Hum. Reprod. Advance Access published online on April 16, 2007
Human Reproduction, doi:10.1093/humrep/dem095
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First trimester maternal serum ischaemia-modified albumin: a marker of hypoxia-ischaemia-driven early trophoblast development
1 Fetal Medicine Unit, Division of Obstetrics and Gynaecology, St. George's University of London, London SW17 0RE, UK 2 Department of Clinical Biochemistry, St George's Hospital NHS Trust, London SW17 0QT, UK
3 Correspondence address. Tel: +44 20 8725 0071; Fax: +44 20 8715 0079; E-mail: basky{at}pobox.com
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Abstract |
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BACKGROUND: A hypoxic intrauterine environment is believed to play a pivotal role in physiological trophoblast development. Ischaemia-modified albumin (IMA) is used in the measurement of cardiac ischaemia. We aimed to test the hypothesis that maternal serum IMA may be elevated in early pregnancy as a measurable manifestation of intrauterine ischaemia.
METHODS: Prospective observational study in healthy women with singleton pregnancies (n = 66) and non-pregnant controls (n = 26). Maternal serum IMA levels were measured at 11–13 weeks of gestation and in non-pregnant women.
RESULTS: The median IMA level in the pregnant group [115.14 kU/l; interquartile range (IQR) 102.33–124.71 kU/l] was significantly higher (P < 0.001) than in non-pregnant controls (73.71 kU/l; IQR 60.38–82.78 kU/l). During pregnancy, absolute values of IMA were higher than the concentration used for the diagnosis of myocardial ischaemia (>95 kU/l) in 86% of women.
CONCLUSIONS: In early pregnancy, IMA levels were above the concentration used for the diagnosis of myocardial ischaemia in most women, and should therefore not be used as a marker for cardiac ischaemia in pregnancy. Maternal serum IMA is elevated to supra-physiological levels in early normal pregnancy supporting the hypothesis that normal trophoblast development is associated with a hypoxic intrauterine environment, although other mechanisms leading to an IMA increase cannot be excluded.
Key words: first trimester/hypoxia/serum/ischaemia-modified albumin/trophoblast
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Introduction |
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There is experimental evidence that pregnancies normally develop in a relatively hypoxic intrauterine environment and that subsequent reperfusion and oxidative stress is important for physiological trophoblast development (Roberts and Hubel, 1999
; Redman and Sargent, 2000; Jauniaux et al., 2001; Jauniaux et al., 2003a). Oxidative damage as a consequence of increased ischaemia and reperfusion is implicated in the pathogenesis of pre-eclampsia, although the precise mechanism is not known (Roberts and Hubel, 1999; Redman and Sargent, 2000). Ischaemia-modified albumin (IMA) is a protein used in the measurement of cardiac ischaemia in acute coronary syndromes (Anwaruddin et al., 2005). There is evidence that reactive oxygen species (ROS), produced during ischaemia/reperfusion, can generate the highly reactive hydroxyl radical, resulting in site-specific modification to the N-terminus of the albumin moiety, especially at the N-Asp-Ala-His-Lys sequence, thereby producing IMA (Marx and Chevion, 1985; Gidenne et al., 2004; Roy et al., 2006). This occurs within minutes of the tissue insult, and its relatively short half-life makes it a marker of ongoing ischaemia. Measurement of this protein has a very high sensitivity and negative predictive value, making it a useful biomarker of myocardial ischaemia (Anwaruddin et al., 2005). The aim of the present study was to determine whether the proposed relative intrauterine hypoxia of early pregnancy influences maternal circulating IMA levels. |
Materials and Methods |
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Venous serum samples were collected from healthy women with singleton pregnancies attending for routine first trimester ultrasound dating at 11–13 weeks gestation, and from non-pregnant healthy women of reproductive age. In all cases, 5 ml of maternal blood were drawn into a non-heparinized tube, allowed to clot and centrifuged. Women with a known medical condition (e.g. diabetes mellitus, connective tissue disease, essential hypertension and cardiac disease) or a history of recurrent miscarriage were excluded from the study. Local ethical committee approval was obtained for the study, and all women gave written, informed consent. All pregnancy outcomes were obtained from the main delivery suite database and checked by review of the individual patient records. Normal pregnancy outcome was defined as delivery after 37 weeks, where the neonatal birth weight was above the 10th centile for gestation, and where there was no occurrence of hypertensive disease of pregnancy.
IMA was measured by the albumin cobalt-binding test (ACB test) on the Roche Cobas MIRA PLUS instrument. Serum specimens collected immediately after the Doppler ultrasound scan were frozen at –20°C or colder within 2 h. Frozen samples were gently vortexed after thawing. Specimens handled in this way showed no significant difference in assay results from the fresh specimens. In the ACB test, 95 µl of a patient sample and 5 µl of cobalt chloride (Co(II)) are incubated for 5 min. During incubation, the Co(II) binds to the N-terminus of unaltered albumin in the sample; albumin for which the N-terminus is altered as a result of ischaemic processes binds to the Co(II) to a far lesser extent. After incubation, 25 µl of dithiothreitol (DTT) is added to the mixture. DTT forms a coloured complex with Co(II) that is not bound at the N-terminus of albumin, and this complex is measured spectrophotometrically at 500 nm. Duplicate IMA values were obtained with the mean recorded as the result of the assay. In our laboratory, the ACB test within-run duplicate coefficient of variation % of patient samples averaged 1.9% (range 0.0–6.5%). In order to exclude any concurrent cardiac ischaemic process in these women, cardiac troponin T (cTnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were measured in these women, using standard laboratory techniques previously described (Hallermayer et al., 1999; Collinson et al., 2004). Median (interquartile range) was used to express data and the Mann–Whitney and Fischer's exact test were used, as appropriate to compare groups. Two-sided P-values are reported.
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Results |
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Maternal serum IMA was measured in 66 pregnant women at a median gestational age of 12.4 weeks (range 11.9–13.7 weeks), and from 26 non-pregnant controls. The median IMA level in the pregnant group 115.14 kU/l [interquartile range (IQR) 102.33–124.71 kU/l] was significantly higher (P < 0.001) than in non-pregnant controls (73.71 kU/l; IQR 60.38–82.78 kU/l, Fig. 1). During pregnancy, absolute values of IMA were higher than the concentration used for the diagnosis of myocardial ischaemia (>95 kU/l) in 57 (86%) women, while in the non-pregnant group this was the case in only one (4%) case (P < 0.00001). Serum cTnT and NT-proBNP concentrations were all in the normal range for a healthy adult.
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Discussion |
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The N-terminus of circulating albumin is altered during tissue ischaemia, to produce IMA. This protein is used as a sensitive and specific marker for cardiac ischaemia, where circulating IMA levels are known to be increased (Anwaruddin et al., 2005
). IMA has a relatively short half-life, but it is produced continually during an ischaemic event thereby maintaining serum high IMA levels. Rarely, IMA has been shown to be elevated in conditions associated with tissue ischaemia other than acute coronary syndromes such as extreme endurance physical exercise (Apple et al., 2002; Apple, 2005; Lippi et al., 2005). The data of this study represent the first demonstration of elevated IMA levels in a physiological condition, namely the first trimester of normal pregnancy. At the time of examination all women were healthy and asymptomatic and had no history of hypertension or ischaemic cardiac disease. In addition, cTnT and NT-proBNP were within the normal range in all cases indicating that underlying cardiac conditions were unlikely to be the cause for the supra-physiological IMA levels.
The finding that IMA levels in the first trimester of pregnancy are well above the normal range used to diagnose cardiac ischaemia may be indicative of uterine ischaemia present in early pregnancy, in support of the hypothesis that early human placentation is associated with a hypoxic intrauterine environment. In early pregnancy, the highly muscular, small-caliber uterine spiral arteries are plugged by endovascular trophoblast derived from the extravillous trophoblast cells of the anchoring villi. The plugging of decidual vessels by the endovascular trophoblast produces a relatively hypoxic uterine environment, and this has been confirmed by direct measurement of low partial pressures of oxygen within the placenta during first trimester termination of pregnancy. The hypoxic intrauterine environment is thought to protect the trophoblast from the oxidative stress-induced damage of a normoxemic environment (Jauniaux et al., 2003b). Although the placental oxygen tension rises after the development of a feto-maternal circulation from about 14 weeks, the partial pressure of intraplacental oxygen remains low compared to the maternal circulation (Jauniaux et al., 2001). The oxidative stress that results from the post-ischaemia reperfusion may well have a physiological role in normal pregnancy development (Caniggia et al., 2000). Poor trophoblast invasion and subsequent spiral artery vasoreactivity is thought to result in a more ischaemic environment. Subsequent reperfusion and exaggerated oxidative stress is thought to predispose to the development of pre-eclampsia in later pregnancy (Burton and Jauniaux, 2004). The latter hypothesis forms the rationale for the use of anti-oxidants to prevent pre-eclampsia in women at high risk. Despite initial optimism, two recent randomized placebo controlled trials of vitamins C and E did not reduce the frequency of pre-eclampsia (Poston et al., 2006; Rumbold et al., 2006), and there was a suggestion of harm, with among others, higher risks of delivering a low-birthweight neonate and stillbirth (Poston et al., 2006). It could be hypothesized that this was due to inappropriate suppression of oxidative stress required for normal placentation in pregnancies that were destined to be normal.
Alternatively, the observed increase in IMA concentrations at 11–13 weeks may be transiently produced by the ROS generated in association with the rise in oxygen concentrations induced by the onset of maternal blood circulation in the intervillous space (Jauniaux et al., 2003a). Before this event, the trophoblasts growing in a hypoxic environment are not necessarily hypoxically stressed, as they are adapted to this condition and employ unusual metabolic pathways to maintain their energy levels (Jauniaux et al., 2005). However, it has been recently hypothesized that IMA production itself, by increasing the concentration of biological active free cobalt in plasma, might increase the activity of hypoxia-inducible factor-1
(Xi et al., 2004), and represent therefore not just an indicator of hypoxic stress but rather an efficient endogenous mechanism of response to ischaemia (Lippi et al., 2006). The relationship between trophoblast hypoxia and IMA production would therefore be better clarified by future research investigating IMA variations before and after the onset of the intervillous circulation, and later in the second and third trimester. Moreover, our interpretation of the findings is based on the assumption that maternal serum IMA levels reflect intrauterine hypoxic processes. It must be kept in mind, however, that some well described systemic adaptations to pregnancy, such as the inflammatory response evident since the fourth week (Sacks et al., 1998, 2004), might be associated with raised IMA. Ideally, our assumption should be invasively verified by comparing peripheral venous blood concentrations of IMA to those obtained by selective uterine venous blood sampling.
Another manifestation of placental development is uterine artery blood flow, which can be measured non-invasively by the use of Doppler ultrasound. It has been shown that the proportion of decidual vessels with endovascular trophoblast invasion is significantly less in women with high uterine artery resistance when compared to those with low resistance. Any correlation with maternal serum IMA would make this an early useful marker of impaired placentation, but also suggest an etiological expression of abnormal oxidative stress. There is mounting interest in the ability to identify women at risk of this pregnancy complication in the first trimester, as there is evidence that the prophylactic properties of low-dose aspirin are particularly useful when treatment is started early (Vainio et al., 2002; Chiaffarino et al., 2004). Whether elevated maternal serum IMA in early pregnancy is different in pregnancies that are subsequently complicated by pre-eclampsia, remains to be established. If abnormal fluctuations in the ischaemia/reperfusion of early pregnancy are etiological in the development of pre-eclampsia, IMA may well be a marker for high-risk pregnancies.
IMA is elevated to supra-physiological levels in the first trimester of normal pregnancy and should therefore not be used as a diagnostic marker of acute coronary syndromes in pregnant women. Although other mechanisms leading to IMA increase cannot be excluded, the high levels of IMA support the hypothesis that normal trophoblast development is associated with a hypoxic intrauterine environment. As pre-eclampsia is associated with abnormalities of the hypoxia, reperfusion and oxidative stress cycle, the role of IMA as a marker of the subsequent development of the disorder should be investigated.
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F.P. was supported by a Marie Curie Reintegration Fellowship of the European Community under contract number MERG-CT-2004-006365.
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Submitted on October 16, 2006; resubmitted on December 20, 2006; accepted on January 26, 2007.
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