Biochemical markers of preeclampsia

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BIOCHEMICAL MARKERS OF PREECLAMPSIA

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

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• 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

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• 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

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• 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 .

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• 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.

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• 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 .

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• . 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 (lt34weeks of
  gestation) and late onset PE (gt34 weeks of
  gestation), have been used to further
  characterize PE, but the overall classification
  still lacks stringency

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Risk factors for pre eclampsia

• Primi gravida
• Family history
• Placental abnormalities
• Obesity
• Pre existing vascular disease
• thrombophilias

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Etio pathological factors for pre eclampsia

• 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

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• 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

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PREDICTIVE BIOCHEMICAL MARKERS

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

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Pregnancy associated protein A (PAPP-A)

• 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

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• 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

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• 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 .

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Fetal hemoglobin and a1-microglobulin

• 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 .

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• 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).

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• 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 .

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• 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 .

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• 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

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Placental protein 13 (PP13)

• 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.

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• 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 .

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• 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
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• 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

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 Soluble fms-like tyrosine kinase 1(sFlt-1) and
soluble endoglin (sEng) 

• 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 .

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• 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 .

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Placental growth factor (PIGF) sFlt

•  
• 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 .

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• 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.

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METABOLOMICS

• 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.

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• 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.

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Cystatin C

• 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.

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• 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.

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Other biochemical markers

• 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

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• 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.

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• . 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.

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• 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.

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Discussion

• 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.

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• 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.

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• 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.

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• 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.

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conclusion

• 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 .

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• 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.

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• 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.

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• 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

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Cystatin-C

• 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

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• 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.

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• 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.

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• 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.

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• 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

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