Pregnancy-Associated Plasma Protein-A and its Clinical Utility
by Kevin Spencer, DSc, FRSC, FRCPath, Consultant Clinical Biochemist, Clinical Biochemistry Department, Harold Wood Hospital, Romford, Essex, and Director of Biochemical Screening, Fetal Medicine Foundation, London, UK

Pregnancy-associated plasma protein-A (PAPP-A) was first described by Lin et al. in 1974 as a high molecular weight component of serum obtained from individuals in late pregnancy.¹ It has since been shown to be a large, dimeric, zinc-containing metalloglycoprotein with a molecular weight of 800 kDa and an a₂ electrophoretic mobility. Each subunit consists of 1,547 amino acid residues and, in pregnancy, is derived from a larger precursor of placental origin. Originally, circulating PAPP-A was thought to occur as a dimer,² but more recent studies³ have shown it to be present as a component of a heterotetramer consisting of two PAPP-A subunits disulfide-bonded to two subunits of the proform of eosinophil major basic protein (proMBP). It is believed that no free form of PAPP-A exists in pregnancy serum.⁴ proMBP also circulates as two additional complexes, one with angiotensinogen and the other with C3dg (a complement protein of the immune system).⁵ In pregnancy, proMBP concentrations exceed those of PAPP-A by 5- to 10-fold. Both PAPP-A and proMBP contain 13 and 39 percent carbohydrate, respectively.⁴ PAPP-A is produced by the placental syncytiotrophoblast (trophoblastic tissue that develops into the outer layer of the placenta) in an initial proform approximately 80 amino acids longer than the mature subunit.⁶ ProMBP, on the other hand, is synthesized in the extravillous cytotrophoblast (trophoblastic tissue that develops into the chorion or outermost extraembryonic membrane).⁷ The genes coding for PAPP-A reside on chromosome 9 and those for preproMBP on chromosome 11.^8,9

The biological function of PAPP-A is still unclear. It has been shown to bind heparin¹⁰ and to be a noncompetitive inhibitor of human granulocyte elastase (a tissue-degenerative enzyme released when tissue inflammation occurs), which has led to postulation that it may have a role in modulating the maternal immune response and be associated with implantation and growth of the placenta. Recently, it has been identified as an insulin-like growth factor-dependent protease that cleaves insulin-like growth factor binding protein-4.¹¹ The identification of PAPP-A and proMBP in several reproductive and nonreproductive tissues,¹² albeit at much lower levels than in placental tissue, has led to further speculation that they may have a role in cytokine-mediated response. The recent observation of PAPP-A in unstable atherosclerotic plaques, along with increased circulating PAPP-A levels, may suggest a new role for PAPP-A as a marker of acute coronary syndromes.¹³ To date, however, its major clinical usefulness appears to be limited to three main areas:

	As a marker of chromosomal aneuploidy
	As an indicator of early pregnancy failure and pregnancy complications
	As a marker of Cornelia de Lange syndrome.

PAPP-A and chromosomal aneuploidy
The natural frequency of chromosomal abnormalities at birth, in the absence of any prenatal screening, has been estimated at 6 per 1,000 births.¹⁴ The most common of these is trisomy 21 (Down syndrome), its risk increasing dramatically with maternal age and compounded by changing demographics in the pregnant population because of a common trend among couples to delay having families; the second trimester incidence of fetal trisomy 21 is now 1 in 500. Other common autosomal trisomies include trisomy 18 (Edwards syndrome) and trisomy 13 (Patau's syndrome), which have birth incidences of 1 in 6,500 and 1 in 12,500, respectively.¹⁵ The incidence of both syndromes increases with maternal age; and in all three trisomies, due to spontaneous fetal loss, the first- and second-trimester incidences are much greater than the birth incidence.¹⁶

Since the early 1990s, prenatal screening, initially instituted for the detection of trisomy 21, has become a standard part of obstetric practice—largely through the measurement of maternal serum biochemical markers in the second trimester (15 to 20 weeks gestation).¹⁷ These markers include a combination of two or three of the following: alphafetoprotein (AFP), total hCG, free b-hCG and unconjugated estriol. In pregnancies with fetal trisomy 21, maternal serum levels of AFP and unconjugated estriol tend to be lower than normal (median MoM 0.7), while levels of free b-hCG or total hCG are increased (2.2 and 2.0 MoM, respectively). Using a combination of maternal age and maternal serum biochemistry, detection rates of 65 to 70 percent can be achieved when screening the entire pregnant population at a 5 to 6 percent false-positive rate.^17,18

In some centers, screening for trisomy 18 is also conducted using the same marker protocol,¹⁹ with fetal trisomy 18 maternal serum levels of all markers being, on average, lower than normal. In general, screening can identify approximately 60 percent of cases at a 0.5 to 1 percent false-positive rate.¹⁹

Much of the research in the past decade has been focused on screening earlier in pregnancy (10 to 14 weeks),²⁰ with the emergence of three candidate markers in the early 1990s. In 1992, Spencer et al.²¹ showed that maternal serum free b-hCG was increased (median MoM 1.98) in the first trimester at a time when total hCG levels are normal (median MoM 1.33).²² Also in 1992, Nicolaides et al. described the use of ultrasound to measure fetal nuchal translucency (NT) thickness and showed that the increased thickness was related to increased risk for fetal aneuploidy (median MoM 2.02).²³ Brambati et al., however, were the first to show that levels of PAPP-A were reduced in pregnancies with fetal trisomy 21 (median MoM 0.45).²⁴

Since the early landmark studies, many others have confirmed these distinguishing serum values. The largest study to date (including some 210 cases with trisomy 21) has shown that, at 10 to 14 weeks, the detection rate using this three-marker combination can be as high as 89 percent at a 5 percent false-positive rate.²⁵ Screening in one-stop clinics^26,27 (sometimes referred to as OSCAR, one-stop clinics for early assessment of fetal risk) where both biochemical screening and ultrasound are completed within a one-hour visit, confirms the high level of detection using such an approach.

PAPP-A, when measured in the second trimester, shows results in trisomy 21 cases that are very similar to those in normal pregnancies.^28,29 This change in the clinical discrimination of PAPP-A between the first and second trimester is an example of a relatively unappreciated phenomenon of the temporality of marker levels.³⁰ It is now clear that the clinical discrimination of all biochemical markers changes across the first and second trimester. For PAPP-A, large-scale studies have shown an increasing linear trend of the median MoM across the first and second trimester in pregnancies with trisomy 21.³⁰ Similar temporality explains why total hCG is a poor first-trimester marker but an adequate second-trimester marker. Free b-hCG, on the other hand, has a relatively stable median MoM from 10 to 18 weeks, but prior to 10 weeks the median levels fall.³⁰ Thus, while the best clinical discrimination for PAPP-A may be as early as 8 weeks, the clinical discrimination for free b-hCG during the early weeks of pregnancy is poor (median close to 1.2 MoM). Consequently, the optimum time for measuring both PAPP-A and free b-hCG together is in the first trimester between 10 and 13 weeks—approximately the time frame when NT should be measured (11 to 14 weeks).²⁵ This type of screening can be readily accomplished in a one-stop clinic.^26,27

Low serum PAPP-A is not just an indicator of trisomy 21. In cases of trisomy 13 and trisomy 18, levels of PAPP-A are also reduced in the first trimester.^31,32 When used in conjunction with free b-hCG (when levels are reduced to around 0.3 MoM) and NT (when levels are increased), suitable algorithms can detect 90 percent of trisomy 13 and 18 cases at a 1 percent false-positive rate.³³ PAPP-A levels are also very low in triploidy cases.³⁴

PAPP-A serum levels remain low into the second trimester in cases of trisomy 18. Currently, PAPP-A may be its best biochemical marker.³⁹ It has been suggested that a two-stage screening program employing PAPP-A as a second-line test could identify 80 percent of trisomy 18 cases at a 0.1 percent false-positive rate.^35,36

Pregnancy complications
Much of the initial work with PAPP-A in the early 1980s was based on the finding that low levels of PAPP-A were associated with poor fetal viability.³⁷ This correlation is being demonstrated in prenatal screening programs, as low serum PAPP-A values in the absence of an ultrasound examination raise suspicions of fetal death.³⁸ Studies have also shown that lower maternal serum PAPP-A is associated with women who subsequently miscarry, develop pregnancy induced hypertension and develop growth restriction.^39,40 The clinical sensitivity and specificity of PAPP-A is low; hence, PAPP-A is unlikely to be a useful predictor of subsequent pregnancy complications.

Cornelia de Lange syndrome
Cornelia de Lange syndrome is a developmental malformation characterized by mental and growth developmental delay, limb reduction abnormalities, a distinct facial appearance and congenital heart defects. The incidence is estimated at 1 in 40,000 births with a 1 percent recurrence risk. Case reports from the early 1980s indicated low levels of PAPP-A in maternal serum collected between 20 and 35 weeks. More recently, in an analysis of 19 cases, Aitken et al.⁴¹ were able to confirm low PAPP-A levels (median MoM 0.21) in the second trimester, and produced estimated odds of an affected fetus based on PAPP-A levels.

PAPP-A as a marker of acute coronary syndromes
A potential new use for PAPP-A measurement has been suggested as a result of PAPP-A identification in atherosclerotic plaques and corresponding increases in circulating PAPP-A levels.¹³ In this context, high-sensitivity assays are required. One study,¹³ which used a threshold level of 10 mIU/L (i.e. 2,000 times less than levels in an 11-week pregnancy), showed that sensitivity and specificity for identification of acute coronary syndromes were 89.2 and 81.3 percent, respectively. The sensitivity was 94.1 percent in cases of myocardial infarction, and 85 percent in cases of unstable angina. Further studies are required to assess the value of PAPP-A for cardiac conditions.

Optimization of PAPP-A assays
Early assays for PAPP-A were either RIAs employing purified and labeled PAPP-A with a short shelf-life and questionable specificity or ELISAs employing polyclonal antibodies. The specificity of the polyclonal antibodies has been a source of controversy for several years. A widely used commercial polyclonal antibody (DAKO) has been shown to crossreact with SP1, proMBP and haptoglobin,⁴² which may have led to underestimates of the clinical utility of PAPP-A.^43,44 Now that robust commercial monoclonal assays are available for PAPP-A, the problem of assay specificity has most likely been resolved, although lack of a suitable International Reference Preparation for assay standardization is hindering attempts to reconcile values from different assay systems. (International organizations such as the WHO are currently evaluating new materials that may become the future standard.)

PAPP-A is relatively stable at room temperature, with levels being unchanged after a period of 7 days. Similarly, freezing and thawing up to five times has no effect on serum levels. For screening purposes, serum is the best sample medium. Collection into EDTA tubes results in the removal of zinc moieties at the center of the PAPP-A molecule.⁴⁵ This produces a conformational change in the protein, making it almost invisible in most assay systems. Collection into heparin also results in lower PAPP-A values.⁴⁵

Conclusion
The clinical value of PAPP-A continues to grow as new data become available. While its established utility as a risk assessment tool for fetal abnormalities is recognized throughout Europe, the latest findings suggest that PAPP-A may also be predictive of cardiovascular events. The versatility of this analyte is still in the throes of debate, but, without doubt, it has already proved deserving of the attention granted by investigators and clinicians.

References
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