First-trimester abnormal placentation may result in adverse pregnancy outcomes including preterm birth, fetal growth restriction, and preeclampsia.1,2 In placenta development and functioning, growth factors including vascular endothelial growth factor (VEGF) and placental growth factor play a key role in the remodelling process of the maternal endothelium in the spiral arteries. Both VEGF and placental growth factor bind to soluble fms-like tyrosine kinase-1, a splice variant of the VEGF receptor 1 and primarily localized to the syncytiotrophoblasts.3,4 Soluble fms-like tyrosine kinase-1 reduces free circulating levels of the proangiogenic factors, VEGF, and placental growth factor and thereby blunts the beneficial effects of these factors.
A second important cascade in placental development and functioning is the fibrinolytic system. Fibrinolysis is activated by conversion of plasminogen to plasmin, which is responsible for fibrin degradation.5 Uncomplicated pregnancy is associated with a reduction of fibrinolytic activity contributing to a state of hypercoagulation.5,6 Reduced fibrinolytic activity is even more profound in pregnancies complicated by abnormal placentation as in the case of preeclampsia and intrauterine fetal growth restriction.5,7 Plasmin depends on a balance between plasminogen activators and plasminogen activator inhibitors (PAI). Plasminogen activator inhibitor-2 is produced by the trophoblast. It increases with gestational age until term in plasma of pregnant women with uncomplicated pregnancies and declines to undetectable levels 6 weeks after delivery.5,8 A decreased production of PAI-2 is thought to be a result of impaired placental function.9
Previously, an association between higher soluble fms-like tyrosine kinase-1 and lower placental growth factor circulating blood concentrations in women with preeclampsia was described.10 Similarly (anti-) angiogenic profiles have been described in women with pregnancies complicated by fetal growth restriction.11 The pathogenesis of preeclampsia is described as abnormal placenta development. The first (placental) stage of the disease might result in a failure in trophoblast invasion. Episodes of placental hypoxia result in oxidative stress and release of various components into the maternal circulation. The second stage of the systemic maternal disease is associated with exaggerated endothelial activation and a generalized hyperinflammatory state compared with normal pregnancy.1 We hypothesized that high soluble fms-like tyrosine kinase-1, low placental growth factor, and low PAI-2 concentrations in blood act as a sign of the first placental stage, resulting in the second stage of systemic maternal disease or placental related diseases, including preterm birth, fetal growth restriction, and preeclampsia. We therefore investigated the associations of soluble fms-like tyrosine kinase-1, placental growth factor, and low PAI-2 concentrations in the first and second trimesters of pregnancy with the uterine artery resistance index in the second trimester, placental weight, and birth weight. Second, we studied the associations of these factors with the risks for complications including preterm birth, fetal growth restriction, and preeclampsia.
MATERIALS AND METHODS
This study was embedded in the Generation R Study, a population-based prospective cohort study from fetal life until young adulthood in the city of Rotterdam, The Netherlands.12 The Generation R Study examines early environment and genetic determinants of growth, development, and health in fetal life, childhood, and adulthood. Enrollment was aimed in the first trimester but possible until birth of the child. Women were enrolled in the study at their routine ultrasonographic examination in pregnancy after written consent was obtained. Assessments in pregnancy, including anthropometrics and questionnaires, were planned in the first, second, and third trimesters. All children were born between April 2002 and January 2006. Of all eligible children in the study area, 61% participated at birth in the study. The Medical Ethical Committee of the Erasmus Medical Centre, Rotterdam, approved the study.
Maternal nonfasting venous blood samples were drawn in the first trimester (weeks in median with 90% range 13.4, 10.5–17.2) and second trimester (weeks in median with 90% range 20.4, 18.8–22.9). Details of processing procedures have been described previously.13 Blood samples were stored at −80°C. Plasma soluble fms-like tyrosine kinase-1 and placental growth factor concentrations were analyzed by the Department of Clinical Chemistry of the Erasmus Medical Centre. Soluble fms-like tyrosine kinase-1 and placental growth factor concentrations were analyzed using an immunoelectrochemoluminence assay on the Architect System. The between-run coefficients of variation for plasma soluble fms-like tyrosine kinase-1 were 2.8% at 5.5 ng/mL and 2.3% at 34.0 ng/mL. The coefficients for plasma placental growth factor were 4.7% at 24 pg/mL and 3.8% at 113 pg/mL. Plasma PAI-2 concentrations, analyzed by the Department of Laboratory Medicine of the Radboud University Nijmegen Medical Centre, were determined by enzyme-linked immunosorbent assay with the same experimental setup as described previously.14,15 For calibration, recombinant PAI-2 generously provided by Biotech Australia was used. The analytical sensitivity, defined as the amount of PAI-2 giving a signal in the enzyme-linked immunosorbent assay greater than two standard deviations above blank values, was 11 pg/mL, whereas the functional sensitivity was 32 pg/mL. For estimation of the accuracy of the method, a reference preparation was used in each microtiter plate. The mean PAI-2 concentration in this preparation was 88.4 ng/mL, whereas the intra-assay variation, the between-plates variation, and the interassay variation amounted to 3.4%, 2.9%, and 8.4%, respectively.
Maternal and neonate outcomes including hypertensive disorders in pregnancy (pregnancy-induced hypertension and preeclampsia), gestational age at birth, birth weight, and placental weight were obtained from medical records, completed by community midwives, and obstetricians. The occurrence of hypertension and hypertension-related complications (including preeclampsia, proteinuria, eclampsia, and hemolysis, elevated liver enzymes, and low platelets syndrome) were crossvalidated using hospital registries.16 Preeclampsia was defined after completion of the pregnancy according to the International Society for the Study of Hypertension in Pregnancy criteria.17 Preeclampsia was defined as a de novo hypertension (an absolute blood pressure 140/90 mm Hg or greater) after the 20th gestational week with concurrent proteinuria (0.3 g or greater in a 24-hour urine specimen or 2+ or greater [1 g/L] on a voided specimen, or 1+ or greater [0.3 g/L] on a catheterized specimen). Superimposed preeclampsia was defined as chronic hypertension (an absolute blood pressure 140/90 mm Hg or greater preconceptionally or before the 20th week of pregnancy) and new-onset proteinuria. Early-onset preeclampsia was defined as preeclampsia with a delivery before a gestational age of 34 weeks.18 Preeclampsia was diagnosed in 167 women (2.2%) and superimposed preeclampsia in 25 women (0.3%). Early-onset preeclampsia was diagnosed in 23 women (0.3%). Preterm birth was defined as a delivery started spontaneously before a gestational age of 37 weeks. Fetal growth restriction was defined a sex-specific gestational age-adjusted birth weight below the 2.3th percentile of the study cohort.
Ultrasonographic examination to assess uterine artery resistance index was performed in midpregnancy (20.5 weeks of gestation in median with 90% range 19.4–22.1). For each measurement, three consecutive uniform waveforms were recorded by pulsed Doppler ultrasonography and the mean was used for further analyses.19 Doppler measurements were available in 54% of the women. The latter was because Doppler measurements could only be performed within one of the research centers. Characteristics of these women did not differ from those without a Doppler measurement.
Information on maternal characteristics was obtained directly in one of the research centers or by a self-administered questionnaire at enrollment. In this questionnaire, ethnic background was extracted from information from the country of birth of the woman herself and her parents and classified as follows: European and non-European. Education (highest completed educational level of the mother) was categorized into three levels: low (no education or primary school), mid (secondary school), and high (college or university). Maternal comorbidity was defined as a mother having a chronic disorder, including chronic hypertension, chronic heart disease, diabetes, hypercholesterolemia, thyroid disease, and systemic lupus erythematosus. Maternal smoking and alcohol consumption habits were assessed by repeatedly applied questionnaires in pregnancy.20,21 Maternal height and weight were measured at enrollment. Body mass index (BMI) is calculated as weight (kg)/[height (m)]2.
Of the total 8,880 mothers who were enrolled during pregnancy, 76% (n=6,748) were enrolled before a gestational age of 18 weeks (first trimester).12 Blood samples from 95% of these mothers (n=6,398) were collected before 18 weeks of gestation. Before a gestational age of 25 weeks, 93% (n=8,241) mothers were enrolled. Blood samples between 18 and 25 weeks of gestation (second trimester) were collected in 92% (n=7,616) of the women. With respect to mothers with multiple pregnancies in the Generation R Study, we only included the first pregnancy to avoid clustering. Women with a stillbirth were also excluded. Consequently, the present analysis was limited to singleton live births delivered at a gestational age of more than 22 weeks. Soluble fms-like tyrosine kinase-1, placental growth factor, PAI-2, or all of these were analyzed in the remaining 7,519 women who had at least one measurement of soluble fms-like tyrosine kinase-1 or placental growth factor in less than 18 weeks of gestation or between 18 and 25 weeks of gestation (Fig. 1).
Power calculations in the Generation R Study are based on 7,000 patients in the whole cohort. For a normally distributed continuous outcome, it is possible to detect with a type I error of 5% and a type II error of 20% (power 80%) a difference of 0.11 standard deviation in the whole cohort if 10% of all patients had the relevant exposure. For dichotomous outcomes with the same type I and II errors, it is possible to detect a relative risk of 1.39 in the whole cohort if 10% of all patients have the relevant exposure and the 1-year incidence of the outcome of interest is 10%. Rates of most dichotomous environmental and genetic exposures in the Generation R Study are expected to vary generally between 10% and 20%.12
Standard deviation scores were created for each biomarker. A linear regression model was used to assess the associations of the maternal characteristics with the biomarker. To enable comparisons of the effect estimates between biomarkers and risk factors, we analyze our results as change per standard deviation score. These scores enable adjustment for gestational age avoiding the inclusion of nonlinear functions of gestational age in models. Our approach for developing reference models for constructing standard deviation scores was based on the LMS model of Cole and Green22 as implemented in the GAMLSS software of Rigby and Stasinopoulos23 and previously applied and reported by us.24 Subsequently, all factors associated with the biomarkers (P<.05) were included in the multivariable model to assess the associations between the biomarkers and placental function and adverse pregnancy outcomes. We assessed individual changes in soluble fms-like tyrosine kinase-1 and placental growth factor (δ soluble fms-like tyrosine kinase-1 and δ placental growth factor, n=4,849 and 4,868, respectively) and we assessed soluble fms-like tyrosine kinase-1–placental growth factor ratios (first trimester and second trimester, n=5,495 and n=6,768, respectively). Because soluble fms-like tyrosine kinase-1 and placental growth factor concentrations were not normally distributed (evaluated in a histogram), we applied a logarithmic transformation for these analyses. To assess the associations of biomarkers concentrations, deltas, and ratios, with maternal and fetal outcomes, we created deciles of each biomarker, the δ and ratio. Furthermore, because high soluble fms-like tyrosine kinase-1 and low placental growth factor or PAI-2 concentration may represent potential risk factors for adverse outcomes, we additionally used for soluble fms-like tyrosine kinase-1 the 10th deciles and for placental growth factor and PAI-2 the first deciles as cutoff values. For soluble fms-like tyrosine kinase-1 in the first trimester, concentrations 9.83ng/mL or less and greater than 9.83 ng/mL and in the second trimester, concentrations 11.12 ng/mL or less and greater than 11.12 ng/mL were considered as low (reference) and as high (10th deciles), respectively. For placental growth factor in the first trimester, concentrations 21.30 pg/mL or less and greater than 21.30 pg/mL and in the second trimester, concentrations 108.09 pg/mL or less and greater than 108.09 pg/mL were considered as low (first deciles) and as high (reference), respectively. For PAI-2 in the first trimester, concentrations 23.61 ng/mL or less and 23.61 ng/mL or greater were considered as low (first deciles) and as high (reference), respectively. A multivariable linear regression model was used to assess the associations between soluble fms-like tyrosine kinase-1, placental growth factor, and PAI-2 concentrations and placental vascular resistance, placental weight, and birth weight. Likewise, multivariable logistic regression models were used to study the associations between soluble fms-like tyrosine kinase-1, placental growth factor, and PAI-2 and preterm birth, fetal growth restriction, and preeclampsia. The consideration of confounding variables was based on literature.20,25 – 28 These possible confounding factors included maternal age, BMI, parity, maternal ethnicity, maternal education, smoking, the use of alcohol and folic acid use as well as sex of the newborn. In the multivariable models, missing data were completed using multiple imputation (missing: maternal ethnicity 7%, maternal education 14%, maternal comorbidity 12%, parity 1%, BMI 1%, smoking 13%, alcohol use 13%, and folic acid use 25%). Data were imputed according to the Markov Chain Monte Carlo method assuming no monotone missing pattern. Five imputed data sets were created. Subsequently, multiple regression analyses were performed on each imputated data set and thereafter combined to one pooled estimate.29 – 31 Because there were no major differences in the observed results between analyses with imputed missing data or complete cases only, only results including imputed data are presented.
Baseline characteristics are depicted in Table 1. Maternal age, BMI, multiparity, European ethnicity, smoking, and the male sex of the neonate were associated with lower soluble fms-like tyrosine kinase-1 concentrations (P<.05). Maternal age, BMI, and the use of alcohol were associated with lower placental growth factor concentrations (P<.05). Multiparity, low education, non-European ethnicity, smoking, and no use of folic acid supplements were associated with higher placental growth factor concentrations (P<.05). Body mass index, multiparity, smoking, and male sex of the neonate were associated with lower PAI-2 concentrations (P<.05).
As shown in Table 2, placental weight and birth weight were significantly correlated (r=0.63, P=.01). High soluble fms-like tyrosine kinase-1 concentrations in both the first (greater than 9.83 ng/mL) and second trimester (greater than 11.1 ng/mL) were associated with a lower uterine artery resistance index in the second trimester (5.2% and 3.1%, respectively), an increased placental weight (5.8% and 4.5%, respectively), and an increased birth weight (1.6% and 3.1%, respectively). Women with low first-trimester and second-trimester placental growth factor concentrations (less than 21.3 pg/mL and less than 108 pg/mL, respectively) had an increased uterine artery resistance index in the second trimester (6.1% and 3.9%, respectively), a decreased placental weight (4.6% and 5.4%, respectively), and a decreased birth weight (3.4% and 4.1%, respectively). Lastly women with low concentrations of PAI-2 (23.61 ng/mL or less) in the first trimester had an increased uterine artery resistance index in the second trimester (1.9%), a decreased placental weight (3.6%), and a decreased birth weight (2.7%).
In Table 3 the concentrations of soluble fms-like tyrosine kinase-1, placental growth factor, and PAI-2, the delta's of soluble fms-like tyrosine kinase-1 and placental growth factor between the first and second trimesters, and the ratios of soluble fms-like tyrosine kinase-1 and placental growth factor in the first and second trimesters are presented. Table 4 shows the results of the associations between the biomarkers and adverse pregnancy outcomes. A high δ soluble fms-like tyrosine kinase-1 (difference between first- and second-trimester concentration, greater than 0.096 ng/mL) was associated with an almost twofold increased risk of preterm birth (odds ratio [OR] 1.86, 95% confidence interval [CI] 1.27–2.74). A trend, although not significant, toward a lower risk of fetal growth restriction was observed for high second-trimester soluble fms-like tyrosine kinase-1 (greater than 11.12 ng/mL) concentrations (OR 0.62, 95% CI 0.38–1.02). First- and second-trimester soluble fms-like tyrosine kinase-1 concentrations were not associated with the occurrence of subsequent preeclampsia. Low placental growth factor (108.1 pg/mL or less) concentrations in the second trimester were associated with a two to almost four times increased risk of preterm birth (OR 1.64, 95% CI 1.16–2.31), fetal growth restriction (OR 2.55, 95% CI 2.55–4.10), and preeclampsia (OR 3.71, 95% CI 2.55–5.40). In addition, low placental growth factor (108.1 pg/mL or less) concentrations in the second trimester were considerably associated with early-onset preeclampsia with an almost 12 times increased risk (OR 11.9, 95% CI 4.20–33.3). Likewise, the δ of placental growth factor between the first and second trimester was associated with a substantially increased risk of early-onset preeclampsia (OR 9.35, 95% CI 3.12–28.0). Lastly, low first-trimester PAI-2 (23.6 ng/mL or less) was associated with higher risk of fetal growth restriction (OR 2.22, 95% CI 1.39–3.55).
In this large observational study we show strong associations between angiogenic, placental growth, and fibrinolytic factors and placental development and function with subsequent risks for significant pregnancy outcomes. High soluble fms-like tyrosine kinase-1 concentrations were associated with lower placental vascular resistance with subsequently higher placental weight and birth weight. Second, a high δ of soluble fms-like tyrosine kinase-1 between the first and second trimester was associated with a decreased risk of preterm births. These results are in line with earlier studies showing increased soluble fms-like tyrosine kinase-1 concentrations in twin pregnancies compared with singletons, suggesting a positive association between trophoblastic mass and soluble fms-like tyrosine kinase-1 concentrations. Each trophoblast does not seem to be programmed to produce more of the antiangiogenic protein soluble fms-like tyrosine kinase-1; however, more soluble fms-like tyrosine kinase-1 is produced because of a greater number of trophoblastic cells. From this we hypothesize that in women with higher soluble fms-like tyrosine kinase-1 blood concentrations, these elevations might be the result of a greater amount of trophoblastic mass.
With respect to preterm birth, Smith et al33 described a high soluble fms-like tyrosine kinase-1 in the first trimester associated with a decreased risk of preterm births. Previously expression of VEGF has been demonstrated in human myometrium and VEGF peaks have been associated with cervical ripening in rats. High soluble fms-like tyrosine kinase-1 could result in a decreased VEGF expression and hence protect for a preterm birth.
Previously, increased soluble fms-like tyrosine kinase-1 concentrations were associated with preeclampsia, but only up to 5 weeks before onset of preeclampsia.10 Other studies, however, presented elevated soluble fms-like tyrosine kinase-1 in the first and second trimester as a risk factor for preeclampsia.34 In contrast to this, Kusanovic35 and Smith33 showed no association between first-trimester soluble fms-like tyrosine kinase-1 and preeclampsia. Previous studies showing an association between a high sFlt1 and preeclampsia were relatively small and did not always adjust for the gestational age at time of sampling. We, like other larger studies, were also unable to confirm a positive association between soluble fms-like tyrosine kinase-1 concentrations in early pregnancy and preeclampsia.35
The upregulation of soluble fms-like tyrosine kinase-1 release has been described as a result of a hypoxic environment. Soluble fms-like tyrosine kinase-1 is highly expressed in the first trimester and it seems plausible that low first-trimester release reflects low angiogenic activity and thereby impaired placental development. One consequence of the placental impairment may be fetoplacental hypoxia followed by a strong subsequent angiogenic activity. High concentrations of soluble fms-like tyrosine kinase-1 may be a marker of this activity.36 High soluble fms-like tyrosine kinase-1 concentrations appearing later in pregnancy lead to endothelial dysfunction and mediate a preeclampsia.37 In the presence of a placenta with an appropriate size for gestational age, predisposing cardiovascular and metabolic syndrome-like disorders might also set off a cascade of placental oxidative stress, resulting in late-onset preeclampsia.38 Finally, soluble fms-like tyrosine kinase-1 is mainly produced by placenta, although other sources have been described as well such as peripheral blood monocytes and vascular endothelial cells.39
Low placental growth factor was associated with a higher uteroplacental vascular resistance in midpregnancy, a lower placental weight and birth weight, and considerably high risks for preterm births, fetal growth restriction, and preeclampsia. The significant associations between low first-trimester concentrations and high uteroplacental vascular resistance in midpregnancy may indicate early placental insufficiency. Specific binding of soluble fms-like tyrosine kinase-1 to placental growth factor has been suggested as an explanation of decreased placental growth factor in pregnancies with adverse outcomes. However, in our study, the soluble fms-like tyrosine kinase-1–placental growth factor ratio was not associated with placental weight and birth weight, suggesting another underlying mechanism.3 Our first- to second-trimester δ of placental growth factor concentrations showed an association with a low placental weight and birth weight. It has been suggested that remodeling of the spiral arteries probably begins in late first trimester after which it is completed by 18–20 weeks of gestation.40,41 This may imply that the second-trimester placental development is also important in relation to adverse pregnancy outcome.
With respect to preeclampsia, a decrease of placental growth factor concentrations 9–11 weeks before the development of preeclampsia with a considerable decrease 5 weeks before the actual onset has been described.10 We demonstrated strong associations between low placental growth factor concentrations in the first and second trimester and a low δ of placental growth factor and the risk of (early-onset) preeclampsia up to 10%. An explanation for development of preeclampsia would be a primary unknown trigger resulting in an abnormal placentation with a decreased release of placental growth factor starting early and in the second trimester and with less effect on the soluble fms-like tyrosine kinase-1 concentration in the first and second trimesters. Furthermore, placental insufficiency with low placental growth factor concentrations in early pregnancy is even more profound in early-onset preeclampsia.
The associations between PAI-2 and birth weight are in agreement with earlier studies.42,43 Because villous cells are the source of PAI-2, its concentrations may reflect trophoblastic mass and therefore explain the association with birth weight. The association between PAI-2 concentrations and the risk for preterm birth has not been described before. Only one study assessed the association between PAI-2 and preterm birth through an association with PAI-2 polymorphism.44 In accordance with our results, Estelles et al42 showed that low placental expression of PAI-2 was shown to be associated with fetal growth restriction. Finally, low PAI-2 concentrations in the third trimester have been associated with preeclampsia.9 However, it is uncertain whether the altered concentrations of PAI-2 precede the clinical onset. Clausen et al45 reported increased PAI-2 concentrations at 18 weeks of gestation in women who developed preeclampsia. Like in our study, Akolekar et al9 found no association between PAI-2 concentrations in the first trimester and preeclampsia.
This was a large population-based prospective cohort study with an extensive data collection. Although participation rates for were relatively high and the ethnic distribution differs only moderately from that of the eligible population in the study area, The Generation R Study is characterized by a rather highly educated and healthy study population compared with available city data.12 In cohort studies, missing data analysis is always a critical issue. We attempted to deal with this by using multiple imputation for missing covariables. However, with respect to some outcomes such as here, complete placental weight information was missing in almost 27% of the women. Characteristics of these women did not differ from those with a known placental weight. For this reason, we do not expect that this has significantly influenced our results, although we are aware that bias cannot be fully excluded.
First-trimester soluble fms-like tyrosine kinase-1 concentrations do not seem to be a valuable predictor for adverse pregnancy outcomes. Lower placental growth factor concentrations, however, are associated with increased risks of preterm birth, fetal growth restriction, and preeclampsia. Lower PAI-2 concentrations in the first trimester are associated with a higher risk of fetal growth restriction but not preeclampsia.
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