Objective
We designed a large prospective study to explore the relationship between maternal homocysteine concentrations and related B vitamins and birthweight.
Study Design
Blood was sampled from pregnant women at 30-34 weeks of gestation and their newborn infants (n = 366).
Results
Concentrations of all analytes were higher in umbilical cord compared with maternal samples. Birthweight was related negatively to maternal homocysteine (r = –0.12) but not related to maternal cobalamin, methylmalonic acid, and folate (r = 0.02, r = 0.06, and r = 0.04, respectively). Regression analysis revealed smoking (β = –313; 95% confidence interval [CI], –479 to –149), gestational age (β = 150; 95% CI, 118–182), female sex (β = –146; 95% CI, –256 to –35), and parity (β = 104; 95% CI, 37–171) as strong determinants of birthweight. Maternal homocysteine, cobalamin, methylmalonic acid, and folate were not determinants of birthweight in multivariate analysis.
Conclusion
Maternal homocysteine and B vitamins are not related to birthweight in a multivariate model that was adjusted for potential confounders.
Homocysteine and methionine and related B vitamins like cobalamin and folate have been shown to play a critical role in fetal nutrition, growth, and development. Several authors have described relationships among total homocysteine (tHcy), B vitamins, and pregnancy complications or adverse pregnancy outcomes. Elevated tHcy and low B vitamin concentrations are associated with increased risk of neural tube defects and pregnancy complications, such as preeclampsia, recurrent early pregnancy loss, and abruptio placentae.
Revealing possible and treatable factors for low birthweight (LBW) is of the utmost importance because LBW increases the risk of morbidity and death in infancy. Associations between LBW and chronic disease in adulthood, such as hypertension, coronary heart disease, stroke, or diabetes mellitus, have been described. Several studies demonstrated an association between high maternal tHcy concentrations and risk of having LBW offspring and negative correlations between maternal homocysteine and birthweight. In contrast, others have found either no differences or even a positive association between maternal tHcy and birthweight. However, some of these studies are relatively small. Others use birthweight <2500 g as a cut-off point instead of birthweight related to or corrected for gestational age. Also, the different timing of maternal blood sampling might contribute to inconsistent results.
Several studies determined cobalamin and/or folate concentrations together with tHcy concentrations in relation to birthweight. However, none of these studies determined methylmalonic acid (MMA) concentration, which is a sensitive marker of intracellular cobalamin, deficiency.
We collected plasma samples from pregnant women and their offspring and prospectively explored whether maternal tHcy, folate, cobalamin, and MMA are risk factors for LBW.
Subjects and Methods
Participants
In the Netherlands, pregnant women visit either a midwife or a gynecologist, depending on medical and obstetric history or complications during pregnancy. Delivery can take place at home or in the hospital supported by the midwife (uncomplicated pregnancies and deliveries) or in the hospital supported by a gynecologist. In the Netherlands, approximately 70% of pregnant women deliver in the hospital.
Patients were included in our study when they visited the outpatient clinic of the department of Obstetrics and Gynecology at the Canisius Wilhelmina Hospital in Nijmegen, the Netherlands, from 2002-2004. All women who attended the clinic were potentially eligible. Our aim was to study the relationship between maternal homocysteine concentrations in the general population; therefore, we have included pregnant women from this large regional hospital. Women with high-risk pregnancies or serious illnesses themselves were referred to a tertiary hospital, mostly at <30 weeks of gestation. They were asked to participate when the time schedule of the clinician, the nurses, and/or the administrative worker allowed this. Approximately 25% of all women who delivered in this hospital during the study period were included. To check whether the included women were representative for the entire population that visited this hospital, we randomly selected 500 additional women from the same period and found comparable results for maternal age, gestational age, birthweight, and newborn infant sex.
In the Netherlands, women are evaluated routinely for irregular antibodies between 30 and 34 weeks of gestation. After written informed consent had been obtained, 478 maternal blood samples were drawn during routine blood sampling of women between 30 and 34 weeks of gestation. Only 8 mothers refused participation after being asked. Of mothers with blood samples, 386 women visited the hospital for the delivery of their child and delivered a total of 406 babies from whom venous umbilical cord blood was drawn (including 20 twin pairs). We decided to exclude twin pregnancies because of a higher risk of lower birthweight in twins, which left 366 mother-baby pairs.
All mothers were asked to fill out a questionnaire. The study protocol was approved by the local medical ethics committee.
Blood sampling
Maternal venous blood was collected in tubes that contained 3.5 mL ethylenediaminetetraacetic acid (EDTA). Immediately after birth, the umbilical cord was clamped, and venous cord blood was collected into a vacuum tube that contained 3.5 mL EDTA. Samples from mothers and cords were immediately placed on ice. After centrifugation (within 2 hours after sampling), plasma was separated, and EDTA plasma and blood cells were stored separately at –20°C until analysis.
Biochemical analyses
tHcy, folate, cobalamin, and MMA acid concentrations were determined in both maternal and umbilical cord EDTA plasma. Plasma tHcy was determined by liquid chromatography-mass spectrometry-mass spectrometry. Deuterated homocysteine was added as an internal standard, and samples were treated with dithioerythritol followed by acid precipitation. Supernatants were analyzed by reversed-phase chromatography. The between-day coefficient of variation was approximately 5%. The method has been validated against existing methods according to the protocol described by Nexo et al and shows excellent correlation with a method based on high performance liquid chromatography and fluorescence detection. Plasma folate concentrations were determined by a Lactobacillus casei microbiologic assay, and plasma cobalamin concentrations were determined by a L rhamnosus microbiologic assay. Both the folate and the cobalamin assays were adapted to a microtiter plate format and carried out by a robotic workstation (Microlab AT plus 2; Hamilton Bonaduz AG, Bonaduz, Switzerland). Plasma MMA values were measured with a liquid chromatography tandem mass spectrometry method.
Statistical analyses
Results are presented as median values with the interquartile range, except when otherwise stated. After logarithmic transformation, Z scores for variables were calculated before further analysis. Linear regression was used to determine predictors of birthweight. Because birthweight and gestational age were strongly related, we calculated birthweights that were standardized for gestational age. Again, linear regression analyses were performed with the standardized birthweights as dependent variables. To reveal nonlinear relations, association curves were constructed to show associations between maternal concentrations and birthweight, with the use of Gaussian generalized additive regression models. Correlations between variables were calculated by Pearson correlation after logarithmic transformation of data. STATA statistical software (version 10.0; Stata Corporation, College Station, TX) was used for all statistical analyses.
Results
Population characteristics
Population characteristics that include blood indices are presented in Table 1 . Babies were born at or near term. All maternal analytes were lower, compared with umbilical analyte concentrations.
| Characteristic | Mother | Child |
|---|---|---|
| Age at delivery, y a | 33.3 (30.7–35.5) | — |
| Gestational age, wk a | — | 39.7 (38.4–40.7) |
| Birthweight, g a | — | 3425 (3075–3855) |
| Total homocysteine, μmol/L a | 5.5 (4.5–6.7) | 5.8 (4.6–7.2) b |
| Methylmalonic acid, μmol/L a | 0.16 (0.13–0.22) | 0.28 (0.24–0.37) b |
| Folate, nmol/L a | 9.1 (6.1–16.4) | 30 (21.7–45.9) b |
| Cobalamin, pmol/L a | 179 (134–219) | 208 (156–307) b |
| Creatinine, μmol/L a | 45 (40–50) | 54 (48–61) b |
| Primiparity, n/N | 172/358 (48%) | |
| Smoking, n/N | 50/350 (14%) | |
| Use of folic acid, n/N | 198/347 (57%) | |
| Male sex, n/N | — | 159/363 (44%) |
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