Significance of pregnancy-associated plasma protein A ( PAPPA ) concentration determination in the assessment of final outcome of pregnancy

Background/Aim. Pregnancy-associated plasma protein A (PAPP-A) is high molecular matrix metalloproteinase originally isolated in the serum of pregnant women. The aim of this study was to analyze the values of concentration of PAPP-A in assessment of progress and outcome of pregnancy in pregnant women diagnosed with threatening preterm delivery, preeclampsia and fetal growth restriction in relation to physiological pregnancy of the same gestational age. Methods. The study included 60 pregnant women that were divided into three groups according to gestational age and the diagnosis of imminent premature birth upon reception, preeclampsia and fetal growth restriction as follows: the group I from 28 to 32 weeks of gestation, a total of 25 pregnant women, the group II from 33 to 36 weeks of gestation, a total of 23 pregnant women, and the group III from 37 to 41 weeks of gestation, a total of 12 pregnant women. The control group consisted of 60 pregnant women without complications of pregnancy that were identically divided into three groups according to gestational age as in the sample. We performed quantitative determination of PAPP-A from the venous blood of patients by using commercial tests of the company Diagnostics Product Corporation (DPC), Los Angeles, California, USA. Results. There was a statistically significant difference in PAPP-A values in the examined groups in all gestational ages (p < 0.01). The value of the PAPP-A concentration in different gestational ages with equal statistical significance indicated the possibility of complications, which was examined during pregnancy in relation to the control group of pregnant women with physiological pregnancies. This study confirmed that there was a statistically significant difference in fetal body weight at birth (p < 0.05), Apgar score in 5 min after birth (p < 0.05), and gestational age at birth (p < 0.05), as parameters of the outcome of pregnancy course, between the examined groups of pregnant women in relation to the value of PAPP-A concentration. The age of pregnant women was not statistically different in the examined groups (p > 0.05). Conclusion. Differences in PAPP-A concentration should point out to the obstetrician the need for more intensive antepartum fetal surveillance in order to increase the chances of favorable perinatal outcome, regardless gestational age.

nia, USA.Results.There was a statistically significant difference in PAPP-A values in the examined groups in all gestational ages (p < 0.01).The value of the PAPP-A concentration in different gestational ages with equal statistical significance indicated the possibility of complications, which was examined during pregnancy in relation to the control group of pregnant women with physiological pregnancies.This study confirmed that there was a statistically significant difference in fetal body weight at birth (p < 0.05), Apgar score in 5 min after birth (p < 0.05), and gestational age at birth (p < 0.05), as parameters of the outcome of pregnancy course, between the examined groups of pregnant women in relation to the value of PAPP-A concentration.The age of pregnant women was not statistically different in the examined groups (p > 0.05).Conclusion.Differences in PAPP-A concentration should point out to the obstetrician the need for more intensive antepartum fetal surveillance in order to increase the chances of favorable perinatal outcome, regardless gestational age.

Introduction
Pregnancy-associated plasma protein A (PAPP-A) is high molecular matrix metalloproteinase originally isolated in serum of pregnant women.PAPP-A is a glycoprotein, macroglobulin, of molecular weight of 800,000 with alpha 2electrophoretic mobility, and it is produced in syncytiotrophoblast cells of the placenta 1 .Determination of PAPP-A is performed by radioimmunoassay method (immune test with isotope).The first radioimmunoassay determination of PAPP-A was carried out in 1980.Using RIA method it is possible to determine its presence already 3-4 weeks after conception and no later than the 6th week of gestation.The maximum level PAPP-A has at the term delivery.PAPP-A exerts an inhibitory effect on the enzyme elastase, a protease located in the granules of neutrophils granulocytes and participates in processes that lead to the destruction of proteins.By direct immunofluorescence, the presence of PAPP-A in spermatozoid's heads is determined at about 2%.PAPP-A exerts an inhibitory effect on fixation of both complements and coagulation system, as well as on the affinity to heparin.It is assumed that suppressed level of PAPP-A reduces the zinc ion that is required in the fetal organogenesis, which represents one of the factors for the occurrence of congenital malformations.During pregnancy, PAPP-A concentration in maternal blood increases.Decreased concentration is related to increased incidence of chromosomal abnormalities in early gestation and in later pregnancy course because of the associated placental insufficiency.It is characterized by the appearance of fetal growth restriction, preeclampsia, preterm delivery and stillbirth.PAPP-A is a regulator of bioactivity of insulin-like growth factor 2 .Testing of the role of PAPP-A in other tissues of the organism has started recently.Increased values of PAPP-A were found in patients with acute coronary syndrom in contrast to healthy population and those with a diagnosis of stable angina pectoris.It is important to mention that PAPP-A, which is in circulation of patients with coronary disease is circulating in free form, whereas in pregnant women a complex of PAPP-A and the proform of eosinophil major basic protein is present [3][4][5] .This brings into question the adequacy of the used substrates that were synthesized for the detection of complex form of PAPP-A.PAPP-A represents a useful biomarker in clinical monitoring of pregnancy course.However, new prospective studies are needed by using appropriate substrates for the detection of PAPP-A in order to assess the proper role of metalloproteinase in clinical practice.The aim of this study was to analyze the value of PAPP-A concentration in assessing the final outcome of pregnancy in pregnant women diagnosed with threatening preterm delivery, preeclampsia and the fetal growth restriction in relation to physiological pregnancies of the same gestational age.

Methods
A prospective, observational study was conducted at the Gynecology and Obstetrics Clinic, Clinical Center Kragujevac, Kragujevac, Serbia, in 2010.During examination the clinical-experimental model of study was used.Quantitative measurements of PAPP-A levels were determined from venous blood of patients using the commercial tests of the company Diagnostic Product Corporation (DPC), Los Angeles, California, USA (DPC-USA).The tests, based on an analytical principle of immunochemiluminescence, were implemented using the automated analyzer Immulite 2000.The manufacturer of the analyzer is also DPC-USA.
The study included 60 pregnant women that were divided into three groups according to gestational age and the diagnosis of imminent premature birth upon reception, preeclampsia and fetal growth restriction as follows: the group I, from 28 to 32 weeks of gestation, a total of 25 pregnant women; the group II, from 33 to 36 weeks of gestation, a total of 23 pregnant women; the group III, from 37 to 41 weeks of gestation, a total of 12 pregnant women.
The criterion for inclusion of pregnant women in the study included the previously established all three diagnoses that were listed as complications of pregnancy course according to the following standards: preterm delivery before the end of 37th week of pregnancy; the diagnosis of preeclampsia based upon the blood pressure above 140/90 mmHg, proteinuria in 24 hour urine of 0.3 g / per day; intrauterine growth restriction (IUGR) of fetus was diagnosed on the basis of ultrasonographic growth parameters: biparietal diameter (BPD), transverse trunk diameter (TTD), head circumference (HC), abdominal circumference (AC), femur length (FL) and differences in the measured parameters below the 10th percentile than expected for gestational age.The control group consisted of 60 pregnant women without complications of pregnancy that were identically divided into three groups according to gestational age as in the sample.All the obtained results of research were entered into a single database with valid logic control.Statistical analysis included calculating the average values and standard deviations (SD) for each numerical parameter and analysis of the obtained value in relation to the subgroups (t-test, Mann-Whitney) by using the statistical software SPSS 17.

Results
There was a statistically significant difference of PAPP-A values in the examined groups in all gestational ages (p < 0.01) (Table 1 and 2 and Figure 1).The mean values and standard deviations of PAPP-A concentration (mU/mL) in a total sample of pregnant women diagnosed with threatening preterm delivery, preeclampsia and intrauterine growth restriction were shown in Table 3.
It is found that pregnant women of 28-32 gestational weeks diagnosed with threatening preterm delivery and preeclampsia, showed significantly lower values PAPP-A than in healthy pregnant women ( p = 0.001).
Pregnant women of 33-36 gestational weeks diagnosed with threatening preterm delivery and preeclampsia, showed significantly lower values of PAPP-A than in healthy pregnant women (p = 0.01) (Table 3 and 4).
Pregnant women at term and the diagnosis of preeclampsia, show significantly lower values of PAPP-A than healthy pregnant women (p = 0.01).Healthy pregnant women at 28-32 gestational weeks, showed significantly higher values than pregnant women diagnosed with preeclampsia and intrauterine growth restriction in the same gestational age.The same comment goes for the t-test in pregnant women of 33-36 gestational weeks and for a group of pregnant women with normal term pregnancies (Tables 3  and 4).

Discussion
The aim of this study was to determine the relative risk for preeclampsia and intrauterine growth restriction at different PAPP-A levels in different gestational ages.According to the literature data, low levels of PAPP-A during the first trimester are associated with the occurrence of preeclampsia later in the pregnancy 6 .PAPP-A levels in maternal serum between 11th and 13th week of gestation in 224 singleton pregnancies, which later developed preeclampsia, were compared to those of 47,770 normal singleton pregnancies that resulted in live born children after 37 weeks of gestation with body weights at birth greater or equal to the 10th percentile in physiological pregnancy 7 .Correlation between the level of this enzyme and the incidence of preeclampsia was estimated by comparing the relative concentration of PAPP-A at different gestation.
In the preeclampsia group, the median PAPP-A MoM was significantly reduced (0.772 MoM, p < 0.0001).With decreasing level of PAPP-A, a probability ratio for preeclampsia was growing.At the 5th percentile of the normal (PAPP-A MoM 0.415), the probability rate was increased 4 times 8,9 .In our sample there was a statistically significant difference in values of PAPP-A in the examined groups at all gestational ages (p < 0.01).We showed that the value of PAPP-A concentration in different gestational ages with equal statistical significance indicates the possibility of complications examined during pregnancy course in relation to the control group of pregnant women with normal pregnancies.A probability factor of preeclampsia on any of PAPP-A MoM levels we consider useful in advising women with low levels of PAPP-A.The use of low PAPP-A in the prediction of preeclampsia and growth restriction for selection of women who will be suggested an intensive surveillance of pregnancy and therefore significantly reduce the incidence and mortality morbidity of mother as well as fetus.PAPP-A is a "protease" for insulin-like growth factor binding proteins 4 and 5.This means that it has the ability to help release insulin-like growth factor from these proteins so that they can freely interact with their cellular receptors.It is considered that insulin-like growth factor plays an important role in trophoblast invasion and hence in the early development and vascularization of the placenta 10,11 .These early events in the formation of the placenta are extremely important for the outcome of pregnancy, and when abnormal, they are associated with miscarriage, fetal growth restriction, hypertensive disorders induced by pregnancy (preeclampsia), fetal death or preterm delivery.It is assumed that low levels of PAPP-A, leading to reduced release of insulin-like growth factor, could be a path to placentation abnormalities, culminating in the adverse outcomes of pregnancy.Spencer et al 8 in their study on 54,722 normal singleton pregnancies examined the role of PAPP-A in the course of pregnancy.At the 5th percentile of PAPP-A (0.415 MoM), the probability rate for the fetus loss before 24 weeks was increased 3.3 times and above 24 weeks 2.8 times.In other words, there was three times increased risk of fetal loss with low levels of PAPP-A.Cowans and Spencer 11 have recently confirmed a link between low PAPP-A and low fetal weight at birth in relation to the expected for gestational age.In their research they found a linear association of fetal growth restriction and reduced level of PAPP-A, in other words, the lower level of PAPP-A, the lower level of fetal birth weight of any gestational age 12 .
Several other studies confirm the association of other "complications of pregnancy" listed above with low levels of PAPP-A [13][14][15] .For example, as additional results of risk assessment in the first and second trimester (FASTER) study, it was found that women with concentration of PAPP-A below the 5th percentile" were significantly more likely to experience fetal loss before or at the 24th week, low fetal weight at birth, preeclampsia, gestational hypertension, preterm delivery (p < 0.001), stillbirth, preterm premature rupture of fetal membranes and placental abruption (p < 0.02) 16 .
Our research confirmed the allegations of these studies since we found statistically significant difference in body weight of the fetus at birth (p < 0.05), Apgar score 5 minutes after birth (p < 0.05), and gestational age at the time of delivery (p < 0.05), as parameters of the final pregnancy outcome between these groups of pregnant women in relation to the value of the concentration of PAPP-A The age of pregnant women in our study was not statistically different in the examined groups (p > 0.05).Despite this association, the positive predictive value of low level of PAPP-A for one of these outcomes is still relatively low.

Conclusion
PAPP-A concentration in the pregnant women of 28-36 gestational weeks had significantly lower values with the diagnosis of preterm delivery and preeclampsia, than in the control group.PAPP-A concentration in the pregnant women diagnosed with preeclampsia in term pregnancy was significantly lower than in he healthy pregnant women at term delivery.PAPP-A concentration is significantly higher in physiological pregnancies of 28-36 gestational weeks compared to the concentrations in pregnant women diagnosed Lon ar D, et al.Vojnosanit Pregl 2013; 70(1): 46-50.

Table 1 Pregnancy-associated plasma protein A (PAPP-A) in the examined pregnant women according to fetal age
p <