Objective
To determine maternal plasma levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), placental growth factor (PLGF), and soluble endoglin (sEng) in monochorionic diamniotic (MC/DA) twin pregnancies complicated by twin-to-twin transfusion syndrome (TTTS) or selective intrauterine growth restriction (sIUGR).
Study Design
A longitudinal cohort study of pregnant women with MC/DA twins who were classified into 3 groups: (1) uncomplicated MC/DA twins (n = 22), (2) TTTS (n = 23), and (3) sIUGR (n = 15). Maternal plasma samples were obtained between 13-20 and 21-28 weeks of gestation and cord blood samples were collected at delivery. Maternal plasma concentrations of sVEGFR-1, PLGF, and sEng, as well as cord blood levels of sVEGFR-1 were measured by enzyme-linked immunoassay.
Results
Maternal plasma levels of sVEGFR-1 and sEng were significantly higher in patients with TTTS at the early and late second trimester compared with normal monochorionic pregnancies ( P < .01). In contrast, in the sIUGR group, sVEGFR-1 and sEng levels were significantly higher only at the late second trimester ( P < .05). PLGF levels were significantly lower at the early and late second trimester in both TTTS and sIUGR compared with controls ( P < .01). Plasma concentrations of sVEGFR-1 were significantly higher among TTTS pregnancies compared with sIUGR at the late second trimester ( P = .027). Cord blood levels of sVEGFR-1 were significantly higher in the smaller intrauterine growth restricted twin compared with the normal cotwin.
Conclusion
Monochorionic pregnancies complicated by TTTS and sIUGR are characterized by decreased angiogenic activity. The disparity in severity of the antiangiogenic state between TTTS and sIUGR suggests that these 2 conditions may represent a continuum.
Monochorionic twin pregnancies are associated with increased perinatal morbidity and mortality compared with their dichorionic counterparts and singleton pregnancies. The increased risk of monochorionic pregnancies is largely attributable to the presence of vascular anastomoses connecting the 2 fetal circulations. The intertwin anastomoses are nearly always present and account for a range of pregnancy complications, including twin-to-twin transfusion syndrome (TTTS), twins anemia polycythemia sequence (TAPS), and neurologic injury to the surviving twin in the event of intrauterine demise of its cotwin. TTTS is the result of an unbalanced transfusion of blood across placental vascular anastomoses from one twin (donor) to the other (recipient). However, even though almost all monochorionic twin placentas have vascular anastomoses, TTTS develops only in 10-15% of these pregnancies. Therefore, although the presence of vascular anastomoses is mandatory for the development of TTTS, it’s mere presence is not sufficient and other additional factors may play a role in the pathophysiology of this condition
In addition, the single monochorionic placenta has to nourish 2 fetuses and is often not equally shared, which may lead to growth restriction and severe discordant birthweight. Selective intrauterine growth restriction (sIUGR) affects about 12-25% of monochorionic twin pregnancies and is increasingly recognized as an important complication of monochorionic twin pregnancies. Indeed, sIUGR is associated with an increased fetal and neonatal mortality and morbidity. The ability to predict monochorionicity-associated complications of pregnancy is quite limited and is mainly based on first trimester sonographic markers. Moreover, the differentiation between TTTS and sIUGR could be challenging as both might be manifested by discordant growth, oligohydramnios in one sac and absent or reverse end-diastolic velocity in the umbilical artery of the small twin. The importance of distinctions between these 2 conditions stems from the fact that the management of these complications is different: laser coagulation of the vascular anastomoses is the treatment of choice for TTTS, whereas the current management options in monochorionic twin pregnancies with sIUGR include expectant management with close surveillance or selective umbilical cord occlusion in cases of high risk of in-utero fetal demise, although laser coagulation of anastomoses has also been used in certain cases.
Angiogenesis plays a central role in normal placental development. It has been shown that angiogenic factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) and 2 antiangiogenic peptides produced by the placenta, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) and soluble endoglin (sEng), contribute to the pathogenesis of preeclampsia as well as IUGR, both associated with aberrant placentation.
Because there is no clear explanation why only certain monochorionic twins are complicated by TTTS or sIUGR, new mechanisms, which play a role in the pathophysiology of these conditions, must be explored. We hypothesized that altered angiogenesis mediated by sVEGFR-1, sEng and PLGF might be the mechanism linking abnormal placentation with pregnancy complications of monochorionic twins.
The aim of this study was to determine maternal and fetal plasma levels of sVEGFR-1, PLGF, and sEng in monochorionic diamniotic (MC/DA) twin pregnancies complicated by TTTS or sIUGR compared with uncomplicated monochorionic twins.
Materials and Methods
This was a prospective cohort study of patients with MC/DA twin pregnancies who were enrolled between November 2010 and May 2012 at a single tertiary center. The study was approved by the local institutional ethics committee, and all patients provided a written informed consent. The patients were classified into 3 groups: (1) uncomplicated monochorionic pregnancies (n = 22), (2) TTTS (n = 23), and (3) sIUGR (n = 15). All patients had a first trimester ultrasound, which confirmed the diagnosis of monochorionicity and established an accurate gestational age. The diagnosis of TTTS was based on the internationally accepted ultrasound criteria: an MC twin pregnancy with polyhydramnios of ≥8 cm deepest vertical pocket in the recipient (or ≥10 cm from 20 weeks of gestation onwards) and oligohydramnios of ≤2 cm deepest vertical pocket in the donor. Selective IUGR was defined as an estimated fetal weight below the 10th percentile in 1 twin and estimated fetal weight discordance of 25% or greater. Patients with a combined pathology of TTTS and sIUGR were classified in the TTTS group. The inclusion criteria for uncomplicated monochorionic pregnancies included: (1) appropriately grown fetuses; (2) estimated fetal weight difference of less than 25%; (3) normal amniotic fluid volumes; (4) normal Doppler velocimetry in the umbilical arteries; and (5) similar measurements of the middle cerebral artery-peak systolic velocity among both twins. Patients with chronic hypertension and pregestational diabetes, as well as pregnancies complicated with congenital anomalies or chromosomal abnormalities or intrauterine fetal death of 1 of the twins at presentation were excluded. Patients whose pregnancies were complicated by severe TTTS (defined as TTTS stage ≥2) were treated by fetoscopic laser ablation (n = 20). Patient with TTTS stage 1 (n = 3) were managed conservatively. Demographic data, ultrasound findings, and perinatal outcomes were entered into a computerized database. Serial samples of peripheral blood were obtained throughout pregnancy starting at the patient’s first visit to our fetal medicine clinic and thereafter every 6 weeks. All the maternal samples used for analysis were taken before laser treatment or selective termination. Cord blood samples were obtained at delivery. Blood samples were collected in tubes containing EDTA, centrifuged at 4°C for 10 minutes and stored at −70°C until further analysis. Maternal plasma levels of sVEGFR-1, sEng, and PLGF and cord blood levels of sVEGFR-1 were determined by enzyme-linked immunoassays (R&D Systems, Minneapolis, MN). All samples were assayed in duplicate at the same time using the same standard curve to minimize interassay variation. The calculated interassay coefficients of variation for sVEGFR-1, sEng, and PLGF were 7.4%, 7.1%, and 8.3%, respectively. The calculated intraassay coefficients of variation for sVEGFR-1, sEng, and PLGF were 2.4%, 2.6%, and 4.8%, respectively.
Normality of the data was tested using Kolmogorov-Smirnov test. Because the data did not fit a normal distribution, values of the factors tested are presented as median and interquartile range. The 3 groups were compared using Kruskal-Wallis test, and comparison of continuous variables between every 2 groups was conducted using Mann-Whitney U test. The χ 2 test was used for comparison of categorical variables. Significance was accepted at P < .05. Statistical analyses were conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS v.19; IBM Corporation Inc, Armonk, NY).
Results
Sixty patients were included in the study; 22 were uncomplicated monochorionic twins, 23 were diagnosed with TTTS, and 15 with sIUGR. The demographic and clinical characteristics of the study population are shown in Table 1 . Of the 23 patients with TTTS included in this study, 3 had a stage 1 disease and were followed conservatively, 12 had a stage 2 disease, and 8 presented with TTTS stage 3-19 of them underwent fetoscopic laser ablation and 1 patient had selective termination. Four of the patients in the TTTS group had a combined pathology of TTTS and sIUGR. Twelve of the 15 patients with sIUGR had abnormal Doppler in the umbilical artery; 10 had persistent AEDV, and 2 had intermittent AEDV. Five of the patients in this group underwent selective termination with radiofrequency ablation. All 3 groups were comparable in terms of maternal age, parity, maternal pregestational body mass index, and the rate of smokers. None of the pregnancies in our cohort were complicated by preeclampsia. As expected, patients with TTTS and sIUGR delivered earlier compared with the uncomplicated MC twins (32.2, 32.5, and 36.2 weeks of gestation respectively, Kruskal-Wallis P = .009).
| Characteristic | Normal (n = 22) | TTTS (n = 23) | sIUGR (n = 15) | P value |
|---|---|---|---|---|
| Maternal age, y | 32 (21–41) | 28 (21–44) | 28 (22–35) | NS |
| Primigravida, % | 22.7 | 26.1 | 33.3 | NS |
| Prepregnancy BMI, kg/m 2 | 22.4 (19–27) | 22.8 (18–30) | 22.5 (20–32) | NS |
| Smoking, % | 4.5 | 13 | 0 | NS |
| Gestational age at blood draw (early second trimester, wk) | 17.7 (13–20) | 17.5 (15–20) | 19 (16–20) | NS |
| Gestational age at blood draw (late second trimester, wk) | 23.5 (21–27) | 22 (21–28) | 24.2 (21–27) | NS |
| Gestational age at delivery, wk | 36.2 (36–37) | 32.2 (24–36) | 32.5 (24–38) | .009 |
| Sample storage time, mos | 11 | 8 | 10 | NS |
The gestational age at blood sampling did not differ among the 3 groups ( Table 1 ).
We have divided the time frame in which maternal blood samples were analyzed into 2: early second trimester (13-20 weeks of gestation) and late second trimester (21-28 weeks of gestation). Patients with TTTS had significantly higher median plasma levels of sVEGFR-1 (3835.2 pg/mL vs 1359.9 pg/mL, respectively, P < .01, Figure 1 , A), and higher median plasma levels sEng (13 ng/mL vs 6.6 ng/mL, respectively, P < .01, Figure 2 , A) compared with normal MC twins at 13-20 weeks. Similarly, patients with TTTS had significantly higher median plasma levels of sVEGFR-1 (6068.7 vs 1927 pg/mL, respectively, P < .01 Figure1 , B), and higher median plasma levels of sEng (22.8 vs 7.1 ng/mL, respectively, P < .01, Figure 2 , B) at 21-28 weeks of gestation. In contrast, median plasma levels of sVEGFR-1 (1671.4 vs 1359.9 pg/mL, respectively, P = .13 Figure 1 , A), and sEng (9.6 vs 6.6 ng/mL, respectively, P = .27, Figure 2 , A) did not differ significantly between patients with sIUGR and controls at 13-20 weeks of gestation. However, at 21-28 weeks of gestation patients with sIUGR had significantly higher median plasma levels of sVEGFR-1 and sEng compared with normal MC twins (3237.2 pg/mL vs 1927 pg/mL, P = .035; 18.8 ng/mL vs 7.1 ng/mL, P < .01, respectively, Figures 1 , B, and 2 , B).
Both patients with TTTS and sIUGR had significant lower median plasma levels of PLGF compared with normal MC twins at the early second trimester (140.3 and 281.9 vs 514.8 pg/mL, P < .01, respectively, Figure 3 , A) as well as at the late second trimester (263.7 and 350.1 pg/mL vs 956.8 pg/mL, P < .01, respectively, Figure 3 , B).
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