We conducted a prospective study that focused on the changes in Hcy during pregnancy, risk factors for Hcy and the impacts of Hcy on PE because there were controversies about Hcy, MTHFR genes and PE.
Changes in Hcy
The average value for Hcy in the subjects in this study was 4.36±1.05 μmol/L in the first trimester (10-14 weeks), 5.95±1.46 μmol/L in the second trimester (24-28 weeks), and 5.95±1.74 μmol/L in the third trimester (30-34 weeks). Hcy in the second and third trimesters was significantly higher than that in the first trimester, similar to the results of Rolf[5] and MW Wallace[6], which may be due to physiological changes and endocrine hormone changes during pregnancy such as HCG. Boxmeer[7] found that after HCG administration, Hcy level had been significantly lower than before during the menstrual cycle. This research found that pregnancy folic acid and vitamin B12 gradually decrease with increasing gestational age, which could explain the changes in Hcy during pregnancy.
Mark Walker[8] found that Hcy drops in the second trimester and then rises in the third trimester, which is different from the results of this study, and the possible reasons are as follows: (1) The detection of gestational weeks selected by the study is different. Gestational weeks chosen by Mark Walker to detect Hcy were 8-16 weeks, 20-28 weeks and 36-42 weeks. (2) Different methods were used in the study. Mark Walker selected pregnant women at different gestational weeks during the same period for the test, while this study conducted a prospective longitudinal study of the changes in the same population at different periods. (3) Due to the different times of the study, the living standards and nutrition intake of the people in 2019 changed greatly compared with those in 1999, which could lead to different results of the two studies. (4) Based on the Chinese population's perception of the timing of multivitamin (containing folic acid) supplementation, some pregnant women take multivitamins before conception and in the first trimester and quit for the second and third trimesters, which could explain why Hcy in this study was significantly higher in the second and third trimesters. After adjusting for risk factors affecting folic acid and vitamin B12, we found that Hcy, folic acid and vitamin B12 were negatively correlated in the first (r=-0.17 for FA, r=-0.15 for Vitamin B12), second (r=-0.17 for FA, r=-0.06 for Vitamin B12) and third trimesters (r=-0.25 for FA, r=-0.11 for Vitamin B12) (p<0.05). With an increase in gestational age, folic acid and vitamin B12 decreased significantly in the second and third trimesters of pregnancy, along with increasing Hcy levels, further indicating the possible reasons for the changes in Hcy during pregnancy in this study.
In this study, MTHFR was found that it might be another factor causing the increase in Hcy, and the mutation rate of the MTHFR gene involved in this study was similar to the results of Moll[9] and Wu. X[10]. Mutations in the MTHFR gene can significantly weaken the function of tetrahydrofolate reductase, and the activity of tetrahydrofolate reductase of heterozygous MTHFR rs1801133 can be reduced to 65% of that of normal people, while the activity of homozygous individuals is only 30%[9]. The enzyme activity of homozygous MTHFR rs1801131 was 60% of normal[9]. Therefore, mutations in the MTHFR gene might be one of the risk factors affecting Hcy in pregnancy[11], which was similar to result in this study. At the same time, we also found that the G mutation of MTHFR rs17367504 had had a protective effect on Hcy during pregnancy, and the G mutation might cause Hcy decline. This might explain Thomsen’s findings[12]. Thomsen[12] first found that the G mutation at MTHFR rs17367504 was a protective factor for PE (OR=0.65, 95% CI 0.53-0.80). In addition to the MTHFR gene, creatinine and protein levels in pregnancy were also possible factors that influence Hcy in pregnancy. The reason might be that the increase in creatinine affects the metabolism of Hcy and reduces urine excretion, which further causes the increase in Hcy in blood[11].
Impact of Hcy on PE
At present, there have been many studies on the correlation among Hcy, MTHFR genes and PE, and the findings are controversial. One study found that the Hcy of the T mutants at MTHFR rs1801133 was significantly higher than that of the non-mutants[13], which further increases the risk of the occurrence of PE[14]. Sunkara[15] found that in the PE group, the T mutation rate of MTHFR rs1801133 was significantly higher than that in the normal group. Furthermore, Dickerson[16] found that the T mutation of MTHFR rs1801133 was not significantly correlated with PE. There was no significant correlation among the three MTHFR genes referred to in this study and the risk of PE (including early-onset PE and severe PE) (p>0.05). Therefore, this study indicates that there is no direct correlation between the MTHFR genes and PE, and mutations in the MTHFR gene are risk factors for elevating Hcy during pregnancy which might directly or indirectly lead to vascular endothelial dysfunction, oxidative stress response, stimulate vascular smooth muscle cell proliferation, disrupt the body's coagulation and fibrinolytic system, and ultimately increase the risk of PE[17].
This study found that elevating Hcy might be correlated with the risk of PE in the third trimester (OR=1.2, 95% CI 1.01,1.42), especially in early-onset PE (OR=3.63, 95% CI 1.71, 7.71, p=0.001) and severe PE (OR=1.46, 95% CI 1.21, 1,76, p<0.001), but not late-onset PE (p>0.05). There was no correlation between Hcy in the first trimester and PE (p>0.05). However, most researchers believe that Hcy in the PE group was significantly higher than that in normal pregnancies, and the increasing Hcy level, especially in the first trimester, significantly increases the risk of PE, which is different from the results of this study[2,18-20]. In the Finnish study, Hietala[3] found no significant differences in Hcy between the PE group and normal group. Such differences may be due to different gestational weeks detecting Hcy. Kahn[21] has found that Hcy detection at 22-26 weeks of gestation showed no significant difference between the PE group and the normal group, while Hogg[22] found that the same group, with no difference at 26 weeks, had a significant difference in Hcy detection at 37 weeks. This is similar to the results of this study, and the possible reasons for such differences are as follows: (1) The gestational weeks detected in this study were 10-14 weeks, 24-28 weeks, and 30-34 weeks, and it can be inferred that Hcy before 28 gestational weeks had no significant correlation with PE. The detection of Hcy at 30-34 gestational weeks was significantly associated with PE, especially early-onset PE and severe PE. (2) The sample size of this study is limited, and the number of cases of PE is small, which is not enough to reflect the predictive value of Hcy in the first trimester for PE. (3) The detection results of Hcy are greatly affected by blood lipids[23], which are prone to increase during pregnancy, especially for patients with PE who usually have abnormal lipid metabolism[24,25], and it may have a certain influence on the detection results. (4) This study find that Hcy in the third trimester was more significantly correlated with early-onset PE, possibly because its detection of gestational weeks were very similar to the onset of the disease, or the test samples were collected at the onset of early-onset preeclampsia. (5) This study find that Hcy in the third trimester was correlated with early-onset PE, other than late-onset PE, possibly due to the different pathogenesis between early-onset PE and late-onset PE. Early-onset PE arises owing to defective placentation which is similar to the pathogenesis mechanism of adverse pregnancy outcomes caused by elevated Hcy , whilst late-onset PE may center around interactions between normal senescence of the placenta and a maternal genetic predisposition to cardiovascular and metabolic disease.[26] Therefore, although the correlation between Hcy in the third trimester and PE is statistically significant, further studies are necessary to confirm whether Hcy can be used as an early indicator of clinical disease.
Strengths and limitation
One of the main strengths of this study is that this was a prospective study by testing the same patient at different gestational ages. We analyzed the correlation between Hcy levels in the first and third trimester of the same patient who finally progressed to PE and took MTHFR genes variation into account.
The limitation of this study regards assessments of folic acid supplementation dose, gestational age, and dietary intake of folate and vitamin B12. Because the dietary structure and living habits of the included subjects are quite different and the use of multivitamins during pregnancy is not the same throughout the gestational period, it is difficult to conduct a quantitative study on the intake of folic acid and vitamin B12 during pregnancy, but a maternal blood test was taken to measure folic acid and vitamin B12 levels instead. On the other hand, the sample size of this study is limited, and the number of cases of PE is small, which is not enough to reflect the predictive value of Hcy in the first trimester for PE.