November 28, 2004
Uterine Artery Doppler and Mid-Trimester Maternal Plasma Homocysteine in Subsequent Pre-Eclampsia
Objective: To investigate whether mid-trimester maternal plasma homocysteine concentration is elevated in women who develop pre- eclampsia and in those women identified at high risk by abnormal uterine artery Doppler examination.
Methods: This was a multicenter study involving healthy women undergoing screening for preeclampsia by uterine artery Doppler velocimetry at 22-24 weeks' gestation. Abnormal uterine artery Mood flow was defined as a mean pulsatility index (PI) above the 95th centile (1.6). Controls (mean PI
Results: In total, 683 women were recruited. Maternal plasma homocysteine concentration did not vary with gestation. Maternal plasma homocysteine concentration in women who subsequently developed pre-eclampsia (n = 80, 12%) was nor significantly different from women with uncomplicated pregnancies (n = 536, 78%) (median 5.1, range 2.7-14.1 mol/l vs. median 5.5, range 1.9-27.9 mol/ l, p = 0.44). There were no significant differences in the maternal plasma homocysteine concentration in women with abnormal uterine artery Doppler findings (n = 275) compared with controls (n = 408), (median 5.6, range 2.6-17.7 mol/l vs. median 5.4, range 1.9-27.9 mol/ l, p = 0.13).
Conclusion: Mid-trimester maternal plasma homocysteine concentration is not elevated in women who developed pre-eclampsia even in those at high risk defined by abnormal uterine artery Doppler velocimetry.
Keywords: HOMOCYSTEINE; PRE-ECLAMPSIA; UTERINE ARTERY; DOPPLER
The accurate prediction of women who are at risk of developing pre-eclampsia has been a focus of research for many years, bur lew antenatal investigations have proved useful. The most promising is mid-trimester uterine artery Doppler velocimetry1. Biochemical markers such as activin A and inhibin A have been shown to be associated with pre-eclampsia2. There is also evidence that combining these markers with second-trimester uterine artery Doppler screening may enhance sensitivity for the prediction of pre- eclampsia3. However, the search for other biochemical markers continues.
Homocysteine is a sulfur-containing amino acid primarily derived from demethylation of dietary methionine, which is abundant in protein of animal origin. Hyperhomocysteinemia results from inherited errors of metabolism, in particular methyltetrahydrofolate reductase deficiency4, hut more commonly in a milder form, due to dietary folate depletion. Folates promote methionine synthase activity, therefore in folate depletion, accumulation of homocysteinc occurs, and this is associated with oxidative stress and subsequent vascular endothelial damage. In non-pregnant adults with cardiovascular disease, hyperhomocysteinemia has been linked to endothelial cell dysfunction and thrombus formation5-7.
In pregnancy, maternal serum homocysteine concentration normally decreases with gestation8, but in folatedepleted women, homocysteine accumulates and high concentrations of maternal homocysteine become useful indicators of even occult folate depletion9,10. Maternal hyperhomocysteinemia has been associated with a number of 'placenta- mediated' diseases including pre-eclampsia11. Most of the studies reporting an association between maternal hyperhomocysteinemia and pre-eclampsia have measured the maternal plasma homocysteine concentration at the time of delivery or immediately afterwards12- 14. However, few studies have investigated mid-trimester maternal hyperhomocysteinemia as a predictor of subsequent pre-eclampsia, and those that have report conflicting results15-17
If elevation in maternal mid-trimester homocysteine concentration did correlate with the development of preeclampsia, this would be a particularly attractive screening test, as there would be potential therapeutic measures, such as continuation of folic acid supplementation, which may improve pregnancy outcome18. Indeed, in one observational study involving 14 women with hyperhomocysteinemia and a history of either pre-eclampsia or intrauterine growth restriction, treatment with folic acid and vitamin B6 improved perinatal outcome in a subsequent pregnancy19.
The aim of this study was to investigate whether maternal plasma homocysteine concentration at 22-24 weeks' gestation was elevated in women who subsequently developed pre-eclampsia and in those women considered to be at high risk by abnormal uterine artery Doppler velocimetry.
Plasma homocysteine concentration was measured retrospectively in women undergoing screening for preeclampsia by uterine artery Doppler velocimetry at 22-24 weeks of gestation. The Doppler screening study was conducted between January 2001 and July 2002. This involved measurement of the mean pulsatility index (PI) in the uterine arteries of healthy women with singleton pregnancies attending for routine ultrasound examination at 22-24 weeks. Recruitment took place in seven hospitals in London, UK20. In the present study, we examined the plasma collected at the time of the Doppler investigation from 275 women with a mean PI above the 95th centiIe, and 408 controls with PI below the 95th centile, matched for gestational age and date of sample collection. Women with pre- existing hypertensive, cardiovascular or renal disease, diabetes mellitus, bleeding disorders or systemic lupus erythematosus were excluded, as were multiple pregnancies and pregnancies complicated by fetal abnormality. Demographic characteristics were collected at the time of the original scan and pregnancy outcome data were obtained from the hospital perinatal computer and examination of individual patient notes. The study was approved by the Multi- Centre Research Ethics Committee as well as the local ethics committees of the individual hospitals. Written consent was obtained from all participants.
Blood sampling and measurement of homocysteine
Blood samples were centrifuged for 10 min at 2500 rpm and plasma was separated and stored at - 20C until analysis. Homocysteine was measured by a competitive chemiluminescent immunoassay method using Immulite 2000 (DPC, Los Angeles, CA, USA). Immulite 2000 performs an initial on-line one-cycle pre-treatment of patient plasma with S- adenosyl-L-homocysteine hydrolase and dithiothreitol. After a 30- min incubation, the treated sample is transferred to a second reaction tube containing an S-adenosyl-L-homocysteine (SAH)-coated polystyrene bead and an alkaline phosphatase-labeled antibody specific for SAH. During a further 30-min incubation, the converted SAH from the sample pre-treatment competes with immobilized SAH for binding alkaline phosphatase-labeled anti-SAH antibody. Unbound enzyme conjugate is removed by a centrifugal wash step. Substrate is added and after a short incubation period the chemiluminescent signal is measured and interpolated against the onboard calibration curve. The assay has a calibration range of 2-50 mol/l and an analytical sensitivity of 0.5 mol/l. Samples were measured over a period of 13 days, with controls (two concentrations) run each day prior to analysis of samples. The between-day precision at a homocysteine concentration of 7.2 mol/l was 5.6% and at 15.7 miol/l was 4.7%. All samples were handled in an identical and blinded fashion.
The outcome measures were pre-eclampsia and fetal growth restriction (FGR). The diagnosis of pre-eclampsia was according to the definition by the International Society for the Study of Hypertension in Pregnancy21. This requires two recordings of diastolic blood pressure of 90 mmHg or higher at least 4 h apart or one recording of diastolic blood pressure of at least 120 mmHg, in a previously normotensive woman, and urine protein excretion of at least 300 mg in 24 h or two readings of 2 + or higher on dipstick analysis of midstream or catheter urine specimens if no 24-h collection is available. FGR was defined as a birth weight below the 5th centile for gestational age22.
Categorical variables were analyzed using the χ^sup 2^ or Fisher's exact test, while continuous variables were compared using the unpaired t test, Mann-Whitney U test and linear regression, where appropriate. Two-sided significance tests are reported and the alpha error was set to 0.05. Statistical analysis was performed using Microsoft Excel 2000 and StatsDirect.
There were no significant differences in the demographic characteristics between the study group and controls (Tahle 1 ). The median gestation at uterine artery Doppler assessment and maternal venepuncture was 23 weeks (range 22-24 weeks). The mean maternal homocysteine concentration did not change significantly with the gestation at which sampling took place (r = 0.0% p = 0.87, Figure 1).
Outcome data were available for all pregnancies studied. In total, 60 (8.8%) women developed pre-eclampsia alone, 20 (2.9%) women developed pre-eclampsia with FGR, and a further 17 (2.5%) women developed FGR without preeclampsia, while 586 (85.8%) pregnancies suffered none of these complications. No significant differences were found when comparing maternal plasma homocysteine concentrations from the 80 women who subsequently developed pre- eclampsia to womenwith normal pregnancy outcomes (median 5.35 vs. 5.49 mol/1, p = 0.41). There were also no significant differences in maternal homocysteine concentrations between pregnancies that were subsequently complicated by pre-eclampsia or fetal growth restriction when compared to those with uncomplicated pregnancies (Table 2, Figure 2).
In the abnormal uterine artery Dopplcr group (n = 275), 56 women (20.4%) subsequently developed pre-eclampsia without FCiR, 20 women (7.3%) developed both preeclampsia and FGR and a further ten women (4%) had pregnancies complicated by FGR. The remaining 189 women (69%) had uncomplicated pregnancies. In contrast, in the normal uterine artery Doppler group (n = 408), 397 women (97.3%) had a normal pregnancy outcome, four (1%) developed pre-eclampsia, and seven (1.7%) had pregnancies with FGR (Table 3).
Table 1 Demographic charaderisties for the two study groups. Values are shown as n (%) or median (range)
Table 2 Characteristics and homocystcine concentrations for the groups by pregnancy outcome
Figure 1 Linear correlarion of marernal plasma homocysteine concentration against gestation
There were no significant differences in maternal homocysteine concentration between the abnormal and normal uterine artery Dopplcr groups (median 5.6 vs. 5.4 mol/l, p = 0.13) (Figure 3). Furthermore, there was no difference in maternal homocysteine concentration in those with an adverse pregnancy outcome compared to those with uncomplicated pregnancies for the two study groups (Table 3).
In the present study, we have confirmed the association between increased mean PI in the uterine arteries at 22-24 weeks and a high incidence of pre-eclampsia and/or FGR20. However, we were unable to demonstrate elevation in the maternal plasma concentration of homocysteinc in either the subgroup that subsequently developed pre- eclampsia or in the women with impaired placentation, as evidenced by increased PI. These findings suggest that increased maternal homocysteine concentration does not precede the clinical onset of the disease.
Figure 2 Spread plot of homocysteine concentrations in women with different pregnancy outcomes. FGR, fetal growth restriction
Table 3 Pregnancy outcomes for the two study groups. Values are shown as n (%) or median (range)
Figure 3 Spread plot of homocysteine concentrations in women with an abnormal uterine artery pulsatility index (PI) compared with those with a normal PI
Previous studies investigating the association between mid- trimester maternal homocysteine concentration in asymptomatic women and the subsequent development of pre-eclampsia have reported variable results. Sorensen and colleagues15 and Cutter and colleagues16 reported that maternal homocysteine concentration at 15- 16 weeks' gestation was significantly higher in women who subsequently developed pre-eclampsia compared with controls who remained normotensive. In contrast, Hietala and colleagues found no significant differences at 16 weeks between 38 women who subsequently developed preeclampsia compared with 68 women who remained normotensive, hut their study included women with less severe clinical manifestations of pre-eclampsia17. In all these studies, maternal blood sampling was performed at around 16 weeks' gestation, as this coincides with serum screening for chromosomal abnormalities. One further study did investigate maternal homocysteine concentration at a more advanced gestation of 26 weeks and reported no differences in 38 women who subsequently developed preeclampsia or FGR compared with controls, but affected women had a higher homocysteine concentration at delivery compared with controls23. The authors of this study concluded that maternal homocysteine concentration may he a useful marker of established disease, but not useful for the prediction of pre-eclampsia.
As with the other studics15-17,23, the women in the present study were not fasted. Ideally, as with all nutrition-dependent markers, measurement of plasma homocysteine should be in the fasted state. However, the influence of diet on homocysteine concentration is minimal24.
The present study suggests that it is impossible to predict which, of any, women would benefit from prolonged or additional folic acid supplementation in pregnancy. If dietary supplementation were beneficial, in countries where there is universal fortification of foods with folic acid one might expect a lower incidence in pre- eclampsia. However, in these countries, the prevalence of pre- eclampsia has remained stable for many decades despite this intervention25. Therefore, although dietary factors in relation to adverse pregnancy outcome are likely to be a continued focus of interest, there is little evidence that maternal hyperhomocysteinemia due to dietary folate depletion plays a central role in the pathogenesis of pre-eclampsia.
The study was funded by the Fetal Medicine Foundation (Registered Charity 1037116). We thank Euro DPC (Ltd) for the gift of homocysteine reagents.
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C. K. H. Yu1, L. Lakasing1, A. T. Papageorghiou1, K. Spencer2 and K. H. Nicolaides1
1 Harris Birthright Research Centre for Fetal Medicine, King's College Hospital, Medical School, London, UK
2 Endocrine Unit, Clinical Biochemistry Department, Harold Wood Hospital, Romford, Essex, UK
Correspondence: Professor K. H. Nicolaides, Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, Denmark Hill, London SE5 9RS, UK
Received 08-01-04 Revised 22-02-04 Accepted 14-04-04
Copyright CRC Press Aug 2004