on
the first trimester biochemical markers, PAPP-A,
beta-hCG, PlGF, desintegrin and ADAM metallo
peptidase domain 12 (ADAM12). Their prediction
of all PE was only 44% at a 5% false positive rate].
 Another first trimester model based on maternal
characteristics, PI and the biochemical markers
PAPP-A, inhibin-A, PP13, ADAM12, free beta-
hCG and PlGF was developed by Audibert et al.

40.  In a large cohort (n ¼ 893) the model showed a
100% prediction rate for early onset PE at a false
positive rate of 10% .
 It is worth noting that PP13 and ADAM12 levels
did not improve the prediction rates.
 In a very recent study by Odibo et al. [65]
maternal characteristics were combined with
serum PP13, PAPP-A and PI in the first trimester.
 In a cohort of 450 patients, the prediction rate
was 68% at a false positive rate of 5%.
Interestingly, PI measurements did not increase
the prediction rate in this study.

41. The ideal biochemical marker for PE should exhibit
the following characteristics:
 1)Play a central role in the pathogenesis and be
specific for the condition.
 2) Appear early or before the clinical
manifestations. Placental factors that can be
detected early in pregnancy are likely to be good
biochemical markers for PE prediction.
 However, placental disorders can cause IUGR
without PE and vice versa, which makes the
clinical evaluation of new markers particularly
hard.

42.  3) Be easy and cheap to measure in maternal blood or urine.
Few of the described factors are easy to measure; most of
them require advanced laboratory system.
 4) Show a high sensitivity and specificity. A small number of
the described biochemical markers fulfill this requirement
and strategies to use them in combination with other markers
and/or, with PI measurements and other clinical parameters
are being investigated.
 5) Correlate with the severity of the condition. As the disease
progresses, several organ systems are affected, which causes
the number of factors to increase throughout pregnancy. A
good candidate marker ought to appear early in pregnancy
andcontinue to rise as the disease progresses.
 6) Be non-detected or expressed at very low levels in norma
pregnancies. Again, a placental factor is favored since the
clinical symptoms disappear after removal of the placenta.

43.  Screening pregnant women with an effective
diagnostic marker for PE IUGR could reduce
unnecessary suffering and major healthcare costs .
 PE is still a dominant problem in the Third World,
where it is often first diagnosed when the women
present with eclamptic seizures.
 Basic equipment for blood pressure monitoring is
often lacking, which requires clinicians to make
careful clinical observations and basic examinations.
 Fetal monitoring with Doppler ultrasound and ECG is
rarely available. Therefore, algorithms that
summarize maternal risk factors are valuable and it
ismost important to develop them further.

44.  Furthermore, the biochemical marker must
be detectable before the disease progresses
into a dangerous stage, so that remote health
care centres can refer their pregnant women
to larger hospitals in timely manner.

45.  Screening for Down syndrome in the first trimester is a good
example where a combination of ultrasound scanning and
biochemical markers are used
 Potential first trimester biochemical markers are PAPP-A , HbF
and A1M .
 Both HbF and A1M play a role in the pathophysiology of PE .
 The biochemical markers appear as early as 10 weeks of
gestation . Furthermore, they can be measured with basic ELISA
techniques and show a high prediction rate at a low false positive
level.
 Maternal plasma concentrations of free HbF have also been
shown to correlate well with severity, i.e. blood pressure, in term
PE pregnancies .

46.  Angiogenic and anti-angiogenic factors are also
very promising biochemical markers.
 Although the combination sFlt-1/PlGF might not
be useful in the first trimester, they are definitely
well evaluated in the second trimester.
 Alterations of sFlt-1 and PlGF about 6 weeks
before the onset of clinical symptoms and
correlate with the severity of the disease.
 PlGF could be a promising biochemical marker
even in the first trimester particularly if combined
with HbF and A1M.

47.  PP13 has shown potential as a biochemical
marker of early onset PE Especially if
combined with Doppler ultrasound uterine
artery PI.
 However, as a general screening marker for
all types of PE, the data is conflicting and
needs further investigation.

48.  New factors should not be viewed solely as
competing biochemical markers for prediction
and diagnosis of PE.
I
 Instead each new factor ought to be welcomed as
a new important puzzle piece that contributes to
illuminating the etiology of PE.
 In the end these advances will hopefully lead to
better prophylactic treatments reducing maternal
and fetal morbidity

49.  It is a marker which has advantages over
serum creatinine
 Cystatin-c is a 13kd non glycosylated protein
 Normal blood level is 0.8 to1.2 mg/L
 It is seen in high concentrations in biological
fluids such as breast milk, tears,& saliva
 It is the most abundant extra cellular cysteine
protease inhibitors

50.  Creatinine is the most widely used biomarker
of kidney function.
 But sometimes it is inaccurate in detecting
mild renal impairment.
 The tubular secretion contributes app. 10% of
the total creatinine excretion by the kidney &
this contribution can increase as GFR
decreases.

51.  Serum creatinine does not increase until GFR
has moderately decreased.
 This insensitivity to moderate decreases in
GFR is called ‘creatinine blind GFR area’
 So serum creatinine may not be a good
parameter for determination of GFR ,
especially at lower levels of glomerular
function.

52.  On the other hand, cystatin-c is produced at
a constant rate & is freely filtered by kidney
glomeruli.
 It is completely reabsorbed ;but degraded in
the tubules ; thus making it an excellent GFR
marker.
 The blood levels are not dependent on age,
sex, muscle mass or inflammatory processes.

53.  It is sensitive to changes in the so called
creatinine blind area of GFR (40-
70ml/min/1.73m2)
 So, serum level of cystatin is a better test for
kidney function(GFR) than serum creatinine
levels.
 Since there is no tubular secretion of
cystatin-c it is extremely sensitive to minor
changes in the GFR in the earliest stages of
chronic kidney diseases