BIOMARKERS FOR PREECLAMPSIA
20180074070 · 2018-03-15
Assignee
Inventors
Cpc classification
G01N2800/368
PHYSICS
International classification
Abstract
The present invention relates to the use of hemopexin, free, non-cell bound fetal hemoglobin and alpha-1-micro-globulin as markers for preeclampsia.
Claims
1-32. (canceled)
33. A method for diagnosing, predicting, or evaluating the risk of developing preeclampsia in a pregnant female mammal, comprising measuring levels of hemopexin (Hpx) and alpha-1-microglobulin (A1M) in a biological sample from the pregnant female mammal.
34. The method of claim 33, wherein the pregnant female mammal is a human, further comprising comparing the measured levels of Hpx and A1M to reference values for Hpx and A1M, wherein the reference values for Hpx and A1M are derived from levels of Hpx and A1M measured in biologicals samples from reference pregnant women who do not develop preeclampsia.
35. The method of claim 34, further comprising determining that the subject has preeclampsia or it at increased risk of developing preeclampsia when the measured level of Hpx is at least 1.1 times less than the reference value for Hpx, the measured level of A1M is at least 1.1 times more than the reference value for A1M.
36. The method of claim 33, wherein the biological sample is selected from blood, plasma, serum, cerebrospinal fluid, urine, placental biopsies, uterine fluid and amniotic fluid.
37. The method of claim 33, wherein the method is for diagnosing, predicting, or evaluating the risk of developing early onset preeclampsia.
38. The method of claim 33, wherein the pregnant female mammal is a human and the biological sample is taken from the subject at a gestational age of 6-20 weeks.
39. The method of claim 33, wherein the pregnant female mammal is a human and the biological sample is taken from the subject at a gestational age of 12-14 weeks.
40. The method of claim 33, wherein the pregnant female mammal is a human and the biological sample is a plasma sample and is taken from the subject at a gestational age of 6-20 weeks, further comprising determining that the subject has preeclampsia or it at increased risk of developing preeclampsia when the measured level Hpx in the plasma sample is 1.0 mg/mL or less, and the measured level of A1M in the serum sample is 15.5 g/mL or more.
41. The method of claim 33, wherein the pregnant female mammal is a human and the method is for diagnosing, predicting, or evaluating the risk of developing late onset preeclampsia.
42. The method of claim 33, wherein the pregnant female mammal is a human and the biological sample is taken from the subject at a gestational age of 34-40 weeks.
43. The method of claim 33, wherein the pregnant female mammal is a human and the biological sample is a plasma sample and is taken from the subject at a gestational age of 34-40 weeks, further comprising determining that the subject has preeclampsia or it at increased risk of developing preeclampsia when the measured level Hpx in the plasma sample is 0.85 mg/mL or less, and the measured level of A1M in the serum sample is 30 g/mL or more.
44. A method for the diagnosis or aiding in the diagnosis of preeclampsia, comprising: (a) obtaining a biological sample from a pregnant female mammal; (b) measuring the levels of hemopexin (Hpx) and alpha-1-microglobulin (A1M) in the sample; and (c) comparing the measured levels of Hpx and A1M with a reference value to determine if the a pregnant female mammal has or has not preeclampsia, or is or is not at increased risk of developing preeclampsia.
45. The method of claim 44, wherein the biological sample is taken at a gestational age of 6-20 weeks.
46. The method of claim 44, wherein the biological sample is taken at a gestational age of 34-40 weeks.
47. A method for monitoring the progression or regression of preeclampsia a pregnant female mammal, comprising: (a) measuring the levels of hemopexin (Hpx) and alpha-1-microglobulin (A1M) in a first biological sample from a pregnant female mammal; (b) measuring the levels of hemopexin (Hpx) and alpha-1-microglobulin (A1M) in a second biological sample obtain from the pregnant female mammal at a later time than the first sample; (c) comparing the values measured in step (a) and (b), and (i) determining that preeclampsia is progressing in the pregnant female mammal when the measured Hpx level in the second biological sample is lower than the measured Hpx level in the first biological sample and the measured A1M level in the second biological sample is higher than the measured A1M level in the first biological sample; or (ii) determining that preeclampsia is regressing in the pregnant female mammal when the measured Hpx level in the second biological sample is higher than the measured Hpx level in the first biological sample and the measured A1M level in the second biological sample is lower than the measured A1M level in the first biological sample.
48. The method of claim 47, wherein the biological sample is selected from blood, plasma, serum, cerebrospinal fluid, urine, placental biopsies, uterine fluid and amniotic fluid.
49. The method of claim 47, wherein the first biological sample is taken at a gestational age of at least 6 weeks.
50. The method of claim 47, wherein the first biological sample is taken at a gestational age of 6-20 weeks.
51. The method of claim 47, wherein the first biological sample is taken at a gestational age of 12-14 weeks.
52. The method of claim 47, wherein the first biological sample is taken at a gestational age of 34-40 weeks.
Description
LEGENDS TO FIGURES
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DISCUSSION OF THE RESULTS
[0213] In the study the inventors have employed PE as a model disease to study the response of the cell-free Hb-defense network in a pathological situation with prolonged elevation of hemolysis. Thus, in order to investigate the physiological relevance and possible pathophysiological importance of cell-free HbF in the disease progression of PE we investigated the impact of cell-free HbF (both free, denoted HbF, and in complex with Hp, denoted Hp-HbF) on the major human endogenous Hb-scavenging systems: Hp, Hpx, A1M and CD163. This allowed us to investigate the potential for HbF, Hp-HbF, Hb-Total, A1M, Hp, Hpx and CD163 as biochemical markers supporting the diagnosis of PE.
[0214] In this study we characterized cell-free HbF and the endogenous Hb- and heme-scavenger systems in pregnant women diagnosed with PE and normal pregnancies (controls) at term. Congruent with previous results, we found a significant increase of HbF in women with PE.sup.11. Furthermore, plasma levels of the Hb- and heme scavenger systems were highly affected, displaying significant reduced levels of the Hb-scavenger Hp and the heme-scavenger Hpx. Interestingly, and in line with previously published studies the extravascular heme- and radical scavenger A1M were significantly increased in plasma of women with PE.
[0215] In this study we also evaluated the diagnostic and clinical usefulness of the investigated biomarkers and found a clear potential of using these as clinical tools in diagnosing women with PE and or HELLP. Furthermore, the biomarkers displayed a clinical utility, enabling the possibility of identifying women and fetus at risk of clinical complications.
[0216] Hemolysis and the subsequent release of cell-free Hb and heme occur in a wide range of clinical conditions and diseases, including HELLP syndrome, transfusion reactions, malaria, hemorrhage, sepsis and sickle cell disease. The release of cell-free Hb and heme causes a range of pathophysiological effects where hemodynamic instability and tissue injury constitutes the major insults. Immediate effects include scavenging of the powerful vasodilator nitric oxide (NO) that leads to increased arterial blood pressure. Furthermore, cell-free Hb and free heme have been described to be accumulated and compartmentalized within the vascular wall and organs, causing subsequent organ failure. Importantly, long-term exposure to cell-free Hb and heme has been described to be associated with NO depletion, inflammation and oxidative stress.
[0217] In a series of recent publications the etiological involvement and importance of cell-free HbF and its downstream metabolites free heme and ROS, in the development of PE-related damage and symptoms, have been characterized. Using the dual placenta perfusion system, May et al. showed that addition of cell-free Hb to the fetal circulation caused a significant increase in perfusion pressure, feto-maternal leakage of extracellular Hb into the maternal circulation and morphological damage similar to what is seen in placentas of women with PE. Using a pregnant ewe PE-model and a pregnant rabbit model, it has been shown that starvation causes hemolysis and an increased amount of extracellular heme and bilirubin in the blood. Furthermore, in these models, severe placenta and kidney damage has also been described. This damage was attributed to be caused by the increased hemolysis and subsequent release of Hb and generation of heme and ROS.
[0218] Here we report, in line with previous studies, that cell-free HbF (both HbF and Hp-HbF) are significantly increased in pregnant women diagnosed with PE. Thus, these women are presented with an increased blood pressure and protein leakage into the urine, both hallmark of pathophysiological exposure to cell-free Hb and heme.
[0219] In order to protect itself against extracellular Hb and free heme, humans have evolved several Hb- and heme-detoxification systems. The most well-investigated Hb-scavenger system is Hp. Hp very efficiently binds extracellular Hb in blood and the resulting Hp-Hb complex is cleared from blood by binding to the macrophage receptor CD163. If Hp becomes depleted as a consequence of large amounts off or prolonged exposure to Hb, excess oxyHb will undergo auto-oxidation reactions resulting in free heme-groups and ROS. Furthermore, excess and non-protein bound Hb will be accumulated within and cause damage to the kidneys, subsequently leading to leakage of proteins into the urine. Depleting, exhausting or overwhelming Hp will allow oxyHb to degrade into its downstream metabolites metHb, free heme and ROS. The major heme-scavenger within the blood stream is Hpx, a highly specific and abundant system that protects cells, vessels and tissue against heme-induces damage. Following binding, Hpx delivers heme via its receptor CD91, preferably expressed on macrophages, hepatocytes, neurons and syncytiotrophoblasts, where it is internalized by receptor-mediated endocytosis and heme is subsequently degraded.
[0220] In this study, a highly significant decrease of both the Hp and Hpx were observed in maternal plasma of women with PE as compare to normal pregnancies. This indicated a prolonged presence of increased levels of both extracellular Hb and heme. Thus, although not presented with very high levels of cell-free HbF, we suggest that a continuous exposure to low or moderate level from early pregnancy, e.g. the study by Dolberg et al. suggest an increased level of cell-free HbF as early as gestational week 10-16, will exhaust the endogenous intravascular Hb- and heme (La Hp and Hpx) protective systems. In addition, in some PE patients we observed a highly significant increase in cell-free HbF (non Hp-bound). Very interestingly, all of these patients were found to be of Hp 2-2 isoform (
[0221] We have previously shown that the radical scavenger A1M binds and degrades heme and protects cells and tissues from oxidation, damage to mitochondrial-, cellular- and tissue structures and cell death. Furthermore, we have shown that plasma A1M concentration is significantly increased in women with PE both at term and early in pregnancy. Analysis of the plasma A1M levels in the present study confirmed previously published data, displaying a significantly increase of the A1M plasma concentration in women with PE as compared to normal pregnancies.
[0222] Why are the A1M-levels increased while the Hp- and Hpx-levels are decreased in the PE patients? It has been shown in several reports that the A1M gene expression is rapidly upregulated in the liver, skin, placenta and other organs as a response to increased levels of Hb, heme and ROS. This will lead to increased secretion of the protein resulting in increased plasma concentrations in pathological situations with increased Hb and ROS loads. Furthermore, no specific receptor-mediated clearance system of A1M has been shown to be triggered during hemolysis or oxidative stress, whereas Hp and Hpx are cleared from plasma upon binding to Hb and heme. As a result, the concentrations of A1M in plasma and extravascular fluids will increase, while Hp and Hpx will be exhausted and hence their plasma concentrations will decrease.
[0223] There is an increased attention towards the use of biomarkers in clinical prediction and diagnosis of PE. Several biomarkers have been suggested so far, but no available guidelines recommend the use of biomarkers in a clinical setting. Recently American Congress of Obstetricians and Gynecologists (ACOG) suggested a definition of severe PE where proteinuria is replaced by the use of biomarkers, currently including thrombocytes (<100,000/microliter), serum creatinine (>1.1 mg/dl) and liver transaminases (twice the normal concentration). Here, we present data suggesting that the biomarkers HbF, A1M and Hpx could be used clinically to support the diagnosis of PE. The combination of HbF, Hpx and A1M displayed the highest correlation to diagnosis (detection rate of 69% at 5% false positives, AUC=0.88,
[0224] Hpx concentration was shown to have a significant negative correlation to the blood pressure (
[0225] Being able to predict fetal and maternal outcomes is of great clinical value as it can help clinicians in the difficult task to optimize timing of delivery. In this study, the correlation between investigated biomarkers and a range of maternal and fetal outcomes were evaluated. The results indicated that HbF, Hp and Hpx correlated with admission to NICU. Furthermore, Hpx was strongly associated to premature birth. However, since all prematurity in this cohort was associated with preeclampsia this strong association could be as result of the strong correlation between Hpx and preeclampsia rather than prematurity itself.
[0226] It is of importance to note that the cohort used in this case-control study is not a normal distributed cohort, i.e. it contains an overrepresentation of women with PE. Consequently, detection and prediction rates reported in this study could therefore be different in a normal distributed cohort, containing 3-8% of PE cases.
[0227] In this study, we have among other things characterized cell-free HbF and the endogenous Hb- and heme-scavenger systems in pregnancies complicated by preeclampsia. Plasma levels of HbF were significantly elevated whereas Hp and Hpx were significantly decreased in women with preeclampsia. The extravascular heme- and radical scavenger, and marker of oxidative stress, A1M was significantly increased in preeclampsia plasma. Furthermore, HbF and the related scavenger proteins displayed a potential to be used as clinical biomarkers for more precise diagnosis of preeclampsia and as predictors that help identifying pregnancies with increased risk of obstetrical complications.
[0228] In the present study the HO-1 concentration was significantly reduced, particularly in the late onset PE group. The low concentration of HO-1 could be due to continuous strain on this system because of elevated heme and HbF levels throughout PE. The HO-1 enzyme is slowly more and more depleted throughout pregnancy and is therefore lower in late onset PE.
[0229] The plasma heme concentration was elevated both in early and late onset PE, however only significantly elevated in late onset PE. The heme concentration obviously correlated well with total Hb concentration. Previously published studies have indicated that the increased levels of HbF throughout the PE pregnancy slowly put a strain on and deplete the maternal Hb and heme scavenging systems including A1M, Haptoglobin and Hemopexin concentration. A constant over-production of HbF in the placenta induces damage to the placenta and the maternal endothelium. The strength of the maternal scavenger and enzyme systems may be important constitutional factors that determine how and when the clinical symptoms present in stage two of PE. The more the systems are strained and/or depleted, the more severe are the clinical symptoms.
[0230] Correlation analysis showed a significantly inverse correlation between Hpx activity and diastolic blood pressure in all the patients.
[0231] Heme oxygenase 1 was also inversely correlated to systolic and diastolic blood pressure. The higher heme load might explain why HO 1 was lower in PE patients. Depletion of HO-1 diminishes the anti-inflammatory properties, which in turn may aggravate maternal endotheliosis and therefore the blood pressure increases. Furthermore, the degradation of heme by HO-1 produces CO, which is a potent vase-dilator. Diminished levels of HO-1 consequently lead to decreased degradation of heme and less production of CO. This could add to the contracted vascular bed seen in patients with PE.
[0232] In this present study, we present a range of potential biomarkers based on HbF and hemoglobin- and heme scavenger proteins and -enzymes. Used in combination, the biomarkers reach a sufficient detection level acceptable for clinical use. The Hpx activity as a single marker was able to detect 30% of PE cases at a 10% FPR. Heme and HO-1 showed similar DRs. Together however, Hpx activity, Hpx, HO-1, Heme and HbF concentrations were able to detect 84% of the PE cases at 10% FPR, which match some of the best biomarkers for PE. Furthermore, several of the biomarkers included in the suggested model correlate with blood pressure and hence with clinical severity of PE.
[0233] By measuring components of the Hb metabolism as potential diagnostic biomarkers, a more precise PE diagnosis can be made.
EXPERIMENTAL
Materials and Methods
Study ISampling at Gestational Age 34-40 Weeks
Patients and Demographics
[0234] At start, 150 pregnant women were included in the study. The patients were randomly retrospectively selected from a currently on-going prospective cohort study. Exclusion criteria were gestational hypertension, essential hypertension and gestational diabetes. In total 5 cases were excluded due to pre-gestational diabetes or pregnancy related diabetes and a total of 145 patients were included 98 developing PE (cases) and 47 with normal pregnancies (controls). Patient demographics are described in Table 1 and 2.
Sample Collection
[0235] The study was approved by the ethical committee review board for studies on human subjects at Lund University, Sweden. The patients signed informed consent after information given orally and written. Maternal venous sample were taken prior to delivery from patients admitted to the Department of Obstetrics and Gynecology, Lund University Hospital, Sweden. The samples were collected as 6 ml blood into EDTA Vacuette plasma tubes (Greiner Bio-One GmbH, Kremsmnster, Austria) and centrifuged at 2000g for 20 minutes. The plasma was then transferred into cryo tubes and stored in 80 C. until time of analysis. Pregnancy outcome for each patient were retrospectively taken from the charts.
[0236] Preeclampsia was defined as de novo hypertension after 20 weeks of gestation with 2 readings at least 4 hours apart of blood pressure 140/90 mmHg and proteinuria 300 mg per 24 hours. This is according to the International Society of the Study of hypertension in Pregnancy's definition.sup.50. Dipstick analysis was accepted if there was no quantification of proteinuria. Furthermore the PE group was further sub-classified as early-onset PE (diagnosis 34+0 weeks of gestation) or late onset PE (diagnosis >34+0 weeks of gestation). There were 3 cases of PE with unknown time of diagnosis and therefore not included in the analyses made with the subgroups of early onset PE and late onset PE.
Reagents and Proteins
[0237] HbF was purified as previously described.sup.16 from whole blood, freshly drawn from umbilical cord blood. Human -chains were prepared by dissociation of purified HbF with p-mercuribenzoate (Sigma-Aldrich, St-Louis, Mo., USA) and acidic precipitation as described by Kajita et al..sup.55 with modifications by Noble.sup.56. The absolute purity of HbF (from contamination with HbA) and of -chains (from contamination with - and -chains) was determined as described previously.sup.11. Mouse antibodies to human -chains, and hence specific for HbF, were produced and purified by AgriSera AB (Vannas, Sweden). Anti-HbF antibodies were conjugated with horseradish peroxidase (Lightning-Link HRP, Innova Biosciences, Cambridge, UK) as described by the manufacturer. Human A1M was purified from urine as described by kerstrm.sup.57. Rabbit polyclonal antibodies were prepared against human A1M.sup.58, mouse monoclonal antibodies against human A1M.sup.59, goat anti-human A1M and goat anti-rabbit immunoglobulin were prepared as previously described.sup.60.
Fetal Hemoglobin (HbF)-Concentrations
[0238] A sandwich-ELISA was used for quantification of uncomplexed HbF. Ninety six-well microtiter plates were coated with anti-HbF antibodies (mouse monoclonal, 4 g/ml in PBS) overnight at room temperature (RT). In the second step, wells were blocked for 2 hours using blocking buffer (1% BSA in PBS), followed by an incubation with HbF calibrator or the patient samples for 2 hours at RT. In the third step, HRP-conjugated anti-HbF antibodies (mouse monoclonal; diluted 1:5000), were added and incubated for 2 hours at RT. Finally, a ready-to-use 3,3,5,5-Tetramethylbenzidine (TMB, Life Technologies, Stockholm, Sweden) substrate solution was added. The reaction was stopped after 20 minutes using 1.0 M HCl and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter (Perkin Elmer Life Sciences, Waltham, Mass., USA).
Haptoglobin-Fetal Hemoglobin (Hp-HbF) Concentrations
[0239] A sandwich-ELISA was used for quantification of Hp-HbF. This ELISA display a high preference for Hp-HbF compared to uncomplexed HbF (>10 recovery of a Hp-HbF calibrator series compared to a HbF calibrator series at the same molar content of HbF). Ninety six-well microtiter plates were coated with anti-Hp-HbF antibodies (HbF-affinity purified rabbit polyclonal; 4 g/ml in PBS) overnight at RT. In the second step, wells were blocked for 2 hours using blocking buffer (1% BSA in PBS), followed by an incubation with Hp-HbF calibrator or the patient samples for 2 hours at RT. In the third step, HRP-conjugated anti-Hb antibodies (HbA-affinity purified rabbit polyclonal; diluted 1:5000), were added and incubated for 2 hours at RT. Finally, a ready-to-use TMB (Life Technologies) substrate solution was added, reaction was stopped after 30 minutes using 1.0 M HCl and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter (Perkin Elmer Life Sciences).
Total Hemoglobin (Hb-Total)-Concentrations
[0240] The concentrations of Hb-Total in maternal plasma were determined using the Human Hb ELISA Quantification Kit from Genway Biotech Inc. (San Diego, Calif., USA). The analysis was performed according to the instructions from the manufacturer and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter.
Alpha-1-Microglobulin (A1M)-Concentrations
[0241] Radiolabelling of A1M with .sup.125I (Perkin Elmer Life Sciences) was done using the chloramine T method. Protein-bound iodine was separated from free iodide by gel-chromatography on a Sephadex G-25 column (PD10, GE Healthcare, Stockholm, Sweden). A specific activity of around 0.1-0.2 MBq/g protein was obtained. Radioimmunoassay (RIA) was performed by mixing goat antiserum against human A1M (diluted 1:6000) with .sup.125I-labelled A1M (appr. 0.05 g/ml) and unknown patient samples or calibrator A1M-concentrations. After incubating overnight at RT, antibody-bound antigen was precipitated by adding bovine serum and 15% polyethylene glycol, centrifuged at 2500 rpm for 40 minutes, after which the .sup.125I-activity of the pellets was measured in a Wallac Wizard 1470 gamma counter (Perkin Elmer Life Sciences).
Haptoglobin (Hp)-Concentrations
[0242] The concentrations of Hp in maternal plasma were determined using the Human Hp ELISA Quantification Kit from Genway Biotech Inc. The analysis was performed according to the instructions from the manufacturer and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter.
Hemopexin (Hpx)-Concentrations
[0243] The concentrations of Hpx in maternal plasma were determined using the Human Hpx ELISA Kit from Genway Biotech Inc. The analysis was performed according to the instructions from the manufacturer and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter.
Hpx Activity
[0244] Plasma Hpx activity was measured in EDTA plasma samples using the Hpx-MCA substrate (synthesized by Pepscan, Lelystad, the Netherlands). The plasma samples (40 l) were diluted 1:4 with the substrate solution (0.2M Tris+0.9% NaCl pH 7.6 (substrate concentration 80 M/L) to a final volume of 200 l. The emission was measured at 460 nm on a Varioskan spectrophotometer (Thermo Fisher) at 37 C. The Hpx activity was measured after 0 min, 30 min (Hpx30), 60 min (Hpx60) and 24 hours. The measured value represented the total amount of serine catabolized by Hpx at the given time point. If the value was <5 after 24 hours of incubation, the activity was considered very low, due to technical problems with either the assay or the samples, and the samples were expelled from further analysis. The area under the curve analysis was based on Hpx30 and Hpx60 measurements (HpxAUC). The measures Hpx30, Hpx60 and HpxAUC mimicked one another and therefore only Hpx30 was used for analysis. In the following Hpx30 is mentioned as Hpx activity.
Cluster of Differentiation 163 (CD163)-Concentrations
[0245] The concentrations of CD163 in maternal plasma were determined using the Human CD163 Duo Set from R&D Systems (Abingdon, UK). The analysis was performed according to the instructions from the manufacturer and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter.
SDS-PAGE and Western Blot
[0246] SDS-PAGE was performed using precast 4-20% Mini-Protean TGX gels from Bio-Rad (Hercules, Calif., USA) and run under reducing conditions using molecular weight standard (precision protein plus dual marker Bio-Rad). The separated proteins were transferred to polyvinylidene difluoride (PVDF) or low fluorescence (LF) PVDF membranes (Bio-Rad). The membranes were then incubated with antibodies against Hp (polyclonal rabbit-anti human Hp, 12 g/ml, DAKO, Glostrup, Denmark). Western blot was performed using HRP-conjugated secondary antibodies (DAKO) and the chemiluminescent substrate Clarity Western ECL (Bio-Rad). The bands were detected in a ChemiDoc XRS unit (Bio-Rad). The relative quantification of A1M bands was performed by densitometry using Image Lab software (Bio-Rad).
Statistical Analysis
[0247] Statistical computer software Statistical Package for the Social Sciences (SPSS Inc., Chicago, Ill.) version 21 for Apple computers (Apple Inc., Cupertino, Calif.) and Origin 9.0 software (OriginLab Corporation, Northampton, Mass., USA) were used to analyze the data.
[0248] ANOVA test was used to compare the groups for clinical parameters such as age, BMI, parity, systolic blood pressure, diastolic blood pressure, proteinuria, gestational age at delivery, birth weight, gestational age at time of sampling and APGAR score at 10 minutes.
[0249] Mann-Whitney test was used to compare Hpx activities, Hpx, HO-1, heme, HbF and total Hb concentrations between PE and controls. Subgroup-analyses were performed for early- and late onset PE.
[0250] The Chi square test was used to compare the groups for fetal gender, labor induction, mode of delivery (e.g. vacuum extraction, caesarean section or vaginal delivery), need of neonatal intensive care unit (NICU) and preterm delivery.
[0251] Mean concentration of the examined variables (henceforth referred to as biomarkers) were evaluated in women with PE compared to the control group using non-parametric statistics. A univariate logistic regression model was developed for the evaluated biomarkers. The gestational age at sampling was adjusted for in the logistic regression model. The biomarkers displaying a significant difference were further evaluated using Receiver Operational Curve (ROC-curve) by analyzing the area under the ROC-curve (AUC) as well as calculating the detection rates at different false positive levels. Parallel analysis was performed for each of the examined biomarker as well as different combinations of them. Furthermore, sub-group analysis of women with PE, i.e. early and late onset PE, compared to the control group was performed. The univariate logistic regression model was also used to further calculate fetal outcomes (i.e. admission to NICU and premature delivery and intrauterine growth restriction (IUGR)) and mode of delivery.
Correlation Analysis
[0252] Correlation analysis (Pearson's correlation coefficient) between biomarkers and diastolic- and systolic blood pressure was performed. A p-value of p0.05 was considered significant in all tests.
[0253] Correlation between Hpx activity and Hpx concentration was calculated using the non-parametric Kendall's correlation coefficient. Furthermore, correlation analysis was performed between Hpx activity and maternal blood pressure (defined as the highest measured blood pressure within 24 hours before delivery).
[0254] Correlation analyses were also done between cell-free Hb (HbF and Total Hb), heme, HO-1 and hemopexin concentrations. Furthermore, heme and HO-1 both were correlated to both systolic and diastolic blood pressure.
Logistic Regression Analysis
[0255] The detection rate was determined by ROC-curve analysis for each of the potential biomarkers Hpx, HO-1 and heme. The detection rates were obtained at 10% and 20% false positive rates. The combined detection potential for the biomarkers was obtained by stepwise logistic regression analysis of the biomarkers and ROC-curve analysis.
[0256] Results
Patient Characteristics
[0257] The characteristics of the included patients are shown in Table 1 and 2. There was a significant difference between women diagnosed with PE and uncomplicated pregnancies (denoted controls) for age, blood pressure, proteinuria, parity, gestational age at sampling, gestational age of delivery and birth weight. Furthermore, for parameters regarding maternal outcome (e.g. mode of delivery incl. induction and instrumental deliveries) as well as fetal outcome (e.g. admittance to NICU and prematurity) a significant difference was observed. A significant difference in the 10 minutes APGAR score was observed between controls and early onset PE but not late onset PE. There was no significant difference between the groups regarding BMI and fetal gender.
TABLE-US-00001 TABLE 1 Patient demographics of PE cases and normal pregnancies (controls). Values are shown as mean (95% confidence interval) or number (%). Statistical comparison vs. controls. p-value <0.05 is considered significant. Normal Early Late pregnancy onset onset (Control; Preeclampsia PE.sup.1 PE.sup.2 Outcome n = 47) (n = 98) (n = 22) (n = 74) Age 29 31** 32 NS 30 NS (28-30) (30-32) (30-34) (29-32) BMI (kg/m.sup.2) 25.0 26.1 NS 27.1 NS 25.9 NS (23.7-26.3) (25.1-27.0) (24.3-29.9) (24.9-26.9) Parity (n) 0.2 0.5* 0.82* 0.37* (0.02-0.32) (0.28-0.64) (0.23-1.41) (0.20-0.54) Systolic BP.sup.3 (mmHg) 123 161** 176** 157** (120-126) (157-165) (167-185) (153-160) Diastolic BP.sup.4 (mmHg) 77 101** 108** 99** (75-79) (99-103) (103-112) (97-101) Proteinuria (g/L) 0.02 2.32** 3.35** 2.08** (0.00-0.04) (2.02-2.61) (2.68-4.02) (1.77-2.39) Gestational age at 282 256** 212** 269** delivery (days) (279-285) (250-262) (199-225) (265-273) Twin pregnancies (n) 0 8 (8%) 2 (9%) 6 (8%) Gestational age at 281 253** 208** 266** sampling (days) (278-284) (247-260) (196-220) (262-270) Gestational Diabetes.sup.5 0 2 (2%) 0 .sup.1 (1%).sup.6 (n) Essential 0 3 (3%) 1 (5%) 2 (3%) Hypertension.sup.7 (n) IVF (n) 1 (2%) 8 (8%) 1 (5%) 7 (10%) ICSI (=n) 1 (2%) 1 (1%) 1 (5%) 0 Egg donor recipient (n) 0 1 (1%) 0 1 (1%) Medication to stimulate 0 2 (2%) 0 2 (3%) ovulation.sup.8 (n) NS: Not significant; *: p = <0.05; **: p = <0.001. .sup.1Early onset PE was defined as diagnosis before 34 + 0 weeks of gestation. .sup.2Late onset PE was defined as gestational week > 34 + 0. .sup.3Highest systolic blood pressure recorded within two weeks prior to delivery. .sup.4Highest diastolic blood pressure recorded within two weeks prior to delivery. .sup.5Gestational diabetes defined according to Swedish definition; fasting P-glucose 7.0 or OGTT with 2 hours P-glucose >12.2 mmol/L. .sup.6Time of diagnosis of PE was not known for one patient with gestational diabetes. .sup.7Essential hypertension was defined as blood pressure 140/90 before 20 weeks of gestation or condition known before pregnancy. .sup.8In one case not known, the other patient medicated with Pergotime.
TABLE-US-00002 TABLE 2 Patient demographics of PE cases and normal pregnancies (controls). Values are shown as mean (95% confidence interval) or number (%). Statistical comparison vs. controls. p-value <0.05 is considered significant. Normal pregnancy Early onset Late onset (Control; Preeclampsia PE.sup.1 PE.sup.2 Outcome n = 47) (n = 98) (n = 22) (n = 74) Birth weight 3602 2834** 1434** 3213** (gram) (3477-3726) (2621-3047) (1105-1764) (3045-3381) Fetal gender 23:24 46:49 NS 7:15 NS 37:34 NS (M:F) HELLP.sup.3 0 7 (7%) 3 (14%) 4 (5%) Eclampsia.sup.4 0 5 (5%) 2 (9%) 3 (4%) Induction (n) 10 (21%) 58** (59%) 2** (9%) 55** (75%) Vaginal 35 (75%) 46* (47%) 3* (14%) 43* (59%) delivery (n) Vacuum 8 (17%) 8* (8%) 0** 8** (11%) extraction (n) Cesarean 12 (26%) 47** (48%) 18** (82%) 27** (37%) section (n) SGA.sup.5 0 1 (1%).sup.5a 0 1 (1%) IUGR.sup.6 0 8 (8%) 5 (23%) 3 (4%) Admitted to 2 (4%) 32** (36%) 14** (82%) 18*** (25%) NICU.sup.7 (n) Neonatal 0 1 (1%) 1 (5%) 0 death Preterm.sup.8 (=n) 0 34** (35%) 20** (95%) 12** (16%) APGAR10.sup.9 9.80 9.75 NS 9.30* 9.90 NS (9.64-9.96) (9.62-9.89) (8.80-9.70) (9.70-10.0) NS: Not significant; *: p = <0.05; **: p = <0.001. .sup.1Early onset PE was defined as diagnosis before 34 + 0 weeks of gestation. .sup.2Late onset PE was defined as gestational week >34 + 0. .sup.3HELLP syndrome (Hemolysis, Elevated Liver enzymes, Low Platelets) diagnosed according to Mississippi classification. .sup.4Eclampsia was defined as seizures occurring during pregnancy and after delivery in the presence of PE. .sup.5SGA (Small for Gestational Age) defined as growth curve on Ultrasonography constant below curve. .sup.5aPatient defined as both SGA and IUGR. .sup.6IUGR (Intra Uterine Growth Restriction) was defined as 2 standard deviations (22%) on Ultrasonography or below 3.sup.rd percentile. .sup.7NICU (Neonatal Intensive Care Unit). .sup.8Preterm was defined as delivery before 36 + 6 weeks of gestation (258 days). .sup.9APGAR (Appearance, Pulse, Grimace, Activity, Respiration) score at 10 minutes.
Cell-Free Hb
[0258] The concentration of cell-free HbF, Hp-HbF and Hb-Total were analyzed in all plasma samples from women with PE and controls (Table 3). A 4-fold increase of the HbF concentration was seen in the PE patients (p-value 0.01) as compared to the controls. When subdividing the PE group into early and late onset PE an almost 5-fold increase in the HbF concentration was observed in the early onset PE group as compared to controls (p-value 0.006). In the late onset PE group, an almost 4-fold increase was observed as compared to controls, that was not statistically significant (p-value 0.17). A statistically significant increase in the mean Hp-HbF concentrations was observed for women with PE as compared to controls (p-value 0.018). This difference was not found when comparing early and late onset PE, although a clear trend towards an increase could be seen in the early PE group (p-value 0.15).
[0259] No significant difference in Hb-Total concentration was observed between PE vs. controls (p-value 0.53) or between early (p-value 0.80) and late onset PE (p-value 0.73) vs. controls.
TABLE-US-00003 TABLE 3 The mean plasma concentrations of the biomarkers in the PE group and normal pregnancies (controls). Statistical comparison vs. controls. Significance was calculated with non-parametric statistics (Mann-Whitney). Values are mean values with (95% Cl). A p-value <0.05 was considered significant. Normal pregnancy Bio- (Control; Preeclampsia Early onset Late onset marker n = 47) (n = 98) PE.sup.1 (n = 2) PE.sup.2 (n = 74) HbF 3.85 15.26 18.72 14.60 (ng/ml) (2.51-5.20) (7.0-23.6) (1.6-39.05) (5.10-24.0) p = 0.01 p = 0.006 p = 0.17 Hp-HbF 0.59 0.61 1.07 0.48 (g/ml) (0.003-1.18) (0.31-0.90) (0.10-2.24) (0.29-0.66) p = 0.018 p = 0.15 p = 0.02 Total-Hb 277 285 290 284 (g/ml) (232-321) (238-331) (152-430) (237-331) p = 0.53 p = 0.80 p = 0.73 Hp 1.17 0.97 1.34 0.89 (mg/ml) (1.04-1.30) (0.75-1.19) (0.39-2.30) (0.77-1.02) p = <0.0001 p = 0.067 p = 0.001 CD 163 461 485 433 508 (g/ml) (408-512) (445-527) (324-543) (465-551) p = 0.37 p = 0.35 p = 0.07 Hpx 0.93 0.69 0.69 0.69 (mg/ml) (0.88-0.98) (0.66-0.73) (0.61-0.77) (0.65-0.73) p = <0.0001 p < 0.0001 p < 0.0001 Hpx 0.80 0.59 0.81 0.54 activity (0.66-0.93) (0.49-0.69) (0.54-1.07) (0.44-0.65) p = 0.019 p = 0.96 P = 0.004 Heme 59.86 75.03 69.54 77.55 (g/ml) (52.34-67.38) (67.43-82.62) (55.07-84.02) (68.37-86.74) p = 0.01 p = 0.26 p = 0.02 HO -1 5.29 4.48 4.67 4.42 ng/ml (4.69-5.9) (4.04-4.93) (3.37-5.97) (4.69-5.89) p = 0.03 p = 0.02 p = 0.01 A1M 29.93 33.50 34.07 33.70 (g/ml) (27.89-31.97) (31.90-35.10) (30.31-37.83) (31.90-35.50) p = 0.035 p = 0.26 p = 0.03 .sup.1Early onset PE was defined as diagnosis before 34 + 0 weeks of gestation. .sup.2Late onset PE was defined as gestational week > 34 + 0
Hp and CD163
[0260] Analysis of the Hp concentration in plasma displayed that the increased HbF concentration in the PE patients was accompanied by a lower Hp concentration (Table 3). The results displayed a highly significant decrease in Hp concentration in plasma samples of women with PE as compared to controls (p-value<0.0001). In addition, late onset PE displayed a significant decrease as compared to the controls (p-value 0.001). In contrast, early onset PE showed a slight but not statistically significant increase in Hp concentration as compared to the controls (p-value 0.067).
[0261] Soluble, shedded CD163, the macrophage receptor mediating elimination of the Hp-Hb complex, was analyzed in plasma.sup.61-63. The analysis displayed a small but not significant (p-value 0.37) increase in the PE group as compared to the controls (Table 3). Subdividing the PE group into early and late onset PE, a small, not statistically significant, increase was observed in the late onset PE group (p-value 0.07 vs. the controls) whereas a small, not statistically significant, decrease was observed in the early onset PE group (p-value 0.35 vs. the controls).
Hpx
[0262] Analysis of the intravascular heme-scavenger protein Hpx displayed a highly significant decrease in plasma Hpx concentration of women with PE (p-value<0.0001) as compared to the controls (Table 3). Subdividing the PE group, displayed a significant decrease in both the early (p-value<0.0001) and late onset PE (p-value<0.0001) PE groups as compared to the controls.
[0263] The blood samples were also analyzed for Hpx activity. Plasma Hpx activity was measured in EDTA plasma samples using the Hpx-MCA substrate (synthesized by Pepscan, Lelystad, the Netherlands). The plasma samples (40 l) were diluted 1:4 with the substrate solution (0.2M Tris+0.9% NaCl pH 7.6 (substrate concentration 80 M/L)) to a final volume of 200 l at 37 C. The emission was measured at 460 nm on a Varioskan spectrophotometer (Thermo Fisher) after 30 min. of incubation (at 37 C.).
[0264] Hpx activity was measured spectrophotometrically at following time points: 0 min, 30 min (Hpx30), 60 min (Hpx60) and 24 hours. The measured value represented the total amount of serine sliced by Hpx at the given time point. If the value was <5 after 24 hours the activity was considered extremely low and the samples was expelled from further analysis due to probable damage to the sample. Area under the curve based on Hpx30 and Hpx60 was calculated.
Hpx Activity
[0265] 11 of the samples (8 controls and 3 PE) showed extremely low value after 24 hours of incubation and were therefore excluded from the analysis.
[0266] Hpx activity was significantly lowered in the PE groups compared to the controls group both after 30 min (p=0.02), 60 min (p=0.05) and HpxAUC (p=0.02) (Table 2). However, when dividing the PE patients into early- and late-onset PE it came clear that in the early-onset group Hpx30=0.81 and identical with the control group (Hpx30=0.80) (Table 4). In contradiction to this the late onset group showed an even more markedly decrease in Hpx activity than PE in general concerning all Hpx activities (Hpx30=0.54) (Table 4).
[0267] Interesting the ratio between Hpx concentration and Hpx activity can be used to evaluate the risk of developing early or late onset PE. As seen from the table above, the ratio for normal pregnancies is 1.16, whereas it is 0.85 for early onset of PE and 1.28 for late onset of PE. Thus, it the ratio is lower compared to control, i.e. 1 or less then there is an increased risk of developing early onset PE, whereas if the ratio is 1.2 or more there is an increased risk of developing late onset PE, and the Hpx activity is measured as described herein as Hpx30.
Results for Hpx.
[0268]
TABLE-US-00004 TABLE 4 Early Late Controls Preeclampsia onset PE onset PE (n = 39) (n = 96) (n = 17) (n = 72) Hpx activity 30 0.80 0.59 0.81 0.54 (0.66-0.93) (0.49-0.69) (0.54-1.07) (0.44-0.65) p = 0.019 p = 0.96 p = 0.004 Hpx activity 60 1.36 1.09 1.33 1.04 (1.09-1.62) (0.97-1.22) (1.06-1.61) (0.89-1.19) p = 0.046 p = 0.92 p = 0.02 Hpx activity 0.74 0.57 0.74 0.53 AUC (0.61-0.87) (0.49-0.65) (0.55-0.93) (0.44-0.62) p = 0.022 p = 0.99 p = 0.007 Hpx plasma 0.93 0.69 0.69 0.69 concentration.sup.1 (0.88-0.98) (0.66-0.73) (0.61-0.77) (0.56-0.73) p < 0.0001 p < 0.0001 p < 0.0001 .sup.1Previously mentioned herein
Correlation Analysis.
[0269] Hpx activity was not correlated to Hpx plasma concentration (p=0.74 for Hpx30). This was neither the case in the early onset group (p=0.17) nor the late onset PE group (p=0.24).
[0270] Hpx30 was significantly correlated to diastolic blood pressure in all patients (p=0.04) and there was a clear tendency towards correlation for Hpx60 (p=0.1) and HpxAUC (p=0.06) (Table 4). When the early-onset patients were expelled from the analysis there was a clear correlation between diastolic blood pressures and each of Hpx30, Hpx60 and HpxAUC (Table 5,
Results for Blood Pressure:
[0271]
TABLE-US-00005 TABLE 5 Hpx30 Hpx60 HpxAUC All patients Systolic blood p = 0.35 NS p = 0.53 NS p = 0.45 NS pressure Diastolic blood CF = 0.18 p = 0.10 NS CF = 0.17 pressure p = 0.04 p = 0.06 NS Late onset PE and controls Systolic blood CF = 0.17 p = 0.17 NS p = 0.11 NS pressure p = 0.07 Diastolic blood CF = 0.25 CF = 0.20 CF = 0.23 pressure p = 0.009 p = 0.04 p = 0.02 CF: Pearson's correlation factor.
[0272] In concordance to previous findings we found decreased Hpx activity in patients with manifest PE. However we did only find Hpx activity to be decreased in patients with late-onset PE but not in early-onset PE. Contrary to this and as described herein, Hpx protein concentration has been shown to be statistically significantly decreased in both early and late onset PE. Correlation analysis showed statistically significant inverse correlation between Hpx30 and diastolic blood pressure in all the patients and there was a tendency towards the same inverse correlation for Hpx60 and HpxAUC (Table 5). When only analyzing the correlation in the controls and late onset groups together there was statistically significant correlation between all of Hpx-activities and diastolic blood pressure and a tendency towards statistically significant correlation to systolic blood pressure.
A1M
[0273] Analysis of plasma levels of the heme- and radical scavenger A1M displayed a significant increase of plasma A1M concentration in women with PE (p-value 0.035) as compared to controls (Table 3). Subdividing the PE group, a statistically significant increase was observed in the late onset PE group (p-value 0.03) and a clear, but not statistically significant, increase was seen in the early onset PE group (p-value 0.26).
Correlation Cell-Free HbF and Hp
[0274] The correlation between plasma cell-free HbF and Hp levels was evaluated. A negative correlation was found, i.e. an increased plasma cell-free HbF concentration was associated with a decreased plasma Hp concentration, when including all patients, controls and women with PE (r=0.335, p-value<0.0001, n=145)(
Association Between Hp Isoforms and Level of Cell-Free HbF, Hpx and A1M
[0275] We identified the predominant Hp-isoform (1-1, 1-2 or 2-2) in the patient plasma samples using Western blot (
Correlation Analysis Between Biomarkers and Disease Severity
[0276] Correlation analysis using Pearson's correlation coefficient showed highly significant inverse correlation between Hpx and blood pressure, both systolic (r=0.511, p-value<0.00001, n=145) and diastolic (r=0.520, p-value<0.00001, n=145)(
Evaluation of Biomarkers as Diagnostic Tools and Clinical Predictors
[0277] A logistic regression models was used to evaluate the usefulness of the described biomarkers as diagnostic markers of PE. Comparing women with PE vs. controls, a significant difference was detected for HbF, A1M and Hpx (p-value<0.0001) but not for Hp and CD163. Each of the significantly altered biomarkers were able to diagnose PE (adjusted for gestational age) but Hpx showed the high level of significance and a diagnostic detection rate of 64% at a false positive rate of 5% with an AUC of 0.87 (Table 6,
TABLE-US-00006 TABLE 6 Sensitivity and specificity values for the combination of 1) HbF, A1M and Hpx, 2) A1M and Hpx and 3) Hpx alone. Detection rates for PE at different false positive rates and AUC for the ROC curve. Calculations are for all PE vs. controls. False positive HbF combined with A1M combined rate A1M and Hpx.sup.1 with Hpx.sup.2 Hpx 5% 69% 66% 64% 10% 69% 67% 70% 20% 81% 81% 75% 30% 83% 85% 79% AUC 0.88 0.87 0.87 .sup.1Based on logistic regression including all three parameters. .sup.2Based on logistic regression including both parameters.
Prediction of Fetal and Maternal Outcomes
[0278] Beside the test of the biomarkers to support diagnosis of PE we examined whether the biomarkers could predict a range of fetal and maternal outcomes. This was done with a logistic regression model similar to the model of PE. The tested fetal outcomes were: admission to NICU, IUGR and prematurity. The tested maternal outcomes were: induction of labor, cesarean section and vacuum extraction. The biomarkers HbF (p-value 0.001), Hpx (p-value 0.008) and Hp (p-value 0.03) each showed potential as predictive biomarkers of admission to NICU. However, in a combined logistic regression model they turned out insignificant. The biomarkers Hpx (p-value 0.0003, AUC=0.71) and CD163 (p-value 0.03, AUC=0.61) showed potential as biomarkers of prematurity. In combination these two biomarkers proved significant with a slightly stronger association to prematurity (p-value 0.001 and p-value 0.025, AUC 0.72).
[0279] None of the biomarkers showed any predictive value concerning induction of labor or vacuum extraction. Hpx displayed a significant association with Cesarean section (p-value 0.009, AUC 0.62).
TABLE-US-00007 TABLE 7 Area under the ROC-curves (AUC) for fetal outcomes (admittance to Neonatal Intensive Care Unit (NICU) and prematurity) and maternal outcomes (risk of cesarean section). The fetal outcome and the maternal outcomes induction of labor and vacuum extraction were not significantly related to any of the biomarkers. All calculations were based on univariable logistic regression analysis. Significance AUC Admittance to NICU HbF 0.001 0.69 Hp 0.03 0.62 Hpx 0.008 0.66 Prematurity Hpx 0.001 0.70 CD 163 0.04 0.61 Combination 0.001 0.72 Hpx + CD 163 0.025 Cesarean section Hpx 0.009 0.62
Study IISampling at Gestational Week 6-20
Patients and Samples
[0280] The study was approved by the ethical committees at St Georges University Hospital, London, UK. All participants signed a written informed consent prior to inclusion. Women attending a routine antenatal care visit at St. Georges Hospital Obstetric Unit, London were recruited during the years 2006 and 2007.
[0281] Gestational length was calculated from the last menstrual period and confirmed by ultrasound crown-rump-length measurement. A maternal venous blood sample was collected at 6-20 weeks of gestation (mean 13.7) in a 5 ml vacutainer tube (Becton Dickinson, Franklin Lakes, N.J.) without additives. After clotting, the samples were centrifuged at 2000g at room temperature for 10 minutes and serum was separated and stored at 80 C. until further analysis.
[0282] All pregnancy outcome-data was obtained from the main delivery suite database and checked for each individual patient. PE was defined as in Study I herein.
[0283] As in Study I, normal pregnancy was defined as delivery at or after 37+0 weeks of gestation with normal blood pressure. The uncomplicated pregnancy (control) samples were recruited as consecutive cases during the same time period.
Measurement of Total Hb, HbF, A1M, Hp and Hpx
[0284] HbF-concentration in serum samples (La cell-free HbF) was measured with a sandwich ELISA using polyclonal antibodies as described in Study I. The A1M concentration was determined by a radioimmunoassay as described in Study I. Hb-Total, Hp and Hpx concentration were serum samples using ELISA Quantification Kit for respective marker as described in Study I.
Statistical Analysis
[0285] SPSS statistics version 21.0 for Apple computers was used along with the statistical software R studio Version (0.98.1062). A p value 0.05 was considered significant in all analyses. Significant differences between the groups for the biomarkers HbF, Hb-Total, Hp, Hpx, and A1M were calculated with one-way ANOVA. Due to differences in gestational age when Doppler ultrasound was performed in the PE and control groups, UtADs were transformed into Multiples of the Median (MoM)-values according to mean values given by Velauthar et al.sup.64.
[0286] Stepwise regression analysis is a commonly used method for developing prediction models but has been criticized.sup.65. We therefore attempted to validate the results also by developing prediction models by two more recently developed statistical methods, Lasso regression and boosted tree regression and compare these methods in terms of their prediction capability. The methods were compared by area under the ROC-curve. In order to validate the prediction results the dataset was randomized into a training cohort () which was used for developing the prediction models and a test cohort () used for testing their predictive ability.
[0287] The final models of the biomarkers and maternal characteristics were built on backwards stepwise logistic regression. Separate analyses were performed for early onset PE and late onset PE. For all regression parameters and the parameters in combination ROC-curves were performed and the prediction rates (PR) at different false positive rates (FPR) were calculated. The optimal prediction rate/FPR was defined as the point in the ROC-curve closest to the upper left corner.
Results
Demographics
[0288] In total, 520 women were included, out of which 86 developed PE (cases), 65 had spontaneous preterm birth (SPTB), 7 were complicated by IUGR, 10 developed pregnancy induced hypertension (PIH), 1 patient had IUGR and placental abruption, 3 had isolated placental abruption (without PE or IUGR), 2 had essential hypertension. 347 women with uncomplicated pregnancies and term delivery (>37 weeks of gestation) were included as controls.
[0289] The maternal characteristics are shown in Table 8. Of the 86 women who developed PE, 28 were delivered before 37+0 weeks of gestation. Out of these, 17 were delivered before 34+0 weeks of gestation showing a significantly lower birth weight compared to controls. The groups essential hypertension without PE (n=2) and abruption (n=3) were excluded from the following analysis due to small sample size.
TABLE-US-00008 Pregnancy Essential hypertension Spontaneous Control Preeclampsia IUGR induced hypertension (without PE) preterm birth Abruption (n = 347) (n = 86) (n = 7) (n = 10) (n = 2) )n = 64) (n = 3) Ethnic origin Caucasian (304) 252 41 4 6 2 34 1 South Asian (70) 36 18 2 1 0 13 0 Black (54) 20 21 0 3 0 10 0 East Asian (4) 3 0 0 0 0 1 0 Mixed (19) 13 2 0 0 0 4 0 Not known (38) 23 4 1 0 0 3 2 p < 0.000001* p = 0.51 NS 0.06 NS p = 0.004* Gravidae 1.46 2.73 3.33 3.44 1.0 2.71 4.67 (1.36-1.56) (2.32-3.14) (1.15-7-82) (0.03-6.86) (1.0-1.0) (2.2-3.21) (3.32-12.65) p < 0.0001 p < 0.0001 p < 0.0001 p = 0.49 NS p < 0.0001 p < 0.0001 Para 0.11 1.14 0.67 2.11 0.0 0.74 0.67 (Mean-95% CI) (0.06-0.16) (0.78-1.14) (0.19-1.52) (0.85-5.07) 1.0 (0.0-0.0) (0.45-1.03) (2.2-3.5) p < 0.0001 P = 0.003 p < 0.0001 p = 0.73 NS p < 0.0001 p = 0.04 Body Mass Index 23.4 26.9 27.4 28.2 20.5 23.5 21.1 (22.9-23.9) (25.5-28.35) (20.9-33.8) (21.7-34.8) (3.3-37.6) (21.9-25.1) (16.2-25.9) p < 0.0001 p = 0.02 P = 0.001 p = 0.36 NS p = 0.88 NS p = 0.37 NS GA at ultrasound 12.5 18.5 20.4 16.3 11.6 20.4 19.0 scanning (12.4-12.6) (17.5-19.5) (15.9-25.0) (12.4-20.3) (7.9-15.2) (19.5-21.4) (5.8-32.3) (Mean-95% CI) p < 0.0001 p < 0.0001 p < 0.0001 p = 0.10 NS p < 0.0001 p < 0.0001 GA at blood 13.5 13.9 13.6 13.8 12.0 14.1 13.2 sampling (Mean- (13.3-13.8) (13.3-14.6) (10.5-16.7) (12.3-15.4) (24.3-48.3) (13.4-14.8) (7.9-18.6) 95% CI) p = 0.17 NS p = 0.94 NS P = 0.66 NS 0.34 NS p = 0.09 NS p = 0.83 Fetal gender Male 185 49 2 4 1 32 2 Female 161 36 4 6 1 32 1 p = 0.44 NS* p < 0.0001 * p = 0.69 NS* NS* p = 0.8 NS* NS* Birth weight 3467 2716 1791 2810 3260 2324 1838 (3415-3520) (2485-2947) (1214-2369) (2090-3531) (3414-3518) (2160-2488) (47-3629) p < 0.0001 p < 0.0001 p < 0.0001 p = 0.56 NS p < 0.0001 p < 0.0001 Prematurity (%) 0% 28 (33%) 7 (100%) 4 (40%) 0 (0%) 60 (100%) 2 (100%) p < 0.0001 p < 0.0001 p < 0.0001 Mean GA at delivery 40.4 36.7 34.8 37.0 39.4 34.6 32.8 (40.3-40.5) (35.7-37.8) (32.6-37.0) (34.2-39.9) (34.8-43.9) (33.8-35.3) (25.6-40.9) p < 0.0001 p < 0.0001 p < 0.0001 p = 0.25 NS p < 0.0001 p < 0.0001 Diabetes Yes 0 3 0 1 0 0 0 No 346 83 7 9 2 65 2
[0290] There was no statistically significant difference between the cases and control groups in terms of time of serum sampling.
Biomarkers
[0291] The serum levels of the biomarkers HbF, Hp, A1M, Hb-Total, Hp and Hpx are shown in Table 9.
[0292] Table 9 shows the mean concentrations with 95% confidence interval of the biochemical markers cell-free HbF, A1M, Hb-Total, Hp, Hpx and Uterine artery Doppler ultrasound Pulsatility Index (UtAD PI) Multiples of the Median (MoM). P-values were calculated with one-way ANOVA as compared to the control group. Analysis of the patient group's pregnancy induced hypertension and IUGR did not show any significant differences to the controls group.
TABLE-US-00009 Spontaneous Controls Preeclampsia preterm birth (n = 346) (n = 86) (n = 65) Biomarker (95% Cl) (95% Cl) (95% Cl) HbF (g/ml) 5.6 10.8 3.5 (4.2-7.4) (5.2-16.5) (2.3-4.8) p = 0.02 p = 0.25 NS A1M (g/ml) 15.5 17.3 14.1 (14.9-16.1) (15.5-19.2) (12.7-15.5) p = 0.03 p = 0.08 NS Hb-Ttotal (g/ml) 297 258 201 (257-337) (160-358) (158-244) p = 0.47 NS p = 0.05 Hp (g/ml) 971 1102 998 (915-1028) (991-1131) (863-1133) p = 0.089 NS p = 0.73 NS Hpx (g/ml) 1143 1062 1061 (1111-1175) (992-1132) (992-1130) p = 0.05 p = 0.05 UtAD PI MoM 0.98 1.18 0.94 (0.92-0.99) (1.04-1.31) (0.87-1.02) p < 0.0001 p = 0.84 NS
[0293] The mean concentration of HbF in the PE group (10.8 g/ml, p=0.02) was significantly higher than in the control group (5.6 g/ml). HbF is total HbF as compared to mainly non-complexed HbF described in Study I. The mean A1M concentration was also significantly increased (17.3 g/ml vs. 15.5 g/ml, p=0.03). The mean Hpx concentration in the PE group was significantly lower, 1062 g/ml, compared to 1143 g/ml in the control group (p=0.05). There was a tendency towards a slightly higher Hp concentration in the PE group (1102 g/ml) as compared to the control group (971 g/ml), however not significant (p=0.089). The PIH or IUGR showed comparable levels to the controls (data not shown). The SPTB group presented significantly lower levels of Hb-Total (201 g/ml vs. 297 g/ml, p=0.05) and Hpx (1061 g/ml vs. 1143, p=0.05). The UtAD MoM values were significantly higher in the PE group than the controls (1.18 vs. 0.95 p<0.0001).
Logistic Regression Analysis
[0294] The abilities of the biomarkers to predict PE were tested in logistic regression models. Corresponding ROC-curves were generated to visualize the prediction values. All biomarkers were individually tested as well as evaluated in combination to find the optimal predictive value. The significant results are outlined in Table 10 and the ROC-curves are shown in
TABLE-US-00010 TABLE 10 Prediction rates (PR) at different false positive rates (FPR) for each of the different biomarkers, the UtAD Pulsatility (PI) MoM values and the maternal characteristics. All prediction values are derived from ROC-curves based on stepwise logistic regression models. Optimal Model AUC (95% CI) 5% 10% 20% 30% (PR/FPR) HbF* 0.65 13% 15% 35% 50% 60%/35% (0.58-0.71) A1M* 0.58 7% 19% 22% 35% 57%/46% (0.5-0.66) Hp 0.58 9% 17% 30% 49% 53%/38% (0.5-0.66) Hpx* 0.58 9% 17% 28% 45% 40%/28% (0.5-0.66) UtAD PI MoM 0.60 18% 25% 39% 49% 48%/27% (0.52-0.68) HbF* + A1M* + 0.73 22% 33% 43% 59% 66%/22% Hp* + Hpx* (0.66-0.8) Maternal characteristics* 0.85 52% 60% 68% 79% 73%/23% (0.8-0.9) UtAD* + Maternal 0.82 51% 57% 69% 80% 78%/27% characteristics* (0.75-0.89) UtAD* + Bimarkers* 0.76 23 40 51 63 61%/24% (0.68-0.83) 26% 42% 58% 67% Biomarkers + 0.83 60% 62% 68% 81% 81%/26% Maternal characteristics* (0.75-0.91) Biomarkers + 0.79 47% 53% 71% 74% 71%/19% Maternal characteristics + (0.71-0.87) UtAD
[0295] Despite a significantly increased serum HbF concentration in patients who subsequently developed PE, it displayed limited predictive value when used alone (PR of 15% at FPR of 10%). A1M showed a similar prediction (PR of 19% at FPR of 10%). Hpx displayed the best individual prediction rates for PE (PR of 42% at FPR at 10%). The optimal prediction rate was obtained by combining A1M, HbF, and Hpx (PR of 62% at FPR of 10%).
[0296] All measures of maternal characteristics were tested alone and in combination using a logistic regression analysis to compare PE and controls.
[0297] The combination of maternal characteristics (parity, diabetes, pre-pregnancy hypertension) and the biomarkers (HbF, A1M and Hpx) increased the PR to 62% at an FPR of 10% (Table 10,
Early-vs. Late Onset Preeclampsia
[0298] We found elevated levels of HbF in both the early- and late onset PE groups (Table 11).
TABLE-US-00011 TABLE 11 The mean concentrations of biomarkers in the sub-groups early onset PE (def.: delivery 34 + 0 weeks of gestation) and late onset PE (def.: delivery >34 + 0 weeks of gestation). P-values were calculated with one-way ANOVA as compared to the control group. Early onset Late onset Controls Preeclampsia preeclampsia (n = 346) (n = 16 (10)) (n = 64) Biomarker (95% Cl) (95% Cl) (95% Cl) HbF (g/ml) 5.6 13.7 10.1 (4.2-7.4) (6.8-34.2) (4.8-15.4) p = 0.05 p = 0.04 A1M (g/ml) 15.5 15.4 17.8 (14.9-16.1) (12.5-18.4) (15.6-20) p = 0.98 NS p = 0.01 HbTotal (g/ml) 297 154 280 (257-337) (67-241) (162-399) P = 0.23 NS p = 0.78 NS Hp (g/ml) 971 1108 1101 (915-1028) (673-1542) (943-1258) P = 0.43 NS p = 0.12 NS Hpx (g/ml) 1143 947 1085 (1111-1175) (757-1137) (1009-1162) p = 0.04 p = 0.22 NS UtAD PI MoM 0.95 1.63 1.06 (0.92-0.99) (1.2-2.06) (0.94-1.19) p < 0.00001 p = 0.06 NS: not significant
[0299] The A1M levels were only significantly higher in the late onset group (p=0.01) (Table 11). The Hpx protein concentration was lower in both groups but only significant in the early onset PE group (p=0.04) (Table 11). UtAD PI MoM was significantly elevated especially in the early onset group (1.63 vs. 0.95, p<0.00001) but only marginally elevated in the late onset group and this difference was not statistically significant (1.06 vs. 0.95, p=0.06). There were no significant differences for Hb-Total or Hp in either of the study groups.
[0300] The logistic regression models for early- and late onset PE for the examined biomarkers showed a prediction rate for HbF of 23% at an FPR of 10%but only in the late onset PE group. A1M was only statistically significant in the late onset group (p=0.01) and Hpx was only statistically significant for the early onset group and showed a PR of 32% at a FPR of 10%.
[0301] UtAD performed best in the early onset group with a PR of 57% at FPR 10% but was even statistically significant in the late onset group.
[0302] None of the biomarkers were statistically significant in combination with each other, with maternal characteristics or UtAD in either of the early- or late onset groups.
Discussion
[0303] The aim of this study was to validate previous findings indicating that serum levels of cell-free HbF and A1M are elevated already in the first trimester of pregnancy and that they are useful as predictive first trimester biomarkers for the subsequent development of PE. The cohort size in this study is larger and reflects the normal incidence of PE better. In addition, the study also evaluates impact of the biologically related heme- and Hb-scavenging proteins Hp and Hpx.
[0304] The main finding in this paper confirms that both HbF and A1M are significantly elevated in serum from pregnant women who subsequently develop PE (Table 9). The increased serum concentrations of HbF are probably caused by a defect placental hematopoiesis reflecting placental oxidative stress. The data indicate that HbF and A1M have a potential as predictive first and early second trimester biomarkers for PE. Furthermore, the heme scavenger Hpx also show good predictive values and is therefore also suggested as an additional potential biomarker for PE. The UtAD indices primarily showed higher PI MoM values in the early onset group. This is in full concordance with previously published results from several research groups. The higher PI in the early onset group reflects increased resistance in the uterine arteries as a result of shallow invasion of the maternal decidual spiral arteriesa hallmark of early onset PE, but less common in late onset PE.
[0305] Interestingly, data showed that cell-free Hb-Total and Hpx were significantly lowered in patients who delivered prematurely. Low enzymatic activity of Hpx is known to attenuate endothelial inflammation. Lower levels of Hpx could therefore contribute to the increased maternal inflammation seen in both PE and preterm birth. Future studies are needed to more carefully decipher the role of Hpx in prematurity.
[0306] Specific first trimester screening for adverse pregnancy outcome is very important as it gives clinicians a tool to target and individualize surveillance of the patients rather than general screening programs later in pregnancy. By identifying high-risk pregnancies, preventive strategies and prophylactic treatment can be initiated. Up to date, the only prophylactic treatment is low dose acetyl salicylic acid (ASA). If the treatment is initiated before 16 weeks of gestation there is a markedly risk reduction (RR=0.47) especially for early onset and severe PE. The number needed to treat (NNT) may be as low as 7 for preventing severe PE in identified high-risk pregnancies. The use of ASA is cheap and has few side effects when given in the low doses recommended (75 mg). The prophylactic treatment should be initiated at the end of first trimester to have the optimal effect. In view of this, it is preferable if PE can be predicted at the end of first trimester or in the beginning of second trimester, possibly combined with other established screening programs for Down's syndrome.
CONCLUSIONS
[0307] HbF, A1M and Hpx measured in maternal serum at the end of first and early second trimester of pregnancy are potential predictive biomarkers for subsequent development of PE. The three proteins are physiologically relevant, since increased amounts of cell-free HbF have been described to be involved in pathogenesis, and potentially consumes the physiological heme-scavenging proteins. Furthermore, the prediction power of the three biomarkers is increased by combination with uterine artery Doppler ultrasound and/or maternal characteristics.
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