BIOCHEMICAL MARKERS OF

PREECLAMPSIA

M.Prasad NaiduMSc Medical Biochemistry,

Ph.D.Research Scholar

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

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

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 .

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.

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 .

. 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

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

Risk 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

Etio pathological factors for pre eclampsia

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

PAPP-A

HbF/A1M all show potential as predictive biochemical markers in the first trimester

PP 13

Sflt-1 & s endoglin

PIGF

Cystatin C

PREDICTIVE BIOCHEMICAL MARKERS

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

Pregnancy – associated protein A (PAPP-A)

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

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 .

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 .

Fetal hemoglobin and a1-microglobulin

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).

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 .

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 .

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

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.

Placental protein 13 (PP13)

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% .

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

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 .

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

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% .

  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% .

Placental growth factor (PIGF) & sFlt

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.

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.

METABOLOMICS

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.

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.

Cystatin C

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.

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

Other biochemical markers

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.

. 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.

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.

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.

Discussion

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.

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.

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.

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 .

conclusion

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.

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.

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

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

Cystatin-C

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.

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.

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.

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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