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ORIGINAL RESEARCH |
From the 1Perinatal Division, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tiqwa, and 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
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ABSTRACT |
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METHODS: A case–control single-center design was used. The sample included 22 women with severe preeclampsia, 22 matched normotensive pregnant women, and 22 nonpregnant women. Enzyme immunoassay was used to measure soluble CD40 ligand.
RESULTS: Significantly higher levels of soluble CD40 ligand were detected in the nonpregnant women (23,767 ± 15,637 pg/mL) and in the women with preeclampsia (21,025 ± 45,386 pg/mL) than in the normotensive pregnant women (8,292 ± 5,926 pg/mL) (P = .026). No significant correlation between soluble CD40 ligand levels and E2 levels was observed.
CONCLUSION: The higher levels of soluble CD40 ligand detected in women with preeclampsia may indicate an exaggerated activation of platelets and endothelial cells.
LEVEL OF EVIDENCE: II-2
which is expressed on T cells, mast cells, polymorphonuclear cells, and natural killer cells.2,3 It was also recently identified on platelets.4 The interaction of CD40 ligand on activated platelets with CD40 on endothelial cells induces endothelial cell expression of adhesion molecules and proinflammatory cytokines and chemokines.4,5 It also leads, within minutes to hours, to proteolytic cleavage of CD40 ligand and the release of the inactive soluble form of CD40 ligand, which inhibits the inflammatory response.6 Platelets are now considered the major source of soluble CD40 ligand in the circulation.4,6,7 Studies have shown that normal pregnancy is associated with increased platelet activation and consumption8 that is controlled by as yet unidentified buffering factors. In preeclampsia, however, there appears to be further, uncontrolled platelet activation, and this phenomenon may be important to the pathogenesis of the disease.12 Preeclampsia has recently been suggested to be a two-stage disorder consisting of an alteration in placental perfusion followed by a maternal syndrome.13 Although vast progress has been made in the understanding of preeclampsia, its etiology remains unknown, and the mechanism linking the two stages is unclear.
Estradiol (E2) also has antiinflammatory properties14 and exerts an inhibitory effect on platelet function.15 Studies have shown that serum E2 levels are lower in preeclamptic women than in women with normal pregnancy.16,17
The purpose of this study was to investigate the involvement of soluble CD40 ligand in normal pregnancy and its possible role as a link between platelet activation and the typical widespread activation of endothelial cells in preeclampsia. We hypothesized that the lower E2 levels in preeclampsia contribute to further platelet activation and higher serum levels of soluble CD40 ligand. Therefore, serum levels of soluble CD40 ligand and E2 were compared among pregnant women with and without preeclampsia and nonpregnant women.
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METHODS |
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The diagnosis of severe preeclampsia was based on findings of systolic blood pressure 160 mm Hg or greater, and diastolic blood pressure 110 mm Hg or greater (2 measurements at least 6 hours apart, at rest) and one of the following: proteinuria 5g or more per 24 hours or 3+ proteinuria occurring for the first time in pregnancy and regressing after delivery, serum creatinine level 1.2 mg/dL or more (unless known to be elevated previously); hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, namely, platelet count less than 100,000/mm3 (thrombocytopenia) or evidence of microangiopathic hemolytic anemia (with elevated lactate dehydrogenase), elevated hepatic enzymes; or intrauterine growth restriction (< 5% percentile of normal growth curve) or oligohydramnios. Symptoms suggestive of significant end-organ involvement, such as headache, visual disturbances, epigastric or right upper quadrant pain, retinal hemorrhage, retinal exudates, or papilledema, pulmonary edema or oliguria less than 500 mL/24 hours were also considered signs of severe preeclampsia.
Blood was collected before the administration of any antepartum treatment agents, such as betamethasone or magnesium sulfate. Peripheral blood was drawn into serum separator tubes and allowed to clot for 30 minutes. The tubes were then centrifuged for 15 minutes at 2,000g. Serum was immediately removed and stored at -20°C. Samples were thawed only once. Complete blood count was measured in all cases at blood collection.
Plasma E2 was measured by a solid-phase competitive chemiluminescent enzyme immunoassay technique using the Immulite 2000 analyzer (Diagnostic Products Cooperation, Los Angeles, CA). The sensitivity was 55 pmol/L, and the intraassay and interassay coefficients of variation were 6.4% and 7.8%, respectively.
We tested serum rather than plasma levels of soluble CD40 ligand because many protocols for plasma preparation, including those recommended by the National Committee for Clinical Laboratory Standards, result in incomplete removal of platelets. Serum measurements by enzyme-linked immunosorbent assay (ELISA) are therefore considered more accurate. We used the enzyme immunoassay technique of R&D Systems (Minneapolis, MN); all samples were analyzed using the same assay. The minimum detectable dose of soluble CD40 ligand was 4.2 pg/mL. The intraassay and interassay coefficients of variation were 5.0% and 6.2%, respectively. Blanks and controls were included in all experiments.
The results are expressed as means and standard deviation for continuous variables and number and rate (percentage) for categorical variables. For statistical analysis, we used the SPSS statistical package 10.0 for Windows (SPSS, Cary, NC).
Log 10 transformation was used for skewed data (for soluble CD40 ligand, skewness was 5.8). Our calculations showed that a sample size of 20 women in the study and control groups was required to detect a difference of 1 unit in the log 10 transformation of soluble CD40 ligand with 80% power. Analysis of variance was used to compare soluble CD40 ligand levels with log 10 transformation in the preeclampsia, normal pregnancy, and nonpregnant groups. Because the matching was done on a one-to-one basis, the paired t test on the log 10 transformation of the continuous data was used to compare results between the preeclampsia and the normal pregnancy groups. The 
2 test was used for categorical data. Multiple linear regression models were fitted to the data to determine the association between serum E2 and CD40 ligand levels. A P value of less than .05 was considered significant.
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RESULTS |
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The clinical characteristics of the women with preeclampsia and their severe manifestations are presented in Table 2.
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Pregnancy outcome was worse in the preeclampsia group than in the normal-pregnancy group (Table 3). Mean arterial pressure was significantly higher, delivery occurred earlier, and birth weight was lower than in the healthy controls.
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Mean platelet volume was significantly higher in the preeclampsia group than in the nonpregnant controls (P = .01). Mean levels of soluble CD40 ligand were significantly different among the three groups (analysis of variance, P = .026). The highest levels (23,767 ± 15,637 pg/mL) were detected in the nonpregnant women and the preeclamptic women (21,025 ± 45,386 pg/mL); the lowest level (8,292 ± 5,926 pg/mL) was found in the normal pregnant women (Table 3).
Mean E2 levels were slightly lower in the preeclampsia group than in the normal pregnancy group. The correlations between log soluble CD40 ligand and log E2 in the preeclampsia and normotensive pregnancy groups, and between log soluble CD40 ligand and log platelet count, were not significant.
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DISCUSSION |
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According to some reports, platelet activation occurs already in the first trimester of pregnancy, weeks to months before the clinical appearance of preeclampsia,11,12,20 suggesting that platelets may be involved in the pathogenesis of the disorder.20 Platelet activation is expressed by platelet adhesion and aggregation and the release of thromboxane A2, which together generate vasoconstriction, further platelet aggregation, and progressive damage to endothelial cells—all characteristics of preeclampsia. An increased inflammatory response has also been implicated in the pathogenesis of preeclampsia10,20,21 The link between the exaggerated platelet activation and inflammation leading to endothelial damage in preeclampsia can be explained by the recently described CD40–CD40 ligand system and the mediatory role of soluble CD40 ligand.4 The results of the present study support this notion.
A relationship between platelet activation and cytokines has been suggested to be a key element in the systemic inflammatory response also in other disorders, such as ischemic heart disease,22 sepsis,23 and myeloproliferative disorders.24 Ovarian hyperstimulation syndrome is also attributed to a massive increase in systemic inflammatory cytokines, and thrombosis is a known complication of severe ovarian hyperstimulation syndrome.25 Together, these data suggest that soluble CD40 ligand is associated with a common inflammatory response pathway in different clinical situations. However, data on whether soluble CD40 ligand is capable of eliciting an inflammatory response remain controversial. Henn et al12 found that even high levels of soluble CD40 ligand failed to induce an inflammatory reaction. They concluded that the CD40 ligand–CD40 interaction leads, over time, to the transformation of CD40 ligand to the biologically inactive soluble CD40 ligand, thereby effectively limiting the inflammatory process in the vascular system. In contrast, Inwald et al7 reported that the ligation of platelet CD40 by soluble CD40 ligand caused CD62P expression and the release of -granules and dense granules which participate in the recruitment and activation of leukocytes. The possible role of soluble CD40 ligand as a proinflammatory or a limiting factor of inflammation needs to be further investigated in prospective studies of patients at risk of preeclampsia before the clinical onset of the disorder.
In normal pregnancy, the increase in platelet activation and consumption and the "balanced" inflammatory response are probably controlled by still unknown buffering factors that maintain hemostasis and prevent further platelet activation.20 The observations that E2 attenuates the activation of the systemic inflammatory response, even at the transcriptional level,14,26 are consistent with findings of the attenuating effect of E2 on platelet activation through nonnuclear receptors.27,28 Together, these data suggest that E2 is important in controlling platelet activation and inflammatory responses in normal pregnancy or even during ovarian hyperstimulation syndrome. The reports that low serum E2 levels were detected in women with severe early-onset preeclampsia compared with controls22,23 led us to examine serum levels of E2 in preeclampsia and their correlation to levels of soluble CD40 ligand. Although the serum levels of E2 detected in the women with preeclampsia were slightly lower than those in the healthy pregnant women, the correlation between soluble CD40 ligand and E2 levels in the preeclampsia and normotensive pregnancy groups was not significant.
In conclusion, the higher levels of soluble CD40 ligand in women with preeclampsia compared with those in normotensive pregnant controls may indicate an exaggerated activation of platelets and endothelial cells in the disorder.
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Footnotes |
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doi:10.1097/01.AOG.0000206206.99212.9e
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REFERENCES |
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