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ORIGINAL RESEARCH |
From the Division of Maternal-Fetal Medicine, The University of Texas Medical Branch, Galveston, Texas; and The University of Nebraska Medical Center, Omaha, Nebraska.
Address reprint requests to: Hassan M. Harirah, MD, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0587; E-mail: hmharira{at}utmb.edu.
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ABSTRACT |
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METHODS: Serum samples from 22 pregnant women diagnosed with HELLP syndrome were compared with sera from 37 healthy women with noncomplicated singleton pregnancies. Serum levels of soluble Fas and soluble Fas ligands were determined by enzyme immunoassay. Student t, 
2, Pearson’s correlation coefficient, and multiple regression tests were used for statistical analyses.
RESULTS: Both soluble Fas and soluble Fas ligand were detected in the sera of normal pregnancies as well as in those with HELLP syndrome. The mean serum level of soluble Fas was significantly higher in women with HELLP syndrome than in healthy gravidas (10.75 ± 0.93 versus 5.81 ± 0.37 U/mL, P < .001). However, there was no significant difference in mean serum soluble Fas ligand levels of the two groups (0.60 ± 0.06 compared with 0.50 ± 0.22 ng/mL, P = .23). In women with HELLP syndrome, there were no significant correlations between serum levels of soluble Fas or soluble Fas ligand with liver transaminases (aspartate and alanine aminotransferase) and platelet count.
CONCLUSION: Serum levels of soluble Fas, but not soluble Fas ligand, are significantly higher in women with HELLP syndrome than healthy gravidas. The source of elevated serum levels of soluble Fas in HELLP syndrome remains to be determined
The syndrome of hemolysis, elevated liver enzymes, and low platelets (HELLP) is a variant of severe preeclampsia, which carries statistically significant perinatal risks to both mother and fetus.1 Elevation of liver transaminases in HELLP syndrome reflects marked derangement in hepatic functions and integrity. HELLP syndrome also shares many clinical and laboratory characteristics with systemic inflammatory response syndrome and disseminated intravascular coagulation.2 The pathogenesis of hepatic damage in cases of severe preeclampsia and HELLP syndrome, in particular, is not fully understood. Autopsy of fatal cases of HELLP syndrome showed periportal hemorrhagic necrosis in the periphery of the liver lobules. However, such lesions are seldom identified by liver biopsy in nonfatal cases.3
Apoptosis or programmed cell death is a requisite physiologic process for normal development and reproductive function in a number of mammalian tissues. It is a process through which individual cells carry out a suicide program in otherwise healthy organs or tissues. The Fas-Fas ligand system is one of the best-studied death systems that can mediate apoptosis. Fas, a member of the tumor necrosis factor receptor superfamily, is a 36 to 45 kDa type I surface protein containing a single transmembrane region and induces apoptosis by Fas-Fas ligand binding.4 Fas ligand is a 40 kDa type II transmembrane protein of the tumor necrosis factor receptor family.5 Soluble Fas (sFas) is produced as the form lacking 21 amino acid residues of the transmembrane domain by alternative splicing of Fas mRNA.6 It protects the cells from Fas-mediated apoptosis by binding to Fas ligand and consequently preventing it from stimulating Fas receptors on cell membranes.7 Soluble Fas ligand, a 26 kDa, is generated by metalloproteases proteolytic cleavage on the membrane-bound form of Fas ligand protein. It is functionally active against cells that are highly sensitive to Fas-mediated apoptosis; however, it has less apoptosis-inducing activity than membrane-bound Fas ligand.8
Apoptosis has recently been reported to occur in normal hepatocytes and in liver damage associated with hepatitis B and C. This was shown by increased expression of Fas and Fas ligand in liver tissue.9 Apoptosis has also been found to be involved in different inflammatory and immune disorders. Our objective was to assess whether serum levels of sFas and sFas ligand are altered in HELLP syndrome. We compared the levels of sFas and sFas ligand in the sera of patients with HELLP syndrome and healthy gravidas.
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MATERIALS AND METHODS |
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= .05. After explaining the study protocol and signing an informed consent, we obtained blood samples from 22 pregnant women complicated with HELLP syndrome. These samples were collected when the diagnosis of HELLP syndrome was clinically determined (rising transaminase concentration and decreasing platelet count) and before the onset of spontaneous or induced labor. Control samples were obtained from 37 healthy gravidas with singletons who had no history of preexisting medical problems. None of these women developed hypertension during the remainder of their pregnancies and all delivered at term.
We collected 5 mL of blood in a sterile vacuum tube and incubated at 37C for 30 minutes to allow clot to form. Serum was separated by centrifugation at 1500 g/minute for 10 minutes at 4C and kept at -80C until the assay was performed. Levels of sFas and sFas ligand were measured by enzyme-linked immunoassays (Oncogene Research Products, Cambridge, MA). The lower limit of detection of this assay was 0.05 U/mL for sFas and 0.02 ng/mL for sFas ligand. The interassay and intraassay coefficients of variation were less than 10%. In the assay, mouse monoclonal antibodies against human Fas and Fas ligand were used to immobilize Fas and Fas ligand proteins onto the surface of the plastic wells. Biotinylated monoclonal antibodies for Fas and Fas ligand were used to detect the respective proteins. Horse-radish peroxidase was used to catalyze the conversion of the colorless chromogenic substrate tetra-methylbenzidine to a colored solution. Levels were quantified by measuring the spectrophotometric absorbance at dual wavelengths of 450 and 595 nm. Maternal liver transaminases (alanine and aspartate aminotransferase), platelet count, and uric acid were measured when the diagnosis of HELLP syndrome was determined.
Student t test was used for comparison of serum levels of sFas, sFas ligand, aspartate aminotransferase, alanine aminotransferase, platelet count, and uric acid between the two groups. 2 was used for analysis of race, gravidity, and parity. Pearson’s correlation coefficient test was used to analyze the correlation among serum levels of sFas and sFas ligand with the transaminases, platelet count, and uric acid. Multiple regression analysis was performed to adjust for maternal and gestational ages. Data are expressed as means ± standard error of the mean. P < .05 was considered statistically significant.
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RESULTS |
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). In normal pregnancies, serum sFas levels correlated with gestational age (r = 0.26, P = .04) but not maternal age (r = 0.07, P > .05). In contrast, sFas ligand levels did not correlate significantly with either gestational (r = -0.06, P > .05) or maternal (r = -0.28, P > .05) ages. In women with HELLP syndrome, the correlations between either serum levels of sFas or sFas ligand and gestational age, maternal age, the two transaminases, and platelet count were not significant. The only significant correlation was between serum sFas and uric acid (r = 0.49, P = .03) (Table 2).
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). However, there was no difference in the serum levels of sFas ligand between the two groups (0.60 ± 0.06 versus 0.50 ± 0.22 ng/mL, P = .23) (Figure 2). In normal pregnancies, significant inverse correlation was noted between serum levels of sFas and sFas ligand even after adjustment for gestational and maternal ages (r = -0.34, P = .04). However, no significant correlation was noted in women with HELLP syndrome (r = 0.35, P = .13).
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DISCUSSION |
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Our finding of elevated serum levels of sFas in HELLP syndrome was consistent with other reports of conditions associated with liver damage such as hepatitis and systemic inflammatory syndromes. In these reports, both serum levels of sFas and sFas ligand were elevated. However, these conditions were associated with more organ damage or failure.6 We did not find a significant change in serum levels of sFas ligand in cases of HELLP syndrome. This lack of change could be explained by the lesser severity of liver damage in HELLP syndrome compared with other disorders. However, our data were consistent with other reports of an absence of correlation between serum levels of sFas and sFas ligand and liver aspartate and alanine transaminases.10 The only correlation we found was between serum levels of sFas and uric acid. This could be due to the decrease of renal perfusion and filtration associated with the disease process, leading to elevated serum levels.
Despite the noted absence of correlation between sFas and sFas ligand with any of the laboratory evidence of hepatic cell damage, hepatocytes may be one of the sources of elevated soluble Fas in women with HELLP syndrome. One explanation is that release of soluble forms of Fas and Fas ligand, protectors against apoptosis, precedes the actual damage of liver cells. Another explanation is that the samples of HELLP syndrome were obtained before significant alteration of liver integrity occurred. Other possible sources of these apoptosis-related molecules include maternal vascular endothelial cells, neutrophils, and trophoblasts. Fas and Fas ligand have recently been detected in the vessel walls, particularly in endothelial cells. In vitro studies also documented the upregulation of Fas expression by endothelial cells when stimulated with tumor necrosis factor-.11 Recent data indicated that delayed maternal neutrophil apoptosis occurs in normal pregnancy and preeclampsia.12 This was explained by release of intracellular presynthesized Fas and Fas ligand molecules from maternal neutrophils.13 Trophoblasts could also be one of the sources. Several studies documented Fas and Fas ligand expression in the uteroplacental unit.14 Other reports showed expression of Fas-Fas ligand system in different gestational tissues.15–17
Although the role of sFas and sFas ligand maternal serum are not well identified, the elevation of sFas may have a clinical value in management of HELLP syndrome. Longitudinal study is warranted to determine the potential of sFas levels in maternal sera for predicting HELLP syndrome. Further studies are also needed to localize the sources of these apoptosis-related molecules.
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Footnotes |
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Received November 13, 2000. Received in revised form March 21, 2001. Accepted March 30, 2001.
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REFERENCES |
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2. Bone RC. Immunologic dissonance: A continuing evolution in our standing of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann Intern Med 1996;125:680–7.
3. Barton JR, Riely CA, Adamec TA, Shanklin DR, Khoury AD, Sibai BM. Hepatic histopathologic condition does not correlate with laboratory abnormalities in HELLP syndrome (hemolysis, elevated liver enzymes and low platelet count). Am J Obstet Gynecol 1992;167:1538–43.[Medline]
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6. Itoh N, Yonehera S, Ishii A, Yonehera M, Mizushima SI, Sameshima M, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991;66:233–43.[Medline]
7. Nagata S, Goldstein P. The Fas death factor. Science 1995;267:1449–56.
8. Hori Y, Wada H, Mori Y, Shimura M, Hiyoyama K, Nakasaki T, et al. Plasma soluble Fas and soluble Fas ligand levels in patients with thrombotic thrombocytopenic purpura and in those with disseminated intravascular coagulation. Am J Hematol 1999;61:21–5.[Medline]
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12. Dadelszen PV, Watson RWG, Noorwali F, Marshall JC, Parodo J, Farine D, et al. Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction. Am J Obstet Gynecol 1999;181: 408–14.[Medline]
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14. Hammer A, Blaschitz A, Daxbock C, Walcer W, Doher G.Fas and Fas-ligand are expressed in the uteroplacental unit of first trimester pregnancy. Am J Reprod Immunol 1999; 41:41–51.
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17. Runic R, Lockwood CJ, LaChapelle L, Dipasquale B, Demopoulos RI, Kumar A, et al. Apoptosis and Fas expression in human fetal membranes. J Clin Endocrinol Metab 1998;83:660–6.
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