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ORIGINAL RESEARCH |
From the Department of Biomedical Science and Advanced Therapy, Section of Obstetrics and Gynecology, Department of Biology, Section of Evolutionary Biology, and Department of Clinical and Experimental Medicine, Section of Nuclear Medicine, University of Ferrara, Ferrara, Italy; and Department of Chemical Endocrinology, University Hospital Nijmegen St Radboud, Nijmegen, The Netherlands.
Address reprint requests to: Adriano Piffanelli, MD, Department of Clinical & Experimental Medicine, University of Ferrara, Section of Nuclear Medicine, Via L. Borsari, 46, 44100 Ferrara, Italy, E-mail: pif{at}dns.unife.it
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Abstract |
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Methods: The study population consisted of 123 single pregnancies at the 17th gestational week, 16 with minor chromosomal abnormalities, 15 aneuploid, and 92 euploid.
Results: Both groups with chromosomal abnormalities had significantly higher serum levels of urokinase plasminogen activator and its complexed form with its type-1 inhibitor compared with euploid pregnancies. In AF, tissue plasminogen activator was significantly lower in the aneuploid than the euploid group, whereas type-1 inhibitor of plasminogen activator was significantly higher in the cases with minor chromosomal abnormalities compared with euploid. At cutoff levels set at 100% sensitivity, the complexed form of urokinase plasminogen activator with its type-1 inhibitor had the strongest specificity (66.3%); after logarithmic transformation, its serum level was 7.53 times higher in aneuploidies than euploidies.
Conclusion: Aneuploid pregnancies appear to be accompanied by abnormalities of the plasminogen activation system, which could lead to impaired placental perfusion and thus to abortion, fetal death, and fetal growth restriction.
The components of the plasminogen activator system are involved in many physiologic and pathologic processes, such as fibrinolysis, ovulation, embryo implantation, and tumor invasion. The functions of the two known plasminogen activators, ie, tissue-type and urokinase-type plasminogen activator, are controlled by two inhibitors, PAI-1 and PAI-2. The former inhibitor has been purified from endothelial cells, smooth muscle cells, lung fibroblasts, hepatocytes, and several transformed cell lines, the latter from placenta, leukocytes, and histiocytic lymphoma cells.1
Several studies have shown that the plasminogen activator system is involved in normal pregnancy. The levels of both activators and their inhibitors are elevated in maternal serum and return to normal soon after delivery. Despite those wide variations, during pregnancy overall plasmatic fibrinolitic activity remains substantially unmodified.2–5
In severe preeclampsia, plasma levels of PAI-1 increase and PAI-2 decrease compared with normal pregnancies.3 An association of fetal growth restriction (FGR) with lower PAI-1 and PAI-2 levels has been reported,5 and alterations in the plasminogen activator system have been found also in gestational trophoblastic disease6 and gestational diabetes.7
It has been hypothesized that vasculopathy could be a pathogenic mechanism possibly in association with other factors that leads to abortion and fetal malformation,8 which are two features of aneuploidy. That hypothesis would be supported by finding differences in behavior of factors involved in angiogenesis, to which the plasminogen activator system belongs, in aneuploid compared with normal pregnancies. The aim of the present study was to measure plasminogen activators, inhibitors, and their complexes in maternal serum and amniotic fluid (AF) during the second trimester of euploid and aneuploid pregnancies.
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Materials and Methods |
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The study population was divided into the following three groups (Table 1
): euploid (n = 92), minor chromosomal abnormality (n = 16), aneuploid (n = 15). Our multiapplicable enzyme-linked immunosorbent assay (ELISA) is suitable for measuring urokinase-type plasminogen activator, PAI-1, tissue-type plasminogen activator, and PAI-2, and complexes urokinase-type plasminogen activator/PAI-1 and tissue-type plasminogen activator/PAI-1 in a single experimental setup.9 This type of ELISA avoids interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in plasma and serum.10
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All measurements were done in duplicate. Standard curves were approximated as polynomials Y = a + bX + cX2, in which X stands for analyte concentration and Y for optical density. The polynomial coefficients a, b, and c were calculated by the least squares method. Statistical analyses were done with the SPSS statistical software package 9.0.0 (SPSS, Chicago, IL). Variables had normal distributions only after logarithmic transformation.
Linear correlation analysis was done to assess whether levels of the components in AF depended on levels in plasma. A one-way analysis of variance with Tukey-HSD test for post-hoc comparison was done to assess differences in levels among the three groups of patients, subdivided as described in Table 1
. Power analysis was done with the method of Pearson and Hartley, as described by Sokal and Rohlf.12 To assess predictive values of the components of interest, ie, the likelihood that a women who shows altered levels of one of the components of the plasminogen activation system or their complexes actually has a fetus with chromosomal abnormalities, the Pearson 
2 test was used.
Significance levels for the rejection of the null hypothesis for all tests were set at P < .05. Data are expressed as mean ± standard deviation (SD) of the variable before the logarithmic transformation in tables. Box and whisker plots are used to summarize distribution of the variable before logarithmic transformation. In those plots the smallest box in each plot represents the mean of the variable, whereas the dispersion of the parameter is represented by ±1 times the standard error (large box), and the whisker around the box indicates 95% confidence interval (CI) of the mean.
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Results |
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2 test indicated that only urokinase-type plasminogen activator and urokinase-type plasminogen activator:PAI-1 in maternal serum had significant predictive value for aneuplodies and minor chromosomal abnormalities (Table 3). That test indicated that a level of urokinase-type plasminogen activator:PAI-1 in maternal serum above 1.16 ng/mL had a specificity of 66.3% at 100% sensitivity in predicting an aneuploid pregnancy. The amount of urokinase-type plasminogen activator in maternal serum was a little less specific for predicting aneuploid pregnancies. At 100% sensitivity, a level above 1.28 ng/mL had a specificity of 52.2%. The sensitivity of those two indices for predicting pregnancies with minor chromosomal abnormalities was lower. A level of urokinase-type plasminogen activator:PAI-1 in maternal serum above 0.95 ng/mL had a specificity of 57.6% at 100% sensitivity, and urokinase-type plasminogen activator levels above 1.13 ng/mL a specificity of 33.7% (Table 3).
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Discussion |
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The results of our study have several pathophysiologic and clinical implications. Pregnancy can be interpreted as a unique vascular event that is under endocrine control. The essence of that event is on the maternal side and must be considered differently than the fetal side. The lack of significant correlation between the analyte concentrations in AF and maternal serum affirms that contention. Whereas de novo formation of vessels is essential for embryoplacental development, in the mother an extensive change in the preexistent vasculature takes place as a consequence of trophoblastic invasion. In the first trimester, degeneration of the internal elastic lamina occurs, with denudation of smooth muscle and elastin in the inner and outer media of the decidual segment of uterine spiral arteries. The architecture of the vessel wall is replaced with hyaline and fibrin, in which trophoblastic cells are embedded. During the second trimester the process is extended into the myometrial segment of the arteries. As a consequence of such profound structural change, maximal blood flow at low resistance is allowed to perfuse the placenta. In preeclampsia, impairment of that process occurs in the early stages of placentation.13 Therefore, supported by the observation of increased incidence of preeclampsia in pregnancies with certain types of fetal malformations,8 it has been hypothesized that inadequate perfusion, whatever the underlying pathogenesis, could be a common ground for abortion, malformation, FGR, and preeclampsia. It is true especially when a chromosomal abnormality exists, depending on the grade and time of onset. For that reason, we are in search of AF and serum analytes that could support the hypothesis of vascular damage in those complications of pregnancy. In that regard, we have previously reported that endothelin-1, a potent vasoconstrictor peptide whose maternal plasma level is increased in preeclampsia, is also significantly higher in AF in fetal aneuploidy.14
In trisomies, growth restriction and smaller placentas have been described.15 The cause of the growth restriction is related possibly to hypovascularity or focal hypervascularity of villi16 and reduced muscular artery counts.17
In human vessels it was shown recently that urokinase induces vascular smooth muscle cell proliferation.18 Therefore, it appears that if the same happens in spiral arteries during early pregnancy, an increase in urokinase-type plasminogen activator above physiologic levels could lead to hyperplastic proliferation of the muscular component of the wall, rather than its weakening, thus impairing placental perfusion.
For the above considerations, our reported increase of maternal plasma urokinase-type plasminogen activator in aneuploid pregnancies might be related to changes in vascularity. The same should be true for urokinase-type plasminogen activator-PAI-1 complex. Binding of uPA by PAI-1 abrogates its proteolytic capacity, whereas this complexed form is still involved in other processes, eg, endometrial angiogenesis.19 We believe the alteration of the plasminogen activator system shown in our data might represent the basis for an impaired perfusion of the trophoblast, which with other factors, might lead to abortion, fetal malformation, and FGR.
A further implication of our results concerns biochemical prenatal screening of aneuploidies. A better performance of current screening methods in detection rate and cost-benefit ratio is needed. In that context, although our population was not representative, our data show a significant capacity of urokinase-type plasminogen activator, particularly in its complex form with PAI-1, for detecting aneuploidies. The mean urokinase-type plasminogen activator and urokinase-type plasminogen activator-PAI-1 serum levels were 2.34 and 7.53 times higher, respectively, in aneuploidies than in normal pregnancies.
The differences in maternal serum levels of components of the urokinase-type plasminogen activator system between euploid and aneuploid pregnancies reported here suggest an association between the plasminogen activator system and fetal chromosomal abnormalities. Based on our results, urokinase-type plasminogen activator in maternal serum, especially in its complexed form with PAI-1, appears particularly suitable for further clinical investigation to verify its potential for improving the detection rate of biochemical screening of aneuploidies.
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Footnotes |
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Received July 31, 2000. Received in revised form October 16, 2000. Accepted October 26, 2000.
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References |
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2. Shimada H, Takashima E, Sona M, Murakami M, Maeda Y, Kasakura S, et al. Source of increased plasminogen activators during pregnancy and puerperium. Thromb Res 1989;54:91–8.[Medline]
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9. Grebentchtikov N, Sweep CGJ, Geurts A, Andreasen P, de Witte H, Schousboe S, et al. ELISA for complexes of urokinase-type and tissue-type plasminogen activators with their type-1 inhibitor (uPA/PAI-1 and tPA/PAI-1). Int J Cancer 1999;81:598–606.[Medline]
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11. Sweep CGJ, Geurts-Moespot A, Grebentchtikov N, de Witte JH, Heuvel JJTM, Duffy MJ, et al. External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type inhibitor (PAI-1) in human breast cancer tissue extracts. Br J Cancer 1998;78:1434–41.[Medline]
12. Sokal RR, Rohlf FJ. Biometry. 3rd ed. New York: WH Freeman and Company, 1995.
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18. Kause SM, Benzakour O, Kantha C, Kost C, Lijnen HR, Preissner KT. Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders. Arterioscl Thromb Vasc Biol 1997;17:2848–54.
19. Sanderg T, Casslen B, Gustavsson B, Benraad TJ. Human endothelial cell migration is stimulated by urokinase plasminogen activator: Plasminogen activator inhibitor 1 complex released from endometrial stromal cells stimulated with transforming growth factor beta 1; possible mechanism for paracrine stimulation of endometrial angiogenesis. Biol Reprod 1998;59:759–67.
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