Vitamin B12 Supplementation in Addition to Folic Acid and Iron Improves Hematological and Biochemical Markers in Pregnancy: A Randomized Controlled Trial

Abstract

Vitamin B12 plays an important role in cell division and is of vital importance during pregnancy. Iron and B12 deficiency increase the risk of neonatal morbidity and the outcome of the overall pregnancy. The aim of our study was to analyze whether the use of vitamin B12, with standard supplements of folic acid and iron among nonanemic pregnant women, will result in improvements of hemogram parameters in terms of hematological and biochemical markers. Study participants were 200 healthy pregnant women, randomized into an intervention group and a control group, recruited from gynecological primary care practices in Split, Croatia. In addition to standard supplementation (350 mg/day ferrous iron, 5 mg folic acid), participants in the intervention group were given 5 μg of vitamin B12 each morning for 100 days. Both biochemical and hematological measurings were conducted in two intervals: 8th–10th week of gestation and then again in the 34th–36th week of gestation. Participants in the control group were given only standard-of-care iron and folic acid supplementation. Significantly lower values of haptoglobin postintervention, compared with baseline, were found only in the intervention group; for erythrocytes, significantly lower values postintervention were found only in the control group. For parameter hematocrit, we found decreased values postintervention, compared with baseline, in both intervention and control group; however, this decrease was within the reference range for the control group, whereas it was above the reference range for the intervention group. The results of this study indicated that intervention with vitamin B12 in pregnancy reduces possibilities of the onset of anemia, but within reference range.

Introduction

Normal pregnancy implies a progressive increase of plasma volume.¹ Plasma volume, which increases by 50%, is proportional to the birthweight of the infant and completed by the 34th week of gestation. Due to the great expansion of plasma volume, the red blood cell (RBC) mass, as well as hemoglobin (Hb) and hematocrit (Hct) concentration proportionally fall during pregnancy. Despite this hemodilution, there is usually no change in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC) during pregnancy.²

The definition of anemia in pregnancy corresponds to the Hb values below 110 g/L in the first trimester and below 105 g/L in the second and third trimesters. Iron (Fe) deficiency is considered the most common nutrient deficiency among pregnant women.³ According to the World Health Organization (WHO) review of nationally representative surveys from 1993 to 2005, 42% of pregnant women suffer from anemia worldwide.⁴ Fe deficiency, as well as delayed neurocognitive development, is associated with maternal anemia, which also increases the risk of low birth weight, premature birth, or fetal growth restriction.⁵ Anemia developing later in pregnancy has a less negative impact on fetal growth then Fe deficiency during the first trimester of pregnancy.⁶

For women who are not anemic, a daily iron supplementation of 27 mg/day is sufficient, which can be obtained from either adequate nutrition or food supplements.³ The WHO has recommended iron supplementation in pregnancy since 1959, and numerous professional associations confirmed this recommendation.⁷ However, when it comes to women with anemia, or women subjected to particular dietary regimens with low Fe levels, including vegetarians or vegans, the required supplementation dosage is higher, and estimated in the literature to be 120 mg/day.⁷

Pregnant women who had vitamin B12 deficiency in early pregnancy stages were up to five times more likely to have a child with several malformations such as spina bifida, which can cause partial paralysis, and anencephaly, a fatal condition in which the brain and skull are severely underdeveloped.^8
–10

Vitamin B12, and especially folate, play an important role in cell division and are needed particularly during infancy and pregnancy. The human body requires folate to produce healthy RBCs and prevent anemia, whereas vitamin B12 plays an important role in supplying essential methyl groups for protein and DNA synthesis.¹¹

Fe supplementation and vitamin B12 supplementation, such as folic acid, are not routinely prescribed during pregnancy and they were suggested for supplementation during pregnancy for the first time in 1967.⁷

In the developed countries, including Croatia, the decision to prescribe or recommend prenatal Fe with folic acid supplementation to women during pregnancy is entirely up to the health care personnel, being based on the individual maternal condition. Cochrane reviews have shown that intermittent or daily use of Fe supplements is associated with lower risk of delivering children with lower birth weight, borderline lower risk of preterm birth before the 34th week of gestation, and higher risk of increased Hb concentration during second and third pregnancy trimesters.¹²

Only a few studies have analyzed the efficiency of combined use of folic acid and Fe supplementation on positive outcomes of pregnancies and newborn children. However, taking only folic acid supplement is associated with marked health-related improvements on outcomes such as number of children with birth weight <2500 g, lower number of preterm births before the 37th week of gestation, neonatal death, and congenital anomalies.¹³ The deficiency of micronutrients, that is, vitamins and minerals, during pregnancy, can have important adverse effects on maternal and birth outcomes.¹³

The aim of our study was to analyze whether the use of vitamin B12, as well as folic acid and Fe supplements among pregnant women who are not anemic, and who only need Fe supplementation, will result in improvements of hemogram parameters in terms of hematological and biochemical markers.

Materials and Methods

Study population

The study was approved by the Ethics Committee of the Split University Hospital, Split, Croatia. The study was conducted in line with all applicable ethical guidelines related to good laboratory and clinical practice, and the Declaration of Helsinki. All participants received information about the study and signed informed consent.

Study participants were 200 healthy pregnant women, recruited from eight gynecological primary care practices in Split-Dalmatian County, Croatia. The average age of the participants was 31.08 ± 4.04. There were 100 women randomized into an intervention group and 100 women randomized into a control group. Both biochemical and hematological measurings in the aforementioned groups were conducted in two intervals: 8th–10th week of gestation and then again in the 34th–36th week of gestation. The intervention group consisted of 100 healthy pregnant women who took vitamin B12 supplementation in addition to standard-of-care Fe and folic acid supplementation. Participants in the intervention group took standard supplementation consisting of 350 mg/day ferrous Fe, 5 mg folic acid, as well as 5 μg of vitamin B12 each morning for 100 days.

The control group consisted of 100 healthy pregnant women who took only standard-of-care Fe and folic acid supplementation. Participants in the control group took 350 mg/day ferrous Fe and 5 mg folic acid each morning for 100 days. All the participants in the study received supplements produced by the same company.

The inclusion criteria included healthy pregnant women with no signs of anemia according to the WHO criteria. Anemia in pregnancy was defined as Hb <110 g/L in the first trimester and Hb <105 g/L in the second and third trimesters of pregnancy. Additional inclusion criteria were unremarkable personal, family and obstetric history, age between 18 and 36, and healthy pregnancy.

Exclusion criteria were diabetes mellitus, heart and liver conditions, Rh and ABO immunization, edema, proteinuria, hypertension gestosis, bleeding, and taking any medications.

In the 34th week of pregnancy, 62 participants in the control group did not show up for final testing and 50 participants in the intervention group did not turn up for final testing. Finally, the number of participants analyzed were 38 in the control group and 50 in the intervention group.

Study procedure

A simple randomization was conducted by tossing a dice. For each participant enrolled in the study, investigators threw a dice to determine to which group a participant will be allocated. Numbers 1–3 of the dice allocated participants into the control group, and numbers 4–6 allocated participants into the intervention group. Randomization sequence was not concealed. Study investigators, gynecologists, and participants were aware of the group allocation.

Participants were invited to participate in the trial during their first gynecological exam in pregnancy, after official confirmation of pregnancy via ultrasound or positive laboratory results using human chorionic gonadotropin in the blood. At the time of enrollment, pregnant women were in the 8–10th week of gestation. According to the standard protocols during pregnancy in Croatia, blood sampling is conducted two times for analyzing blood group; immunization test and hepatitis B test are conducted. These two blood draws are scheduled in the 8–10th week of gestation and then in the 34–36th week of gestation. Some of the blood taken during regular sampling of our participants was used to measure parameters analyzed in this study. Therefore, participants enrolled in this study were not subjected to any additional blood sampling beyond standard clinical practice.

Demographic data, personal anamnesis' details, and history of taking narcotics were collected from participants' gynecologists, whereas additional information such as HIV status, hepatitis B status, and results of Treponema Pallidum Hemagglutination Test were obtained from clinical and laboratory tests results.

Blood sampling and laboratory analyses were conducted two times during the trial: first time in the 8–10th week of gestation and second time in the 34–36th week of gestation. Blood testing was conducted at the Department for Medical Laboratory Diagnostics of the University Hospital Split in Split, Croatia, in the Laboratory for Hematology and Biochemistry.

Blood samples for biochemical and hematological tests were taken in line with the standard protocols. We took 8 mL of blood from cubital vein in two tubes. Blood was stored in tubes with ethylenediaminetetraacetic acid (EDTA) and in tubes without EDTA (the tube for biochemical tests/serum without anticoagulants continuous polystyrene foam for separating serum from the cells).

All tubes were marked with name of the participant, age, and laboratory identification number. Samples were kept at room temperature until processing, which took place within 2 h from blood sampling. Hematological parameters were measured by using hematological analyzer ADVIA 2120 (Global Siemens Healthcare Headquarters Siemens, Erlangen, Germany), whereas biochemical parameters were measured by using automatic biochemical analyzer Beckman Coulter AU680 (Beckman Coulter Ireland, Inc., Mervue, Galway, Ireland).

Outcomes

Main outcome measures were the following biochemical and hematological variables: red blood count, Hb concentration, MCV, mean corpuscular hemoglobin (MCH), MCHC, and Fe.iron.

Secondary outcome measures were values of the following hematological and biochemical parameters: haptoglobin, transferrin, unbound iron binding capacity (UIBC), total iron binding capacity (TIBC), and ferritin.

Statistical methods

Data were analyzed by using statistical software SPSS 19.0 (IBM Corp., Armonk, NY, USA). Depending on data distribution, quantitative variables were described as mean and standard deviation, or median and interquartile range. The normality of the distribution was assessed by Kolmogorov-Smirnov test and by QQ graph.

Differences in mean values between hematological and biochemical parameters in the intervention and control group at the beginning and at the end of the trial were analyzed by using t-test for independent samples, or Mann–Whitney test if the distribution significantly deviated from normal. Same tests were used for analyzing differences in mean changes of parameters between the groups. Significance of differences in mean values of parameters between two time points (baseline and end of treatment) were tested within each group by using t-test for dependent samples or its nonparametric alternative, Wilcoxon test for matched samples. Statistical significance was set at P < .05.

Results

The results are presented in Tables 1 and 2.

Table 1.

Differences in Hematological and Biochemical Parameters Values During Pregnancy Before and After Taking Vitamin B12 in the Intervention Group

Variables/units	Mean ± SD/median (IQR)Before implementation	Mean ± SD/median (IQR)After implementation	95% CI	P
RBC × 10¹²/L	4.22 ± 0.36	3.97 (3.71; 4.19)	(−2.36 to 6.61)	.346
Hem g/L^*	122.02 ± 8.02	116.06 ± 9.82	(−8.45 to −3.47)	<.001
Hct L/L^*	0.37 ± 0.02	0.35 ± 0.03	(−0.03 to −0.01)	<.001
MCV fL	89.00 (85.55; 90.78)	89.20 (85.48; 92.43)	(−0.35 to 2.58)	.131
MCH pg^*	28.98 ± 1.70	29.70 (28.60; 31.05)	(0.15 to 1.19)	<.05
MCHC g/L	329.14 ± 9.76	332.88 ± 15.81	(−1.34 to 8.82)	.145
Fe μmol/L^*	17.65 ± 6.48	12.21 ± 7.16	(−8.37 to −3.34)	<.001
UIBC μmol/L^*	49.16 ± 16.17	83.58 ± 25.57	(28.03 to 41.16)	<.001
TIBC μmol/L^*	67.02 ± 11.99	95.51 ± 22.30	(23.41 to 33.97)	<.001
Ferritin μg/L^*	28.55 (16.23; 51.20)	13.90 (10.83; 21.40)	(−29.08 to −14.37)	<.001
Haptoglobulin g/L^*	1.23 ± 0.45	1.02 ± 0.40	(−0.29 to −0.14)	<.001
Transferrin g/L^*	2.59 ± 0.48	3.73 ± 0.59	(1.00 to 1.27)	<.001

Levels of significance less than 5% (P < .05).

95% CI, 95% confidence interval; Fe, serum iron; Hct, hematocrit; Hem, hemoglobin; IQR, interquartile range; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; SD, standard deviation; TIBC, total iron binding capacity; UIBC, unbound iron binding capacity.

Table 2.

Differences in Hematological and Biochemical Parameter Values During Pregnancy Between the First and the Third Trimester of Pregnancy in the Nonintervention Group

Variables	Mean ± SD/median (IQR) 1st time point	Mean ± SD/median (IQR) 2nd time point	95% CI	P
RBC × 10¹²/L^*	4.29 ± 0.37	3.99 (3.75; 4.17)	(−0.45 to −0.20)	<.001
Hem g/L^*	127.32 ± 9.75	118.74 ± 9.36	(−12.20 to −4.96)	<.001
Hct L/L^*	0.39 ± 0.03	0.36 ± 0.03	(−0.04 to −0.02)	<.001
MCV fL	90.60 (88.70; 92.68)	90.65 (87.73; 93.48)	(−21.67 to 66.22)	.311
MCH pg	29.80 ± 1.46	30.30 (28.95; 30.90)	(−6.96 to 21.37)	.309
MCHC g/L	329.26 ± 11.68	332.24 ± 14.27	(−2.83 to 8.78)	.306
Fe μmol/L^*	22.24 ± 7.46	12.04 ± 4.68	(−13.33 to −7.84)	<.001
UIBC μmol/L^*	41.75 ± 13.70	80.12 ± 21.42	(32.18 to 46.36)	<.001
TIBC μmol/L^*	64.08 ± 11.25	92.16 ± 19.21	(22.67 to 34.55)	<.001
Ferritin μg/L^*	36.30 (24.18; 44.70)	14.15 (11.05; 23.13)	(−39.26 to −13.87)	<.001
Haptoglobin g/L	1.20 ± 0.45	1.13 ± 0.37	(−0.22 to 0.04)	.186
Transferrin g/L^*	2.50 ± 0.49	3.83 ± 0.71	(1.12 to 1.61)	<.001

Levels of significance less than 5% (P < .05).

Statistically significant increase compared with baseline were detected in both control and intervention group for the following three hematological parameters: transferrin, UIBC, and TIBC. At the final endpoint, values of those three parameters were above the reference range (Tables 1 and 2). Postintervention, MCV and MCHC levels were within reference range in both intervention and control group; there were no statistically significant changes in those two parameters between baseline and final end-point. We observed a statistically significant increase in the level of MCH in the intervention group between baseline and final end-point, but this increase was still within reference range.

A statistically significant decrease between baseline and postintervention was found in both intervention and control group for the following three parameters: Hgb, Fe, and ferritin. A decrease of Hgb was outside the reference range for both intervention and control group, whereas for parameter Fe and ferritin decreased postintervention value was still within borders of reference range for both intervention and control group. For parameter Hct we found decreased values postintervention, compared with baseline, in both intervention and control group; however, this decrease was within the reference range for the control group, whereas it was above the reference range for the intervention group.

Postintervention, RBC and haptoglobin were within reference range in the intervention and control group. Significantly, lower values of haptoglobin postintervention, compared with baseline, were found only in the intervention group; for RBC, significantly lower values postintervention were found only in the control group.

Discussion

This study could be important, because the results pointed out that intervention with vitamin B12 in pregnancy could reduce possibilities of the onset of anemia, but within reference range.

In both groups, the difference in values at the beginning and after the intervention shows a statistically significant increase of transferrin, UIBC, and TIBC.

Within the group that received vitamin B12 a decrease of Hb, hematocrit, iron, ferritin, and haptoglobin is evident, even with haptoglobin testing sub-optimally powered at 60%. Within the control group at the beginning and after the intervention, there was a significant decrease of erythrocytes, Hct, Hb, iron, and ferritin. Referent studies show that, physiologically in pregnancy, there is an increase of transferrin and MCH,¹⁴ as well as a decrease of Hb¹⁵ erythrocytes and TIBC,¹⁴ UIBC,¹⁶ Hct,¹⁵ iron,¹⁶ and ferritin.¹⁷ The aforementioned findings are confirmed with this study as well with a somewhat lower power of the MCH finding of 54%, whereas the listed parameters in both groups change during the pregnancy in the expected direction.

This study shows that women who took vitamin B12 during pregnancy did not have a decrease of erythrocytes, even though the previous studies detected a decrease of erythrocytes during physiological pregancies.¹⁴ These results show a possibility that taking vitamin B12 enables a better erythropoiesis of blood cells. This finding is important, because women lose Fe and B12 during pregnancy due to fetal development, and they may lose blood during childbirth. Improved depots of ferritin may prevent adverse outcomes in women and infants. Haptoglobin values were found to be low in 39% of pregnant women, and a direct relationship between these low haptoglobin levels and the Hb concentrations was observed. The cause of the haptoglobin values' decrease in the intervention group (detected at a power of analysis of 60%) was probably due to hemodilution and increased blood estrogen concentrations during pregnancy.¹⁸ Therefore, the more Hb is produced, the more it needs to be eliminated, using haptoglobin, causing less of it to be present in the serum.

Although, within both groups, a decrease of other observed hematological and biochemical values coincides with the pregnancy duration, the results of this study show a slight relative decrease within the group of women who took vitamin B12. In both tested groups, a decrease of Hgb is detected, which has been determined in previous studies.¹⁵ However, in the intervention group, unlike the control group, a less significant decrease of Hgb is demonstrated. According to recent studies, the level of Hb starts to decrease from the 16th week of pregnancy, as a result of an increased plasma volume, which can cause heart failure and alterations in coagulation factors. Our results show that the B12 supplement in 5 μg doses can lead to a smaller decrease of average values in comparison to the control group that did not take vitamin B12.

The decrease of Hct values in both groups, determined in this study, is due to an increase in plasma volume. Variations in Hct are caused by the same factors as in RBCs and Hgb.^19,20 A small decrease of Hct values in the intervention group decreases the risk of cardiac arrest and spontaneous blood clotting²¹ with women who took vitamin B12 during the pregnancy.

Further, within both groups, a decrease of iron and ferritin is detected, showing a small decrease in both parameters. This means that the B12 supplement in 5 μg doses led to a smaller decrease of iron and ferritin compared with the control group that did not take vitamin B12. The lack of iron and ferritin during pregnancy can cause a pregnant woman to experience anemia; lead to deterioration of cognitive functions, tissue enzyme malfunction, and neuromuscular transmission disorder. On the other hand, anemia can lead to small gestational age and miscarriages, whereas low ferritin levels lead to placental hypertrophy, that is, increased angiogenesis.²²

Within this study, the pregnant women received a vitamin B12 supplement, in 5 μg doses, and it led to positive effects on hematological and biochemical parameters. Therefore, future studies should verify whether the increase of vitamin B12 would lead to statistically significant changes in hematological and biochemical indicators of anemia.

The importance of this study is that it demonstrates that supplementation of vitamin B12 during pregnancy can lower the decrease of hematological and biochemical indicators of anemia (although within referent intervals), as well as instigate new research that will continue to study the effects of vitamin B12 in relation to prevention of anemia during pregnancy. The results of this study indicated that intervention with vitamin B12 in pregnancy reduces possibilities of the onset of anemia but within the reference range.

Considering important changes of these parameters in pregnancy, future studies should investigate the importance of various levels of analyzed parameters within reference range and establish whether supplementation with vitamin B12 should be universally recommended to all pregnant women.

This study had several shortcomings. Large dropout rate observed in the study indicated a possible decrease in power and an increase in the risk of attrition bias. However, after performing post hoc power analysis and taking into consideration observed effect sizes for included outcomes, we found that major findings of this study were adequately powered. In addition, due to a fact that all the outcomes were objective laboratory measures, obtained by a blinded technician in Central University Hospital's Medical Diagnostics Laboratory, and given the evidence from the literature of no significant difference in estimates between the studies with less than 20% and those with ≥20% dropout rate, the effect of bias due to attrition in this study might not be too far off. Serum concentrations of vitamin B12 and folic acid were not measured. The possibility of inadequate absorption of vitamin B12 and Fe due to hyperemesis or potential malapsorptive disorders (coeliac disease, Mb. Crohn, ulcerose colitis, etc.) was not taken into consideration. Also, it was not possible to estimate possible patient noncompliance. In future research, it is recommended to determine vitamin B12 levels and folic acid serum levels before the intervention. Future research should be conducted by using a larger number of randomly chosen participants.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this study.

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