Objective
Massive perivillous fibrin deposition (MPFD) is associated with serious complications of pregnancy including recurrent spontaneous abortion, fetal growth restriction, and fetal demise. The aim of this study was to determine whether maternal plasma concentrations of angiogenic/antiangiogenic factors in MPFD differ from those of uncomplicated pregnancies.
Study Design
This retrospective longitudinal case-control study included MPFD cases (n = 10) and control patients (n = 175) with uncomplicated pregnancies who were enrolled in a longitudinal study and delivered at term. Serial plasma concentrations of placental growth factor (PlGF), soluble endoglin (sEng), and soluble vascular endothelial growth factor receptor (sVEGFR)-1 and -2 were determined by an enzyme-linked immunosorbent assay (cases, n = 28 samples; controls, n = 723 samples). Individual analyte concentrations were averaged across gestational age at specimen collection intervals. Linear mixed models were used to test for differences in log-transformed mean analyte concentrations both overall and as a function of time.
Results
The following results were found: (1) patients with MPFD had a lower mean plasma PlGF concentration ( P = .03) and higher mean plasma concentrations of sVEGFR-1 and sEng (both P < .01) than controls, adjusted for potential confounders; (2) the mean plasma concentration of PlGF differed further among cases and controls as a function of gestational age interval ( P < .0001); however, mean sVEGFR-1 and sEng group differences as a function of gestational age interval approached but did not reach significance ( P = .09 and P = .11, respectively); (3) patients with MPFD had lower mean plasma concentrations of PlGF/sVEGFR-1 ( P < .0001) and PlGF/sEng ( P < .001): both of these relationships differed further as a function of gestational age interval (both P < .0001); and (4) differences in mean sVEGFR-1, sEng, and the ratios of PlGF to sVEGFR-1 and PlGF to sEng were observed before 20 weeks of gestation.
Conclusion
An imbalance of angiogenic/antiangiogenic factors is present in patients with MPFD prior to the diagnosis. We propose that these changes participate in the mechanisms responsible for adverse pregnancy outcomes in patients with MPFD.
Massive perivillous fibrin deposition (MPFD), also known as maternal floor infarction (MFI), is characterized by obliteration of the villous trophoblast with extensive deposition of fibrinoid material in the intervillous space. This condition was first described by Benirschke and Driscoll in 1967 and its frequency is 0.09-0.5%. MPFD is associated with recurrent serious adverse pregnancy outcomes including spontaneous abortion, fetal growth restriction, and fetal death. The proposed etiologies include autoimmunity (such as antiphospholipid or antiurokinase antibodies ) and cytotoxicity caused by proliferation of X-cells, which are extravillous trophoblasts that can produce major basic protein similar to that of eosinophil granules. However, the precise mechanisms leading to MPFD are unknown.
Angiogenesis, the development of new blood vessels from preexisting vasculature, is crucial for fetal growth and placental development. Successful pregnancy requires a balance between angiogenic and antiangiogenic factors. A growing body of evidence suggests that an imbalance of angiogenic/antiangiogenic factors is involved in the pathophysiology of preeclampsia (PE), pregnancies with small-for-gestational-age (SGA) neonates, spontaneous preterm labor and delivery, stillbirth, mirror syndrome, twin-to-twin transfusion syndrome, and molar pregnancies. Moreover, changes in the concentrations of the angiogenic factor (placental growth factor [PlGF]) and antiangiogenic factors (soluble vascular endothelial growth factor receptor [sVEGFR]-1 and soluble endoglin [sEng]) in maternal circulation precede the clinical diagnosis of PE, SGA and stillbirth. Because the clinical presentation of MPFD includes conditions associated with derangements of angiogenic and antiangiogenic factors, it is possible that an antiangiogenic state may play a role in the genesis of MPFD.
The objective of this study was to determine whether pregnancies with MPFD have alterations in maternal plasma concentrations of PlGF, sEng, sVEGFR-1, and sVEGFR-2 before the diagnosis of the condition.
Materials and Methods
Study design and patient selection
A longitudinal retrospective case-control study was conducted by reviewing placental pathology records in our institution from 2006 to 2011. Cases consisted of patients with placental pathology meeting the diagnostic criteria for MPFD, which was defined as a placenta with perivillous fibrinoid material (either limited to the maternal floor of the placenta or extending from maternal to fetal surfaces) encasing at least 50% of the villi on a minimum of 1 slide. Controls were women without MPFD in the placenta, who had uncomplicated pregnancies, delivered a term neonate whose birthweight was appropriate for gestational age (10th to 90th percentiles) and had plasma samples available for at least 5 of the following gestational age intervals: 6-9.9, 10-14.9, 15-19.9, 20-23.9, 24-27.9, 28-31.9, 32-36.9, and 37 weeks or longer.
These patients were enrolled in a longitudinal protocol to identify biological markers for the prediction of PE, SGA, and stillbirth. Venous samples were collected every 4 weeks until 24 weeks and every 2 weeks thereafter until delivery. Exclusion criteria were multiple gestations and a congenital fetal anomaly.
All women provided written informed consent before participating in the study and the use of clinical data and collection and utilization of biological samples for research purposes were approved by the institutional review boards of Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services.
Sample collection and immunoassays
Venipuncture was performed serially at regular prenatal visits and admissions to the hospital for all normal and MPFD affected pregnancies. Blood was collected into tubes containing EDTA. Samples were centrifuged and stored at −70°C until used for assay. Sensitive and specific immunoassays (R&D Systems, Minneapolis, MN) were used to determine maternal plasma concentrations of PlGF, sEng, and sVEGFR-1 and -2. All immunoassays utilized the quantitative sandwich enzyme immunoassay technique, and their concentrations in maternal plasma were determined by interpolation from the standard curves.
The inter- and intra-assay coefficients of variation were as follows: PlGF, 6.02% and 4.8%, respectively; sEng, 2.3% and 4.6%, respectively; sVEGFR-1, 1.4% and 3.9%, respectively; and sVEGFR-2, 2% and 4%, respectively. The sensitivity of the assays was as follows: PlGF, 9.52 pg/mL; sEng, 0.08 ng/mL; sVEGFR-1, 16.97 pg/mL; and sVEGFR-2, 19.01 pg/mL.
Statistical analysis
Demographic and obstetrical characteristics
Comparisons between continuous variables were performed by Mann-Whitney U tests. Proportions were compared using either Fisher exact or χ 2 tests as appropriate. A P < .05 was considered statistically significant. A descriptive analysis was performed using SPSS version 15.0 (SPSS Inc, Chicago, IL).
Longitudinal analysis of angiogenic/antiangiogenic factor concentrations
Individual analyte concentrations (PlGF, sEng, sVEGFR-1, and sVEGFR-2) and their ratios (PlGF/sEng and PlGF/sVEGFR-1) were averaged across 4 intervals defined by gestational age at venipuncture (<14 weeks, 14-16 weeks, 17-19 weeks, and 20-30 weeks). Linear mixed models were used to test for differences in log 10 -transformed mean analyte concentrations overall and as a function of time using a robust covariance matrix estimator. Covariables included in adjusted models were selected based on clinical knowledge and factors associated with MPFD and/or analyte concentrations. These included gestational age at venipuncture, body mass index (BMI), maternal age, African American race, and nulliparity.
Model reduction was additionally performed based on the plausibility of regression coefficients, association with independent/dependent variables, magnitude of change in the main effect parameter estimates, and model fit as indicated by the Bayesian Information Criteria. Linear combinations of model parameters comparing differences between cases and controls at each gestational age interval were used to determine the timing of changes in angiogenic/antiangiogenic factors. Longitudinal analyses were performed using SAS version 9.3 (SAS Institute Inc, Cary, NC).
Results
Clinical characteristics
During the study period, 10 pregnancies with MPFD and 175 controls were identified. Table 1 describes the clinical and demographic characteristics of the study population. As expected, the median gestational age at delivery and median birthweights were lower in MPFD-affected pregnancies than in uncomplicated pregnancies (each P < .001; Table 1 ). Pregnancy complications in cases with MPFD included miscarriage in the second trimester (n = 4), fetal growth restriction (n = 4) with abnormal umbilical artery Doppler velocimetry (n = 3), second- and third-trimester fetal demise (in utero: n = 5; intrapartum: n = 1), and abruptio placentae (n = 2). With the exception of 1 patient who delivered at term, all MPFD cases delivered before 31 weeks of gestation and only 2 had viable neonates ( Table 2 ). Three women were evaluated for the presence of anticardiolipin antibody and lupus anticoagulant, and all had negative results.
| Demographic | Uncomplicated pregnancies (n = 175) | MPFD (n = 10) | P value |
|---|---|---|---|
| Maternal age, y | 23 (20-26) | 31 (26-35) | < .001 |
| African American | 151 (86%) | 10 (100%) | .4 |
| Nulliparity | 62 (35%) | 3/10 (30%) | 1.0 |
| BMI, kg/m 2 | 27 (23-32) | 29 (28-35) | .04 |
| Gestational age at delivery, wks | 39 (39-40) | 23 (17-29) | < .001 |
| Birthweight, g | 3330 (3150-3555) | 277 (175-605) | < .001 |
| Stillbirth (>20 wks) | 0 | 4 (40%) | — |
| Miscarriage in the second trimester (<20 wks) | 0 | 4 (40%) | — |
| Fetal growth restriction | 0 | 4 (40%) | — |
| Placental abruption | 0 | 2 (20%) | — |
| Case number | Age, y | Gravida, parity | GA at delivery (weeks+days) | Clinical description | Birthweight, g (percentile for GA) | Prelabor rupture of membranes | Fetal growth restriction | Fetal demise | Second-trimester miscarriage |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 24 | G 4 P 2-0-1-2 | 15+6 | Presented with ruptured membranes and was induced for inevitable abortion | 150 | Yes | No | No | Yes |
| 2 | 27 | G 3 P 0-0-2-0 | 30+0 | Presented with fetal growth restriction, heavy vaginal bleeding/clinical placental abruption, and emergent cesarean delivery was performed | 755 (1%) | No | Yes | No | No |
| 3 | 22 | G 2 P 0-0-1-0 | 22+3 | Short cervix was noted at 20 wks; membranes ruptured with spontaneous labor at 22 wks and delivery of a stillborn infant | 448 (34%) | Yes | No | Yes | No |
| 4 | 28 | G 11 P 0-1-9-1 | 23+6 | Fetus noted to have thickened placenta, multiple placental lacunae, and oligohydramnios at 18 wks; abnormal Doppler parameters, fetal demise was diagnosed, and the patient was induced | 277 (1%) | No | Yes | Yes | No |
| 5 | 43 | G 13 P 3-3-6-4 | 16+4 | Presented with ruptured membranes and fetal demise | Unknown | Yes | No | Yes | Yes |
| 6 | 35 | G 7 P 0-0-6-0 | 17+3 | Cervical length of 0 mm on routine scan; a rescue cerclage was placed but membranes ruptured shortly afterward, induction for inevitable abortion | 160 | Yes | No | No | Yes |
| 7 | 29 | G 3 P 1-1-0-1 | 17+2 | Presented with abdominal pain and vaginal bleeding; fetal demise was diagnosed and the patient was induced | 190 | No | No | Yes | Yes |
| 8 | 35 | G 12 P 8-2-1-8 | 23+1 | Fetus noted to have decreased growth and progressive deterioration of Doppler parameters starting at 20 wks gestation, and fetal demise diagnosed at 23 wks | 274 (1%) | No | Yes | Yes | No |
| 9 | 34 | G 11 P 8-1-1-8 | 28+2 | Fetus noted to have growth restriction and progressive deterioration of Doppler parameters starting at 20 wks; fetal demise diagnosed and the patient was induced | 454 (1%) | No | Yes | Yes | No |
| 10 | 33 | G 10 P 7-1-1-7 | 38+1 | Spontaneous labor at term | 3285 (51.5%) | No | No | No | No |
Longitudinal analysis of plasma sVEGFR-1, sVEGFR-2, sEng, and PlGF concentrations
Patients with MPFD had a significantly lower mean plasma PlGF concentration ( P = .03) but significantly higher mean plasma concentrations of sVEGFR-1 ( P < .01) and sEng ( P < .01) than controls after adjusting for potential confounders ( Figures 1 and 2 ). The mean maternal plasma concentrations of PlGF differed further among patients with MPFD and controls as a function of gestational age interval ( P < .0001). However, the magnitude of the differences in mean plasma concentrations of sVEGFR-1 and sEng did not change significantly with gestational age interval ( P = .09, Figure 2 ; P = .11, Figure 1 , B). There were no significant differences in plasma concentrations of sVEGFR-2 observed overall ( P = .97) or as a function of time ( P = .17) among cases and controls ( Figure 3 ).
Patients with MPFD had significantly lower mean ratio concentrations of PlGF/sVEGFR-1 ( P < .0001) and PlGF/sEng ( P < .001; Figure 4 ) after adjustment for potential confounders; both of these relationships differed significantly as a function of gestational age interval (each P < .0001; Figure 4 ).
As shown in Figures 1-4 , although the differences in mean plasma PlGF concentration among cases and controls became statistically significant at 20-30 weeks of gestation, differences in mean sEng and the ratios of PlGF/sEng and PlGF/sVEGFR-1 among cases and controls became significant from 17-19 weeks of gestation onward. Consistent changes in the mean plasma sVEGFR-1 concentration in cases compared with controls appear to begin early, at 14-16 weeks’ gestation. The mean concentration of each angiogenic and antiangiogenic factor for each gestational age interval in MPFD patients and controls are shown in Table 3 .
| Analyte | Gestational age interval | Study group | N a | Mean | SD | Median | 25th centile | 75th centile |
|---|---|---|---|---|---|---|---|---|
| PlGF, pg/mL | I: <14 wks | Case | 4 | 20 | 7 | 22 | 16 | 24 |
| Control | 110 | 40 | 98 | 23 | 16 | 36 | ||
| II: 14-16 wks | Case | 7 | 106 | 45 | 119 | 76 | 152 | |
| Control | 82 | 106 | 61 | 87 | 59 | 132 | ||
| III: 17-19 wks | Case | 4 | 110 | 63 | 126 | 73 | 148 | |
| Control | 72 | 201 | 106 | 189 | 125 | 260 | ||
| IV: 20-30 wks | Case | 6 | 125 | 103 | 100 | 43 | 206 | |
| Control | 172 | 598 | 409 | 516 | 320 | 775 | ||
| sEng, ng/mL | I: <14 wks | Case | 4 | 8.0 | 2.4 | 7.1 | 6.6 | 9.4 |
| Control | 110 | 7.0 | 2.7 | 6.6 | 5.8 | 7.7 | ||
| II: 14-16 wks | Case | 7 | 8.1 | 2.5 | 7.6 | 6.6 | 8.3 | |
| Control | 82 | 7.1 | 5.7 | 6.5 | 5.3 | 7.2 | ||
| III: 17-19 wks | Case | 4 | 10.5 | 4.6 | 9.8 | 7.6 | 13.5 | |
| Control | 72 | 6.3 | 2.6 | 5.7 | 5.3 | 6.6 | ||
| IV: 20-30 wks | Case | 6 | 16.3 | 12.0 | 10.6 | 7.4 | 26.7 | |
| Control | 172 | 6.1 | 2.9 | 5.8 | 5.0 | 6.4 | ||
| sVEGFR-1, pg/mL | I: <14 wks | Case | 4 | 2200 | 1165 | 2487 | 1510 | 2891 |
| Control | 110 | 1697 | 1276 | 1513 | 986 | 1930 | ||
| II: 14-16 wks | Case | 7 | 2972 | 1439 | 3006 | 1305 | 4102 | |
| Control | 82 | 1912 | 1022 | 1661 | 1249 | 2355 | ||
| III: 17-19 wks | Case | 4 | 4955 | 2961 | 5664 | 2799 | 7111 | |
| Control | 72 | 2276 | 2707 | 1737 | 1295 | 2660 | ||
| IV: 20-30 wks | Case | 6 | 28,526 | 56,386 | 4209 | 1695 | 16,377 | |
| Control | 172 | 2092 | 1610 | 1727 | 1173 | 2456 | ||
| sVEGFR-2, ng/mL | I: <14 wks | Case | 4 | 10.3 | 1.4 | 10.2 | 9.2 | 11.5 |
| Control | 110 | 10.1 | 2.0 | 9.9 | 8.8 | 11.1 | ||
| II: 14-16 wks | Case | 7 | 10.7 | 1.7 | 11.1 | 9.7 | 11.8 | |
| Control | 82 | 10.3 | 1.8 | 10.2 | 9.0 | 11.2 | ||
| III: 17-19 wks | Case | 4 | 11.2 | 1.3 | 11.1 | 10.2 | 12.3 | |
| Control | 72 | 10.6 | 1.9 | 10.3 | 9.3 | 12.0 | ||
| IV: 20-30 wks | Case | 6 | 9.9 | 2.5 | 10.5 | 7.6 | 11.9 | |
| Control | 172 | 10.9 | 2.0 | 10.7 | 9.7 | 12.2 | ||
| PlGF/sEng | I: <14 weeks | Case | 4 | 2.4 | 0.7 | 2.4 | 1.8 | 2.9 |
| Control | 110 | 5.9 | 13.7 | 3.7 | 2.6 | 5.2 | ||
| II: 14-16 wks | Case | 7 | 14.1 | 7.3 | 14.3 | 5.6 | 20.6 | |
| Control | 82 | 16.6 | 9.0 | 14.2 | 9.7 | 22.2 | ||
| III: 17-19 wks | Case | 4 | 11.8 | 8.2 | 11.7 | 6.1 | 17.6 | |
| Control | 72 | 34.1 | 17.4 | 33.6 | 21.3 | 49.2 | ||
| IV: 20-30 wks | Case | 6 | 14.1 | 15.2 | 9.8 | 1.2 | 23.7 | |
| Control | 172 | 102.4 | 67.8 | 98.1 | 55.3 | 125.4 | ||
| PlGF/sVEGFR-1 | I: <14 wks | Case | 4 | 0.01 | 0.01 | 0.01 | 0.01 | 0.02 |
| Control | 110 | 0.02 | 0.03 | 0.02 | 0.01 | 0.03 | ||
| II: 14-16 wks | Case | 7 | 0.04 | 0.03 | 0.03 | 0.03 | 0.03 | |
| Control | 82 | 0.07 | 0.04 | 0.06 | 0.03 | 0.09 | ||
| III: 17-19 wks | Case | 4 | 0.02 | 0.00 | 0.02 | 0.02 | 0.03 | |
| Control | 72 | 0.13 | 0.11 | 0.10 | 0.07 | 0.15 | ||
| IV: 20-30 wks | Case | 6 | 0.04 | 0.05 | 0.03 | 0.00 | 0.05 | |
| Control | 172 | 0.36 | 0.30 | 0.29 | 0.19 | 0.42 |
Results
Clinical characteristics
During the study period, 10 pregnancies with MPFD and 175 controls were identified. Table 1 describes the clinical and demographic characteristics of the study population. As expected, the median gestational age at delivery and median birthweights were lower in MPFD-affected pregnancies than in uncomplicated pregnancies (each P < .001; Table 1 ). Pregnancy complications in cases with MPFD included miscarriage in the second trimester (n = 4), fetal growth restriction (n = 4) with abnormal umbilical artery Doppler velocimetry (n = 3), second- and third-trimester fetal demise (in utero: n = 5; intrapartum: n = 1), and abruptio placentae (n = 2). With the exception of 1 patient who delivered at term, all MPFD cases delivered before 31 weeks of gestation and only 2 had viable neonates ( Table 2 ). Three women were evaluated for the presence of anticardiolipin antibody and lupus anticoagulant, and all had negative results.
| Demographic | Uncomplicated pregnancies (n = 175) | MPFD (n = 10) | P value |
|---|---|---|---|
| Maternal age, y | 23 (20-26) | 31 (26-35) | < .001 |
| African American | 151 (86%) | 10 (100%) | .4 |
| Nulliparity | 62 (35%) | 3/10 (30%) | 1.0 |
| BMI, kg/m 2 | 27 (23-32) | 29 (28-35) | .04 |
| Gestational age at delivery, wks | 39 (39-40) | 23 (17-29) | < .001 |
| Birthweight, g | 3330 (3150-3555) | 277 (175-605) | < .001 |
| Stillbirth (>20 wks) | 0 | 4 (40%) | — |
| Miscarriage in the second trimester (<20 wks) | 0 | 4 (40%) | — |
| Fetal growth restriction | 0 | 4 (40%) | — |
| Placental abruption | 0 | 2 (20%) | — |
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