Hum. Reprod. Advance Access originally published online on February 16, 2008
Human Reproduction 2008 23(4):803-806; doi:10.1093/humrep/den029
Defective endovascular trophoblast invasion in the first trimester is associated with increased maternal serum ischemia-modified albumin
1 Fetal Medicine Unit, Academic Department of Clinical Developmental Sciences, 4th Floor, Lanesborough Wing, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK 2 Department of Clinical Biochemistry, St George's Hospital NHS Trust, London, UK
3 Correspondence address. Tel +44-20-8725-0071; Fax: +44-20-8725-0079; E-mail: basky{at}pobox.com
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Abstract |
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BACKGROUND: Ischemia-modified albumin (IMA), a protein elevated in cardiac ischemia, is also increased to supra-physiological levels in early normal pregnancy. This finding supports the hypothesis that normal trophoblast development is stimulated by a hypoxic intrauterine environment. The aim of this study was to examine whether first trimester IMA levels are further elevated with defective trophoblast development.
METHODS: Prospective study of healthy women with singleton pregnancies undergoing nuchal translucency assessment at 11–14 weeks. First trimester maternal serum IMA concentrations in those subsequently developing pre-term pre-eclampsia (n = 19) were compared to randomly chosen controls with normal pregnancy outcome (n = 69).
RESULTS: Median first trimester serum IMA concentrations were significantly higher in women who subsequently developed pre-eclampsia (median 126.5 kU/L, interquartile range (IQR) 114.33–134.36 kU/L) when compared to those with normal pregnancy outcome (median 115.01 kU/L, IQR 102.29–124.81 kU/L, P = 0.02).
CONCLUSIONS: Maternal serum IMA levels are elevated in the first trimester in women with pre-eclampsia, a clinical manifestation of defective endovascular trophoblast development. This suggests that abnormally high intrauterine hypoxia and subsequent reperfusion oxidative damage may be associated with defective trophoblast development. First trimester serum IMA may be a potential biomarker for abnormal placental development.
Key words: ischemia-modified albumin/trophoblast development/pre-eclampsia
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Introduction |
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During normal placental development trophoblast invasion of the maternal spiral arteries allows the increase of uterine blood supply necessary for maintaining pregnancy. Defective endovascular trophoblastic invasion and inadequate remodeling of the uterine spiral arteries is known to be associated with the development of pre-eclampsia (Pijnenborg et al., 1980
, 1991; Khong et al., 1986; Meekins et al., 1994; Kam et al., 1999; Kliman, 2000; Brosens et al., 2002). This is supported by the use of non-invasive assessment of uterine artery resistance using Doppler ultrasound which has confirmed the associations between poor endovascular trophoblast invasion, defective physiological transformation of the spiral arteries and the subsequent development of pre-eclampsia (Campbell et al., 1983; van den Elzen et al., 1995; Martin et al., 2001; Vainio et al., 2002; Savvidou et al., 2003; Prefumo et al., 2004; Papageorghiou et al., 2004; Gomez et al., 2005). There is now compelling experimental evidence that normal pregnancies develop in a relatively hypoxic environment and that placental oxidative stress plays a pivotal role in the physiological trophoblast development (Roberts et al., 1999; Redman and Sargent, 2000; Prefumo et al., 2007).
During tissue ischemia, reactive oxygen species generate highly reactive hydroxyl free radicals, resulting in a site-specific modification of serum albumin to produce ischemia-modified albumin (IMA). Measurement of IMA has been shown to be a useful marker of cardiac ischemia in acute coronary syndromes (Bar-Or et al., 2000; Anwaruddin et al., 2005). As IMA has a relatively short half-life, it is a marker of ongoing ischemia. We have previously demonstrated that maternal circulating serum IMA reaches supra-physiological levels in normal pregnancy. This supports the hypothesis that normal placental development occurs in the presence of relative intrauterine hypoxia and that exposure of the maternal circulation to the uteroplacental unit results in elevated production of IMA (Prefumo et al., 2007).
Current evidence suggests that increased intrauterine hypoxemia predisposes to defective endovascular trophoblast invasion (Jauniaux et al., 2003), possibly leading to the subsequent development of pre-eclampsia (Prefumo et al., 2004). The latter is especially evident when pre-eclampsia occurs in the pre-term period (Egbor et al., 2006). We hypothesize that first trimester maternal serum IMA concentrations are elevated in pregnancies with defective trophoblast endovascular invasion.
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Methods |
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In order to test our hypothesis, two group of patients were compared: patients who developed pre-term pre-eclampsia (as a surrogate for defective trophoblast invasion) and those with normal pregnancy outcome. The patients for this study were recruited from women attending for routine antenatal care in a hospital obstetric unit. A maternal venous blood sample was collected from women with singleton pregnancies immediately after the routine nuchal translucency assessment at 11–14 weeks of gestation. In all cases, 5 mL of maternal blood were drawn into a non-heparinized tube, allowed to clot and centrifuged. Serum was stored and analysed only when pregnancy outcomes were available. Laboratory staff were blinded to the outcome of pregnancy. Women with pre-existing medical conditions including diabetes mellitus, connective tissue disease, essential hypertension, a history of ischemic heart disease and or recurrent miscarriage were excluded from the study. At the time of the examination, all women included in the study had a blood pressure below 140/90 mmHg. Gestational age was calculated from the last menstrual period and confirmed by crown-rump length measurement. Local ethical committee approval was obtained for the study, and all women gave written, informed consent.
Assay of IMA
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. 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 ischemic 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 colored 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 percentage of patient samples averaged 1.9% (range 0.0–6.5%). The IMA upper limit of normal (95th centile of 111 apparently healthy people) reported by the manufacturer is 85 U/mL and higher levels are considered positive for cardiac ischemia.
Outcomes
All pregnancy outcomes were obtained from the main delivery suite database and checked by review of individual patient records. Pre-eclampsia was defined as a blood pressure >140/90 mmHg on at least two occasions and proteinuria of 
300 mg in 24 h (or two readings of at least 2+ on dipstick analysis of midstream or catheter urine specimens if no 24-h urine collection was available) (Brown et al., 2001). For the purposes of this study, we only included those women where the severity of the disease resulted in delivery occurring before 37 weeks of gestation. Normal pregnancy outcome was defined as delivery after 37 weeks of gestation in normotensive women where the neonatal birthweight was above the 5th centile for gestation (Yudkin et al., 1987).
Statistical analysis
Continuous data were expressed as mean (SD) or median (interquartile range (IQR)) as appropriate, depending on normality of distribution. For inter-group comparisons, Mann–Whitney test, 
2-test and Fisher's exact test were used. All calculations were performed using the Statistical Package for the Social Sciences (release 11.5, SPSS Inc., Chicago, IL, USA) and Prism (release 3.00, GraphPad Software Inc., San Diego, CA, USA) software packages.
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Results |
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Maternal serum IMA was measured at a median gestational age of 12 weeks and 3 days (IQR 12 + 1 to 13 + 1) in 20 women who subsequently developed pre-eclampsia requiring elective pre-term delivery and in 70 women with normal pregnancy outcome. There was no difference in the gestational age at enrolment (median 12 + 3 weeks and 12 + 4 weeks, respectively, P = 0.91). In two cases, one from each group, IMA analysis did not yield results and these were excluded from the subsequent analysis. Maternal characteristics and pregnancy outcomes of the 88 women included in the study are summarized in Table I.
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The median IMA concentration was significantly higher (Fig. 1, P = 0.02) in women who subsequently developed pre-eclampsia (median 126.5 kU/L, IQR 114.33–134.36 kU/L) than in women with a normal pregnancy outcome (median 115.01 kU/L, IQR 102.29–124.81 kU/L). IMA levels in women with normal pregnancy outcome were greater than 85 kU/L (the concentration used for the diagnosis of myocardial ischemia in non-pregnant individuals) in 65 of 69 cases (94%).
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Although there was a significant difference in body mass index (BMI) between the two groups, BMI did not correlate with IMA levels (r = 0.04, P = 0.74).
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Discussion |
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In the first trimester of pregnancy, maternal IMA levels are further increased in women who subsequently developed pre-term pre-eclampsia, a clinical manifestation of defective endovascular trophoblast.
In early normal pregnancy, the successful invasion of extravillous trophoblast is associated with physiological conversion of the spiral arteries and reduction in the resistance to flow in the uterine artery, as reflected by uterine artery Doppler resistance indices. It has previously been hypothesized that this process of placental development and trophoblast invasion occurs in a hypoxic environment (Jauniaux et al., 2003). IMA is known to form under conditions of tissue hypoxia, and this is the basis for its use in acute coronary syndromes. Pregnancy is the first physiological condition where IMA levels were demonstrated to be elevated to supra-physiological levels (Prefumo et al., 2007). In fact, first trimester maternal IMA levels are well above the cut-off used to diagnose cardiac ischemia in non-pregnant individuals, supporting the hypothesis that normal placentation is associated with relative intrauterine hypoxia (Prefumo et al., 2007). This assertion is further supported by direct measurement of low partial pressures of oxygen within the placenta during the first trimester, confirming the existence of a hypoxic intrauterine environment in normal pregnancy (Jauniaux et al., 2001). Although it is possible that IMA may arise from a different source, it is logical to conclude that the developing uteroplacental unit is responsible for the differences in IMA levels observed between pregnant and non-pregnant women.
When there is poor trophoblast invasion, maternal spiral arteries retain their vasoreactivity leading to poorer placental perfusion. The resulting hypoxia leads to oxidative stress from reperfusion-related injury and is thought to pre-dispose to the subsequent development of pre-eclampsia (Burton and Jauniaux, 2004). The finding that uterine Doppler indices (Martin et al., 2001; Plasencia et al., 2007) and placental volume (Hafner et al., 2003) are altered in the first trimester in pregnancies that are subsequently complicated by pre-eclampsia suggests that the normal development of the maternal circulation is modified very early on in this disorder. To date, evidence supporting this hypothesis has only been seen in studies conducted in mid-pregnancy with soluble-endoglin (Levine et al., 2006) or after the development of the clinical disorder in late pregnancy, such as the over expression of hypoxia inducible transcription factors-1
and -2 proteins (Rajakumar et al., 2001) and an excess of the soluble receptor, FMS-like tyrosine kinase 1 (sFlt-1) (Shibata et al., 2005). More recently, Whitley et al. (2007) demonstrated increased trophoblast apoptosis in women with high-resistance first trimester uterine Doppler indices. These data provide compelling evidence that the cellular and biochemical milieu responsible for the subsequent development of pre-eclampsia is already present in the first trimester of pregnancy.
In the present study, first trimester serum IMA levels from women with clinical manifestations of defective endovascular trophoblast invasion were higher than those with normal placentation. This difference in IMA levels is most likely a consequence of increased trophoblast hypoxia. These data provide support for the hypoxia-reperfusion oxidative stress hypothesis of defective trophoblast development. The finding that maternal serum IMA levels are raised so early in pregnancy, months before the onset of clinically evident pre-eclampsia, mandates further prospective evaluation of IMA as a potential biomarker for abnormal placental development related to miscarriage as well as pre-eclampsia.
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F. Prefumo 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 August 9, 2007; resubmitted on December 1, 2007; accepted on January 14, 2008.
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