OBJECTIVE: Abnormalities in circulating angiogenic factors have been reported in diseases of abnormal placentation, such as preeclampsia and intrauterine growth restriction. Our objective was to determine whether circulating angiogenic factors are altered in another placental vascular disease, abruptio placentae.
METHODS: In a nested case-control study of nulliparous pregnancies, we examined levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) in serum collected prospectively from 31 women who later developed placental abruption and from 31 normal control subjects. All serum specimens were collected before the onset of hypertension or abruption and before labor or delivery. Serum angiogenic factors were compared within 3 gestational age windows: early (20 weeks or less), middle (21–32 weeks), and late (33 weeks or more) pregnancy.
RESULTS: During early pregnancy women who developed placental abruption had lower PlGF and higher sFlt-1 concentrations and higher sFlt-1/PlGF ratios than women with normal pregnancies. In mid-pregnancy these differences became greater, reaching statistical significance for PlGF concentration (431 versus 654 pg/mL, P<.01) and the sFlt-1/PlGF ratio (25.3 versus 2.5, P<.01). When the women with placental abruption were subdivided into those who did (n=10) and those who did not (n=21) develop preeclampsia or gestational hypertension, significant alterations in angiogenic factors were noted only in women who later developed hypertension in pregnancy. Among these women, PlGF concentrations were decreased in mid-pregnancy (160 versus 723 pg/mL, P<.001), and the mid-pregnancy sFlt-1/PlGF ratio was increased (70.1 versus 2.3, P=.001).
CONCLUSION: Serum levels of the proangiogenic factor PlGF were decreased, and those of the antiangiogenic ratio sFlt-1/PlGF were increased in nulliparous women who subsequently developed hypertension and placental abruption.
LEVEL OF EVIDENCE: II-2
Serum proangiogenic factor PlGF levels are decreased and the antiangiogenic ratio sFlt-1/ PlGF is increased in nulliparous women who subsequently develop hypertension and placental abruption.
From the 1Epidemiology Branch and the 2Biometry and Mathematical Statistics Branch; Division of Epidemiology, Statistics, and Prevention Research, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland; 3Allied Technology Group, Rockville, Maryland; 4Division of Molecular and Vascular Medicine, Department of Medicine, and the 5Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
This research was supported by the Intramural Research Program of the National Institute of Child Health and Human Development.
Corresponding author: Richard J. Levine, MD, NIH/NICHD, Building 6100, Room 7B03, Bethesda, Maryland 20892; e-mail: LevineRJ@mail.nih.gov.
Normal placentation requires trophoblast invasion of maternal spiral arteries, in which trophoblast and fibrin replace the arterial media and transform the uteroplacental circulation into a high-flow, low-resistance system.1,2 Vascular remodeling occurs under the influence of several angiogenic factors, including vascular-endothelial growth factor (VEGF) and placental growth factor (PlGF).3,4
Considerable evidence indicates that abnormalities in trophoblast invasion are associated with the development of uteroplacental diseases such as preeclampsia and intrauterine growth restriction (IUGR).5–8 Recent studies have demonstrated decreased maternal serum levels of free VEGF and free PlGF during and before the onset of preeclampsia.9–12 Serum PlGF is decreased in women with IUGR accompanying preeclampsia and may be decreased in women with IUGR alone.4,9,13,14 Moreover, maternal serum soluble fms-like tyrosine kinase 1 (sFlt-1), a potent binder and inhibitor of VEGF and PlGF,15 is increased in women destined to develop preeclampsia.9,16 These findings suggest an important association between angiogenic factors and development of uteroplacental disease related to abnormal trophoblast invasion.
Abruptio placentae occurs in 0.5–2% of pregnancies.17,18 As a major risk factor for stillbirth, preterm delivery, and fetal growth restriction, placental abruption accounts for up to 25% of all perinatal deaths.19,20 Although the precise mechanisms underlying placental abruption are unknown, it has been suggested that abruption may result from abnormalities in trophoblast invasion and subsequent rupture of the maternal spiral arteries, causing retroplacental hemorrhage and premature separation of the placenta from the uterine wall.1,21 Histologic examination of placental bed tissue from cases of abruption shows frequent absence of trophoblast invasion in the spiral arteries,21 indicating similarities between abruptio placentae, preeclampsia, and IUGR. In addition, varying degrees of glomerular endotheliosis, a hallmark of preeclampsia, have been reported in patients with abruptio placentae.22,23 Preeclampsia and other hypertensive disorders of pregnancy, including chronic hypertension, are known to be important risk factors for placental abruption.17,20 Because alterations in angiogenic factors have been shown to be related to preeclampsia, we hypothesized that such alterations might also be present in patients with placental abruption.
We examined levels of PlGF and sFlt-1 in serum samples collected prospectively from women who later developed placental abruption and from normal controls in a nested case control study within the trial of Calcium for Preeclampsia Prevention (CPEP) conducted in healthy nulliparous women. Our objective was to determine whether these angiogenic factors are altered in the setting of abruption.
MATERIALS AND METHODS
We conducted a nested case-control study of subjects from the Calcium for Preeclampsia Prevention study, a randomized, double-blind clinical trial performed by the National Institute of Child Health and Human Development during 1992–1996 to evaluate the effects of daily supplementation with calcium or placebo on the incidence and severity of preeclampsia. Healthy, normotensive, nulliparous women with singleton pregnancies were enrolled between 13 and 21 weeks of gestation at 5 participating U.S. medical centers and followed until 24 hours postpartum, using a common protocol and identical data collection forms. Gestational age was determined by ultrasound examination. Serum specimens were requested from participants at 13–21 weeks, at 26–29 weeks, at 36 weeks, and when hypertension or proteinuria was noted. Many women actually provided samples outside the requested gestational periods; all specimens were included.
Enrollment details for the original study are described elsewhere.24 Of 4,589 participants in the Calcium for Preeclampsia Prevention study, we excluded 253 who were lost to follow-up, 21 whose pregnancy terminated before 20 weeks, 13 who were missing maternal or perinatal outcome data, 4 without smoking information, 9 with hypertension not verified by team chart review, 32 with stillbirths, and one woman whose infant had a chromosomal abnormality. These exclusions left 4,257 women with adequate information and a live birth. Of these, 102 were excluded because they lacked a baseline serum specimen; another 524 were excluded because their baseline specimens had label dates more than 2 days after the date recorded by the research nurse on the laboratory specimen forms.
Of the remaining 3,631 women, 2,469 had remained normotensive throughout pregnancy and had delivered an appropriate- or large-for-gestational age infant. We randomly selected 120 of these women to serve as a pool of normal controls. Separately, we identified all 32 women with abruptio placentae (including women with live births and stillbirths), but excluded one woman because her only specimen could not be located in the specimen repository. From the pool of normal controls, we selected one control subject for each abruption case, matched for clinical center, current smoking status (current smoker or quit after the last menstrual period versus never smoked or quit before the last menstrual period), and specimen collection pattern. Matching for specimen collection pattern meant that the control must have had at least as many specimens as the case in the following gestational-age intervals: 20 weeks or less, 21–32 weeks, and 33 weeks or more. If more than one control met all matching criteria for a given case, a random selection was made. Once a control was matched to a case, it could not be used as a match for any other case. All serum specimens collected from subjects before the onset of hypertension or proteinuria and before labor or delivery were included in the present study.
Abruptio placentae was clinically diagnosed by physicians participating in the CPEP study and was defined as the presence at delivery after 24 weeks of gestation of a retroplacental blood clot and/or antecedent vaginal hemorrhage not associated with vasa previa, placenta previa, or uterine rupture. Hypertension was defined as a diastolic blood pressure of at least 90 mm Hg on 2 occasions 4–168 hours apart in a woman with blood pressure less than 135/85 mm Hg before CPEP enrollment at 13–21 weeks of gestation. Preeclampsia required, in addition, proteinuria of at least 1+ (30 mg/dL) on dipstick testing, each on 2 occasions 4–168 hours apart, a protein/creatinine ratio of 0.35 or greater, or a 24-hour urine collection with more than 300 mg protein. Gestational hypertension was the de novo onset of hypertension after 20 weeks of gestation in the absence of proteinuria. A small for gestational age (SGA) infant was defined as an infant whose birth weight was below the 10th percentile according to U.S. tables of birth weight for gestational age that account for race, parity, and the sex of the infant.25. The Office of Human Subjects Research at the National Institutes of Health granted an exemption from institutional review board approval because data and specimens could not be linked to identifiable women.
Never-thawed serum specimens were randomly allocated to batches and analyzed by enzyme-linked immunosorbent assays (ELISAs) for human sFlt-1 (sVEGF R1) and human PlGF, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). We11 and others26 have demonstrated that the ELISA for human PlGF used in our study measures free PlGF. The intra- and interassay coefficients of variation were less than 10%. All samples were run in duplicate by technicians blinded to pregnancy outcome, and the mean values of the duplicate samples were reported.
Chi-square or t tests were used in analyses of maternal or infant characteristics to compare categorical or continuous variables, respectively. Although arithmetic mean concentrations of sFlt-1 and PlGF and levels of the sFlt-1/PlGF ratio are reported in the text and tables, statistical tests for differences in levels of these angiogenic factors were performed after logarithmic transformation. Because the number of samples in each group was relatively small, we performed both matched and unmatched analyses. In the matched analysis, the unit of analysis was the case-control pair; as such, in any gestational-age interval in which one member of the pair had no serum specimen (for example, because she had delivered before that interval), no sample(s) from the other member of the pair were considered in the analysis. In the unmatched analysis, the unit of analysis was an individual specimen; specimens from each subject were retained in the analysis regardless of the availability of a specimen from the same gestational interval from the matched subject. The results of both these analytic methods were similar; the unmatched analysis is presented because, since the numbers were greater, it had greater statistical power. All P values are 2-sided; a value of P<.05 was considered significant. Analyses were conducted with SAS 9.1 (SAS Institute, Cary, NC).
Characteristics of the case and control subjects are presented in Table 1. As expected, women with placental abruption were older than controls and delivered infants at an earlier gestational age and with lower birth weight than control women. Preterm birth was more common among infants of mothers with placental abruption. Abruption and control subjects did not differ significantly on race/ethnicity, body-mass index, or blood pressure at enrollment in the CPEP trial (mean 17.6 weeks). It should be noted that the women with abruption who also developed hypertension in pregnancy tended to have severe forms of hypertensive disease. Among these 10 women, 4 of 7 with preeclampsia developed severe disease, including 1 with hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome and 1 with eclampsia; 5 of the preeclampsia cases were delivered before term; 3 women with preeclampsia and 1 woman with gestational hypertension had infants that were born small for gestational age; and 1 of 3 women with gestational hypertension had severe hypertension.
Maternal serum levels of sFlt-1, PlGF, and the sFlt-1/PlGF ratio were compared in 3 gestational age intervals (baseline, 20 weeks or less; midpregnancy, 21–32 weeks; and late pregnancy, 33 weeks or more; Table 2). At baseline, women who developed placental abruption had lower PlGF and higher sFlt-1 and sFlt-1/PlGF ratio than normal control women. In midpregnancy, the differences became greater, reaching statistical significance for PlGF and the sFlt-1/PlGF ratio (both P<.01). Because of the high rate of preterm delivery (61%) among abruption cases, relatively few case subjects (n=10) contributed a specimen at 33 weeks or more of gestation. Nevertheless, in late pregnancy, sFlt-1 levels and the sFlt-1/PlGF ratio were again higher among cases than controls, but the PlGF concentration was higher among cases. These differences did not reach statistical significance. Two normal controls and 2 women with abruption without hypertension also developed proteinuria. After removing these women from the analysis, angiogenic factor levels were not changed substantially.
Because sFlt-1 and PlGF concentrations have been associated with preeclampsia,9,16 we wished to determine whether women with abruption and hypertension would have greater abnormalities in concentration of these angiogenic factors. We therefore analyzed sFlt-1 and PlGF concentrations separately for the 10 (32%) abruption cases who developed gestational hypertension or preeclampsia during pregnancy and for the 21 cases who remained normotensive (Table 3). In early pregnancy, sFlt-1 levels, PlGF levels, and the sFlt-1/PlGF ratio were not significantly different in nonhypertensive and hypertensive abruption subjects compared with controls, nor were these values in midpregnancy significantly different from controls in the abruptio patients who remained normotensive during pregnancy. However, in midpregnancy in the women with abruptio who developed hypertension, the difference in PlGF was statistically significant (P<.001), as was the increase in sFlt-1/PlGF ratio (P=.001). In late pregnancy the same pattern of angiogenic factor changes was found among the hypertensive abruption cases. Compared with controls, PlGF was decreased and both sFlt-1 and sFlt-1/PlGF ratio were significantly increased (P=.03 and P=.02, respectively).
We were interested in whether the changes in angiogenic factors observed among subjects with both placental abruption and hypertension were comparable to changes observed previously among other complicated pregnancies. Table 4 includes the midpregnancy and late pregnancy sFlt-1 and PlGF levels observed in our study alongside sFlt-1 and PlGF levels reported by Levine et al9 during the same gestational intervals in other CPEP patients with preeclampsia of varying severity and in normotensive controls (unpublished data courtesy of RJ Levine and SA Karumanchi). The analyses in both the Levine study and the current study were conducted by the same laboratory using the same commercially available assay. At 21–32 weeks, sFlt-1 levels were higher and PlGF levels were lower in the subjects with abruption and hypertension than in previously studied women with preeclampsia and a normally grown infant, preeclampsia at term, and even preterm preeclampsia and preeclampsia with a small for gestational age infant. The current results were also very different from previously reported control values. During late pregnancy (33 weeks or more), the observed elevation in sFlt-1 among abruption cases was second only to that previously reported for preterm preeclampsia. Late pregnancy PlGF levels in abruption were not as low as those previously reported for preterm preeclampsia or preeclampsia with an SGA infant, but were substantially lower than those observed in uncomplicated preeclampsia and preeclampsia at term.
Our results show that alterations in angiogenic factors are associated with abruptio placentae in nulliparous pregnancy in those women who develop preeclampsia or gestational hypertension. Specifically, in mid-pregnancy, PlGF is decreased and the sFlt-1/PlGF ratio is increased among women before the development of hypertension and placental abruption. Women who developed abruption in the setting of hypertension had much lower PlGF levels and much higher sFlt-1/PlGF ratios than women with abruption alone, suggesting possible differences in the extent of placental disease. Our findings raise the possibility that angiogenic factors known to be involved in the pathogenesis of the hypertensive disorders may also have a role in the development of abruptio placentae complicated by hypertension in pregnancy. However, we cannot determine whether changes in concentrations of circulating angiogenic factors reflect a direct contribution of angiogenic factors to the pathogenesis of abruptio placentae or an indirect contribution through the induction of hypertension, a well-known risk factor for abruption.
We recently demonstrated that alterations in angiogenic factors increased with increasing severity of preeclampsia.9 The alterations in sFlt-1 and PlGF levels using the same assay among women with hypertension and abruption in our study were comparable with those seen in clinically dangerous forms of preeclampsia: preterm preeclampsia and preeclampsia complicated by fetal growth restriction. They were more pronounced than the alterations in patients with preeclampsia and a normally grown infant or in those with preeclampsia at term. This is not surprising because the hypertensive disorders observed in the women with abruption were frequently severe. These results are consistent with the notion that the severity of the subsequent clinical manifestation of uteroplacental disease may be related to the degree of alteration in circulating angiogenic factor concentrations.
Placental growth factor is produced primarily by trophoblast and influences trophoblast differentiation, survival, and function via autocrine mechanisms.3,15 Placental growth factor also acts in a paracrine manner to support angiogenesis, stabilize vasculature, and protect endothelial cells.15 Soluble fms-like tyrosine kinase 1 is a soluble splice variant of the membrane-bound receptor for PlGF and VEGF that can suppress the biologic effects of both of these angiogenic factors. In vitro studies have shown that PlGF is decreased27 and sFlt-1 is increased28 in trophoblast cells under conditions of reduced oxygen tension. It appears that PlGF deficiency and sFlt-1 excess may result from placental hypoxia associated with incomplete remodeling of maternal spiral arteries. The incompletely remodeled arteries offer persistently high resistance to uterine artery blood flow, and they may be predisposed to vascular rupture in the placental bed, especially after the onset of hypertension.1,21 This could result in retroplacental hemorrhage with premature separation of the placenta from the uterine wall, ie, abruptio placentae. Further study is needed to determine whether the decrease in PlGF and the increase in sFlt-1 that we have detected in pregnancies destined to develop abruptio placentae, and that have been shown previously in preeclampsia,9–12 are a cause or effect of early abnormal placental development.14,15
Some limitations and strengths of our study should be noted. One limitation is the small number of abruption cases, but abruptio placentae is a rare outcome. We were able to analyze samples collected before the onset of symptoms in all abruption cases from a large prospective, carefully monitored cohort. Thus, we were able to minimize selection bias. Furthermore, all specimens were obtained before hypertension or placental abruption developed in any of the subjects. Because of this, we were able to demonstrate that the changes in angiogenic factors preceded, and were not the result of, hypertension or abruptio placentae. Precise information on the abruption-to-delivery interval was not available. Most of the serum samples from abruption cases were collected at 32 weeks of gestation or less, whereas the mean gestational age at delivery (and, presumably, of the abruption) among the abruption group was 35.8 weeks. The small number of subjects available in our study group during late pregnancy is the result of the high rate of preterm delivery in abruption cases.
In summary, we have shown that circulating levels of the proangiogenic factor PlGF were decreased and levels of the antiangiogenic ratio sFlt-1/PlGF were increased in nulliparous women who subsequently developed placental abruption and hypertension, but not in the women who developed abruption alone. Abnormal concentrations of angiogenic factors in the women who developed placental abruption and hypertension were especially evident in midpregnancy and appeared to be of comparable magnitude to levels previously observed in women with severe forms of preeclampsia. The degree of abnormality of circulating angiogenic factors in midpregnancy may reflect the severity of future clinical manifestations of placental disease. Our findings complement those of others in suggesting the possibility of a future diagnostic test for preeclampsia by measurement of plasma or serum concentrations of angiogenic factors. These measurements may indicate not only the likelihood of developing preeclampsia, but also the severity of impending disease, including the possibility of hypertension complicated by placental abruption.
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