Angiogenesis is a critical component of normal placental development. Numerous investigators have reported that an imbalance in circulating levels of proangiogenic factors, such as placental growth factor (PlGF) and anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sFlt-1), may be associated with abnormal placentation and predict the development of uteroplacental diseases such as preeclampsia and intrauterine growth restriction (IUGR) (Chaiworapongsa et al., 2005; Levine et al., 2004; Shibata et al., 2005; Taylor et al., 2003; Thadhani et al., 2004). We recently demonstrated that abnormalities in PlGF and sFlt-1 also precede the onset of hypertension and placental abruption (Signore et al., 2006).

Endoglin is a cell-surface co-receptor for transforming growth factors β1 and β3 that has anti-angiogenic effects and is highly expressed in syncytiotrophoblast (Venkatesha et al., 2006). Elevated serum levels of soluble endoglin (sEng) have been demonstrated in women prior to the clinical onset of preeclampsia (Levine et al., 2006).

Enrollment details for the original study are described elsewhere (Levine et al., 1996). Of 4589 CPEP participants, we excluded 253 who were lost to follow-up, 21 whose pregnancy terminated prior to 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, leaving 4257 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 following the date recorded by the research nurse on the laboratory specimen forms.

Of the remaining 3631 women, 2469 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 from which to select normal controls. We identified all 32 women within the CPEP cohort who experienced abruptio placentae (including women with live births and stillbirths), but excluded one woman since her only serum specimen could not be located in the specimen repository. Ten of the 31 women with abruption developed hypertension during their pregnancies – seven, preeclampsia, and three, gestational hypertension. 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 vs. 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, 21–32 weeks, and ≥33 weeks. 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. Because controls had to be matched on several factors and had to have at least as many samples as the cases, we were able to match only one control to each case. All serum specimens collected from subjects prior to 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, according to the study protocol and manual of operations, and was defined as the presence at delivery after 24 weeks gestation of a retroplacental blood clot and/or antecedent vaginal hemorrhage not associated with vasa previa, placenta previa, or uterine rupture. Evidence of fetal distress and/or sonographic evidence of abruption were present in some cases, but these findings were not required for diagnosis. Hypertension was defined as a diastolic blood pressure of at least 90 mm Hg on two occasions 4 to 168 hours apart in a woman with blood pressure <135/85 prior to CPEP enrollment at 13–21 weeks of gestation. Preeclampsia required, in addition, proteinuria of at least 1+ (30 mg per deciliter) on dipstick testing, each on two occasions 4 to 168 hours apart, a single dipstick indicating ≥2+ proteinuria, a protein / creatinine ratio of 0.35 or greater, or a 24-hour urine collection with >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 10^th percentile according to U.S. tables of birth weight for gestational age that account for race, parity, and the sex of the infant (Zhang & Bowes, Jr. 1995).

Never-thawed serum specimens were randomly allocated to batches and analyzed, using an enzyme-linked immunosorbent assay (ELISA) for endoglin according to the manufacturer's instructions (R & D Systems, Minneapolis, MN). The measured interassay coefficient of variation in our laboratory was 12%. 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 sEng are reported in the text and tables, statistical tests for differences in levels of this factor were performed after logarithmic transformation. Because the number of samples in each group was relatively small, we performed both a matched and an unmatched analysis. 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 prior to that interval), no sample(s) from the other member of the pair was/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. Since the results of both these analytic methods were similar; the unmatched analysis is presented because the numbers were greater, giving it greater statistical power. All P values are 2-sided; a value of P<0.05 was considered significant. Analyses were conducted using SAS v 9.1 (SAS Institute, Cary, NC).

Maternal serum sEng levels were compared in three gestational age intervals (baseline, ≤ 20 weeks; mid-pregnancy, 21–32 weeks; and late-pregnancy, ≥ 33 weeks; Table 2). At baseline, women who developed placental abruption had somewhat higher sEng levels than normal control women. In mid-pregnancy, the difference became greater, reaching statistical significance (P<.01). Because of the high rate of preterm delivery (61 percent) among abruption cases, relatively few case subjects (N=10) contributed a specimen at ≥ 33 weeks gestation. In late pregnancy, sEng was higher among abruption cases than controls, but this difference did not reach statistical significance.

Because elevated sEng concentrations have been associated with preeclampsia, we wished to determine whether women with abruption and hypertension would have greater abnormalities in concentration of this antiangiogenic factor. We therefore analyzed sEng concentrations separately for the 10 abruption cases (32 percent) who developed gestational hypertension or preeclampsia during pregnancy and for the 21 cases who remained normotensive (Table 3). sEng levels in normotensive case and control women were similar at all 3 gestational age intervals. In the group of women with hypertension and abruption, however, sEng levels were strikingly higher compared to control women. The differences reached statistical significance despite the modest numbers in mid- (P=0.002) and late (P=0.04) pregnancy.

We compared concentrations of sEng at 21–32 weeks among women who later developed abruption and hypertension to sEng concentrations in other CPEP subjects who developed preeclampsia and had sEng measurements within the same gestational interval (Levine et al., 2006 and RJ Levine, personal communication). sEng levels in women with abruption and hypertension were as high as in women who subsequently developed preterm preeclampsia. They were, however, greater than levels in women who developed preeclampsia at term (19.3 vs. 8.2 ng/mL, P=0.05; Figure 1).

Only one other study of endoglin concentrations and abruption has been reported to date (Tikkanen et al., 2007). The same commercial assay was used in that study, and the mean sEng concentration in control subjects at an average gestational age of 15.5 weeks (5.5 ng/mL) was comparable to that observed in control subjects from the present study (6.3 ng/mL) in specimens collected at ≤ 20 weeks (mean gestational age 16.1 weeks). However, in contrast to the present study, Tikkanen et al concluded that second-trimester maternal serum sEng failed to predict placental abruption. They also noted that sEng levels in hypertensive women with abruption were not different from those in normotensive women with abruption. Several factors may explain the difference in our findings. In the Tikkanen study, subjects with preeclampsia and small for gestational age infants were included in the control group. As these pregnancy complications have been associated with increased sEng (Levine et al., 2006; Romero et al., 2008), the inclusion of these subjects may have blunted the study's ability to detect a difference in sEng between abruption cases and controls. Additionally, only 4 of the 42 abruption cases in the Tikkanen study had preeclampsia or pregnancy-induced hypertension. With this small number of cases it would be difficult to detect a significant difference in sEng among hypertensive abruption cases. Importantly, Tikannen measured soluble sEng at a single point early in pregnancy (15 – 16 weeks of gestation). In our previous work, we have shown that significant differences in anti-angiogenic factors among women who develop pregnancy complications are rarely apparent prior to 17 weeks of gestation (Levine et al., 2004; Levine et al., 2006; Rana et al., 2007). In the current study, we were able to assess serum sEng at multiple time points in pregnancy, and thus demonstrated that significant elevations in sEng do occur prior to abruption, but later in the second trimester, at 26–27 weeks on average, and that these elevations persist throughout pregnancy. Furthermore, we were able to show that the interval between measurement of sEng at 21–32 weeks of gestation and delivery with abruption and hypertension averaged 7.6 weeks. Thus, abnormalities in circulating sEng occur early enough that they may be useful for the early identification of women at risk.

By comparing soluble endoglin levels reported here to those in a previous publication (Levine et al., 2006), we demonstrated that sEng concentrations at 21–32 weeks in women who later developed placental abruption and hypertension were as elevated as in women who subsequently developed preterm preeclampsia and greater than in women who developed preeclampsia at term. This provides additional evidence linking the extent of abnormalities in circulating angiogenic factors with the severity of clinical manifestations of placental disease (Signore et al., 2006).

Normal placentation requires that chorionic villous cytotrophoblasts acquire an invasive phenotype, enabling them to penetrate and remodel the maternal decidual vasculature. Endoglin appears to play an important role in these processes. Addition of a monoclonal antibody to endoglin or of antisense endoglin oligonucleotides promoted trophoblast outgrowth and migration in placental explant cultures (Caniggia et al., 1997), indicating that endoglin is an important negative regulator of trophoblast invasion and placental development. Endoglin expression is increased on the syncytiotrophoblast of preeclamptic placentae at 25 and 40 weeks compared to age-matched normotensive controls (Venkatesha et al., 2006). Soluble endoglin, a 65 kDa truncated form of endoglin which lacks the transmembrane and cytoplasmic domains, may be produced by the placenta and is known to be elevated in the sera of women with preeclampsia (Venkatesha et al., 2006). sEng inhibits the formation of capillary tubes in vitro and induces vascular permeability and hypertension with focal glomerular endotheliosis in vivo (Venkatesha et al., 2006).

Thus, endoglin and sEng are involved in multiple aspects of placental development, including trophoblast invasion and angiogenesis. sEng appears to be a promising marker for gestational diseases related to poor placentation, such as preeclampsia and placental abruption. We speculate that upregulation of membrane-bound endoglin early in pregnancy, at the time of trophoblast migration and spiral artery invasion, may impair uterine vascular remodeling, leading to persistent high-pressure uteroplacental circulation and placental hypoxia. Upregulation of membrane-bound endoglin causes increased amounts of soluble endoglin to be released into maternal blood.

Some limitations and strengths of our study should be noted. One limitation is the small number of abruption cases; however, abruptio placentae is a rare outcome. 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. We were able to analyze samples collected prior to 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 sEng preceded, and were not the consequence of, hypertension or abruptio placentae.