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Original Article

Prospective Study on Role of Folic Acid and Vitamin B12 in

Early Pregnancy and Spontaneous Abortion
Simmi Kharb1, A. Singh1, J. Bala1, P. Gahlawat2, S. Nanda2
1
Department of Biochemistry, Pt. B.D. Sharma University of Health Sciences, 2Department of Biochemistry, Obstetrics and Gynecology, Pt. B.D. Sharma PGIMS,
Rohtak, Haryana, India

Abstract 
Background: Vitamin B12 maintains normal folate metabolism during pregnancy. Maternal folate status has been associated with various
adverse pregnancy outcomes. The aim of this study is to predict the risk of abortion in early pregnancy by studying the relation between
folate, Vitamin B12 levels, and rate of abortion. This prospective, observational study was carried out on 100 pregnant women with singleton
pregnancy of 6–12 weeks of gestation. Methods: The patients were divided into three groups, namely, Group 1 (control group) comprising of
30 normal pregnant women with no history of abortion; Group 2 (control group) comprising of 35 pregnant females with history of previous
abortion; and Group 3 (study group) comprising of 35 pregnant women were coming with chief complaint of bleeding per vaginum. About 5
ml blood was taken and serum was separated using centrifugation. Serum folate and Vitamin B12 levels were estimated by chemiluminescence.
Data so obtained were computed as mean + standard deviation and SPSS/ANOVA was applied. Results: Serum folate and Serum B12 levels
were comparable in all the three groups and the difference was statistically significant. 26.66%, 37.13%, and 31.42% patients had folate
deficiency in Groups 1, 2, and 3, respectively. Out of which, 3.33%, 5.71%, and 5.71% patients aborted from folate deficient Groups 1, 2, and
3, respectively. Thus, folate deficiency was not found to be associated with abortion (P = 0.944). Patients with Vitamin B12 deficiency were
73.33%, 82.85%, and 37.13% in Groups 1, 2, and 3, respectively; out of which 3.33% patients from Group 1, 8.57% from Group 2, and 2.85%
patients from Group 3 were aborted. Correlation between maternal serum B12 deficiency and abortion was found to be insignificant (P = 0.551).
Conclusion: A positive correlation was seen between serum folate and B12 levels. All females planning for conception should be given folate
and Vitamin B12 supplementation.

Keywords: Abortion, folic acid, pregnancy, Vitamin B12

Introduction below the nonpregnant cutoff level.[6] While others have

Pregnancy is a period of rapid growth and organ differentiation.
Women are prone to develop folate deficiency due to low
dietary folate intake increased or metabolic requirement
for folate. Furthermore, maternal folate status has been
associated with other adverse pregnancy outcomes, namely,
preeclampsia, congenital malformations (orofacial clefts and
neural tube defects [NTDs]), preterm delivery, spontaneous
abortion, fetal growth restriction, and fetal death.[1] Folic acid
supplementation has been known to prevent megaloblastic
anemia during pregnancy and NTDs in the newborn infant.[2‑4]
Pernicious anemia is a known cause of infertility,[5] and
therefore, severe clinical deficiency of Vitamin B12 cannot
coexist with pregnancy. Ball and Giles reported that 20% of
women in mid‑pregnancy had Vitamin B12 concentrations
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DOI:
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reported that 53% of the women had decreased Vitamin
B12 as compared to nonpregnant individuals. Low Vitamin
B12 has been associated with pregnancies affected by
anencephaly.[7] Some studies had reported that maternal
Vitamin B12 status was an independent risk factor for NTD.[8]
It has been observed that low amniotic Vitamin B12 levels
or elevated transcobalamin can occur during NTD‑affected
Address for correspondence: Dr. Simmi Kharb,
 H. No. 1396, Sector-1, Rohtak, Haryana, India.
E‑mail: simmikh@gmail.com
ORCID: http://orcid.org/0000-0002-1773-2422
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How to cite this article: Kharb S, Singh A, Bala J, Gahlawat P, Nanda S.
Prospective study on role of folic acid and vitamin B12 in early pregnancy
and spontaneous abortion. Biomed Biotechnol Res J 2018;2:265-8.
Received: 15.07.18; Revision: 05.08.18; Accepted: 12.08.18; Published: 11.12.18

© 2018 Biomedical and Biotechnology Research Journal (BBRJ) | Published by Wolters Kluwer - Medknow 265
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Kharb, et al.: Folic acid and Vitamin B12/spontaneous abortion
pregnancies.[9,10] Reznikoff‑Etiévant et al. in their study in
patients with very early recurrent abortions (before 5 weeks
of gestation) or early recurrent abortions (before12 weeks)
observed that ten cases (9%) had Vitamin B12 levels lower
than 180 pg/mL compared with controls (1%: women with one
or more children and no history of abortion) and after Vitamin
B12 treatment in 10 cases, 4 went on to normal full‑term
pregnancies.[11]
Medical Research Council study has shown that folic acid is
protective against NTD. Many researchers found that folic acid
supplements also reduced the risk of babies developing cleft
lip or cleft palate.[4] Folate deficiency can result in spontaneous
abortion, recurrent pregnancy loss, and stillbirth.[1] A Liverpool
study in low‑income women after histidine loading, it was
noted that abnormal formiminoglutamic acid excretion was
more frequent in younger gravidas and multiple pregnancies.
They suggested that folate deficiency was closely associated
with placental abruption and recurrent spontaneous abortion.[12]
In a trial of periconceptional supplementation with folic acid,
it was observed that folic acid decreased the risk of NTDs by
causing abortion of the fetuses.[13]
In view of the possible association of maternal folate and
Vitamin B12 levels with progress and outcome of pregnancy,
the present study was designed to analyze Vitamin B12 and
folate levels in early pregnancy (6–12 weeks) to predict the
risk of abortion.
Methods
This prospective observational study was carried out in 100
pregnant women with singleton pregnancy of 6–12 weeks of
gestation admitted in labor room under emergency condition
and attending the Outpatient Department of Obstetrics and
Gynaecology, Pt. B.D. Sharma, PGIMS, Rohtak, for routine
antenatal examination in collaboration with Department of
Biochemistry. Women who were not sure of dates, known case
of diabetes mellitus, hypertension, thyroid disorder, renal or
liver disease, autoimmune disorder, and multiple pregnancies
were excluded. Informed consent was obtained from these
women, and the study was approved by ethical committee of
the institutional postgraduate research board. The proposal was
reviewed and approved by Research and Ethical committee of
the department of Biochemistry, Obstetrics and Gynecology,
Rohtak, Haryana, India. The Ethics Committee Approval
number was IN.Har. Rohtak 2017, and date was 1.1. 2017-
1.2-2018.
Table 1: Biochemical investigations in three groups
Investigations Group I (n=30)
Fasting blood sugar (mg/dl) 90.16±11.23
Serum Vitamin B12 (pg/ml) 118.3±9.72
Serum folate (ng/ml) 10.17±8.13
Data presented as mean±SD. **P<0.001 as compared to Group I, *P<0.01 as compared to Group I, +P+ NS. n: Number of cases, SD: Standard deviation,
NS: Not significantly
266 Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 4 ¦ October-December 2018
The patients were divided into three groups, namely,
Group 1 (control group) comprising of 30 normal pregnant
women with no history of abortion; Group 2 (control group)
comprising of 35 pregnant females with history of previous
abortion; and Group 3 (study group) comprising of 35 pregnant
women were coming with chief complaint of bleeding per
vaginum. A detailed history and thorough general physical
examination was carried out in all the pregnant women.
Routine antenatal investigations were done at the time of first
antenatal visit. Five milliliters blood was drawn aseptically and
serum was separated by centrifugation. Routine investigations
and serum folate and Vitamin B12 levels were estimated in
maternal blood by chemiluminescence. Reference range for
serum folate was 4.5–20 ng/ml and for serum B12 was 118–435
pg/ml. All the patients were followed every 4 weekly till
20 weeks and the outcome was noted in terms of anembryonic
pregnancy, missed abortion, spontaneous abortion, or
continuation of pregnancy.
Statistical consideration
Data thus collected were presented as mean ± standard
deviation and Student’s t‑test was done using SPSS (Statistical
Package for the Social Sciences, version 17.0, SPSS Inc,
Chicago, Illinois, USA).
Results
In the present study, serum B12 levels were significantly higher
in the study group as compared to controls [Table 1]. The
mean values of serum Vitamin B12 in Group 1 were higher
in Group 2 as compared to Group 1 and lower than Group 3
[Table 1]. The difference between the mean values of serum
B12 of women in three groups was statistically significant.
On ccorrelating folate levels with abortion, it was observed
that in Group 1, a total of 26.66% (n = 8) women had folate
deficiency, of these women 23.33% (n = 7) had normal
pregnancy and 3.33% (n = 1) resulted in abortion during
the first trimester. Seventy‑three percent of the women
had normal folate levels; out of which 70% (n = 21) had
normal pregnancy, while 3.33% (n = 1) women aborted
spontaneously. The difference between the women with folate
levels <4.5 ng/ml and those with >4.5 ng/ml was statistically
insignificant (P = 0.439). Similarly, in women with Group 2,
a total of 37.13% (n = 13) had folate deficiency; out of which
31.42% (n = 11) had normal pregnancy, while 5.71% (n = 2)
resulted in abortion. 62.85% (n = 22) women had normal folate
levels out of which 57.14% (n = 20) had normal pregnancy
Group II (n=35) Group III (n=35) Statistical significance
87.6±15.59 85.54±6.73+ 0.293
98.57±15.41 125.31±32.08** 0.001
5.95±3.63 6.75±3.20* 0.01
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Kharb, et al.: Folic acid and Vitamin B12/spontaneous abortion

and 5.71% (n = 2) resulted in abortion. 62.85% (n = 22) women

Table 2: Correlation of serum folate levels with abortion
had normal serum B12 levels out of which 48.57% (n = 17)
Serum folate Group I Group II Group III had normal pregnancy and 14.28% (n = 5) women aborted.
(n=30) (n=35) (n=35) The difference between the women with serum B12
<4.5 ng/ml levels <118 pg/mL and those with >118 pg/mL was found to
Abortion 1 (3.33) 2 (5.71) 2 (5.71) be statistically insignificant (P = 0.599).
Continuation 7 (23.33) 11 (31.42) 9 (25.71)
>4.5 ng/ml
Abortion 1 (3.33) 2 (5.71) 5 (14.28) Discussion
Continuation 21 (70) 20 (57.14) 19 (54.28) In the present study, mean serum folate levels were within normal
Data presented as n (%). n: Number of cases range in all the three groups. The difference between the mean
value of serum folate of women in three groups was statistically
significant (P = 0.01). Reference range for serum folate was
Table 3: Correlation of serum Vitamin B12 levels with
4.5–20 ng/ml. Group 2 and Group 3 showed serum folate levels
abortion
on lower limits of normal range. Lower limits of normal folate
Serum Vitamin B12 Group I Group II Group III values could be due to increased demand during pregnancy,
(n=30) (n=35) (n=35) poor nutritional status, malabsorption, noncompliance of
<118 pg/ml folate supplementation during periconceptional period, less
Abortion 1 (3.33) 3 (8.57) 2 (5.71)
interpregnancy interval, or nausea‑vomiting occurring in the
Continuation 21 (70) 26 (74.28) 11 (31.42)
first trimester of pregnancy and vegetarian status as all the
>118 pg/ml
women were vegetarian in the present study.
Abortion 1 (3.33) 1 (2.85) 5 (14.28)
Continuation 7 (23.33) 5 (14.28) 17 (48.57) In the study, 26.66% (n = 8), 37.13% (n = 13), and
Data presented as n (%). n: Number of cases 31.42% (n = 11) patients had folate deficiency in Groups 1, 2,
and 3, respectively. Out of which 3.33% (n = 1), 5.71% (n = 2),
and 5.71% (n = 2) women aborted. The difference between the and 5.71% (n = 2) women aborted from Group 1, 2, and 3,
women with folate levels <4.5 ng/ml and those with >4.5 ng/ml respectively. Hence, folate deficiency was not found to be
was found to be statistically insignificant (P = 0.571). associated with abortion (P > 0.05) (odds ratio [OR] = 0.84;
In Group 3, a total of 31.42% (n = 11) women had folate 95% confidence interval [CI] 0.13–5.22). These findings were
deficiency; out of which 25.71% (n = 9) had normal comparable with the Hungarian randomized trial and they
pregnancy, while 5.71% (n = 2) aborted. Women with normal had also concluded that folate deficiency has no significant
folate levels were 68.56% (n = 24) out of which 54.28% effects on birth weight, miscarriage, and stillbirth after
periconceptional multivitamin supplementation.[3] George et al.
(n = 19) had normal pregnancy and 14.28% (n = 5) women
reported that low blood folate was a significant independent risk
aborted. The difference between the women with folate
factor for spontaneous abortion (OR = 1.47; 95% CI 1.01–2.14)
levels <4.5 ng/ml and those with >4.5 ng/ml was found to be
after adjustment for a number of known risk factors and
statistically insignificant (P = 0.855, Table 2).
other possible confounding variables but high folate status
Serum Vitamin B12 was correlated with abortion and it was was associated with a nonsignificant decrease in risk
noted that in Group 1, a total of 73.33% (n = 22) women had (OR = 0.74; 95% CI, 0.47–1.10).[14]
Vitamin B12 deficiency out of which 70% (n = 21) had normal
In the present study, difference between the mean values of
pregnancy, while 3.33% (n = 1) resulted in abortion. 26.66%
serum Vitamin B12 of women in three groups was statistically
women had normal B12 levels out of which 23.33% (n = 7)
significant  (P < 0.001). Seventy‑three percent in Group 1,
had normal pregnancy and 3.33% (n = 1) women aborted.
83% in Group 2, and 37% in Group 3 had Vitamin B12 levels
The difference between the women with serum B12
below cutoff (<118 pg/ml), out of which 3.33% (n = 1) women
levels <118 pg/mL and those with >118 pg/mL was found
from Group 1, 8.57% (n = 3) from Group 2, and 2.85% (n = 2)
to be statistically insignificant  (P = 0.439). Furthermore,
women from Group 3, respectively, were aborted. Hence,
in Group 2, a total of 82.85% women were found with
Vitamin B12 deficiency was not significantly associated with
Vitamin B12 deficiency out of which 74.28%  (n = 26) had
abortion (P > 0.05) (OR = 0.61; 95% CI 0.10–3.76). Reference
normal pregnancy and 8.57% (n = 3) aborted. Women with
range for serum Vitamin B12 was 118–435 pg/ml. Hibbard and
normal serum B12 levels were 17.13% (n = 6); out of which
Spencer conducted a study on pregnant females and found that
14.28% (n = 5) had normal pregnancy and 2.85% (n = 1)
53% of the women had Vitamin B12 levels below the normal
women aborted. The difference between the women with serum
range for nonpregnant individuals (150 pg/mL). Furthermore,
B12 levels <118 pg/mL and those with >118 pg/mL was found
14% of the women had levels <50 pg/mL. However, in a
to be statistically insignificant (P = 0.657, Table 3).
3‑year postnatal follow‑up of these women, only four women
In Group 3, a total 37.13% (n = 13) women had Vitamin B12 remained deficient after 6 months. They concluded that Vitamin
deficiency out of which 31.42% (n = 11) had normal pregnancy B12 deficiency was common during pregnancy and in most of

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Kharb, et al.: Folic acid and Vitamin B12/spontaneous abortion
the cases, it was not associated with latent pernicious anemia.
This was in accordance with the present study.[15]
In contrast, another study in pregnant females between 20 and
30 weeks of gestation, attending high‑risk pregnancy clinic,
a below‑normal value of serum B12 was found in 22% of
patients tested.[16] They agreed with the previous work by Ball
and Giles.[6] These findings were in contrast with the study
reported that women who had taken lower limit of Vitamin
B12 as 180 pg/ml instead of 118 pg/ml in their study. Bennett
followed the obstetric histories of women with recurrent severe
Vitamin B12 deficiency  (<180 pg/mL) and observed that
78.5% (n = 11) had frequent fetal loss. They suggested that
Vitamin B12 deficiency leading to raised homocysteine levels
and hypercoagulability could be the cause for the same.[17]
Reznikoff‑Etiévant et al. recruited women with very early
recurrent abortions (VERA – before 5 weeks of gestation), or
early recurrent abortions (ERA – before 12 weeks) were as
cases and women with one or more children and no history of
abortion as controls. 9% (n = 10) of the cases had Vitamin B12
levels lower than 180 pg/mL compared with 1% (n = 1) in the
control group. Of the 10 cases, 4 went on to normal full‑term
pregnancies after Vitamin B12 treatment.[11]
In Group 1, a positive but nonsignificant correlation was seen
between serum folate and Vitamin B12 levels (r = 0.124,
P >  0.05), while a significant positive correlation was seen
between serum folate and Vitamin B12 levels in both Groups
2 and 3. A much stronger correlation was seen between the
two variables in Group 2 compared to Group 3 (r = 0.816 vs.
0.468). Both Vitamin B12 and folate had mean value in the
lower limit of normal range in all the three groups. This might
be related to poor nutritional intake, increased maternal and
fetal demands, hemodilution, increased maternal catabolism,
and renal clearance from plasma.[18]
Conclusion
In the present study, difference between the mean values of
serum Vitamin B12 and folate levels of women in three groups
was statistically significant. On correlating serum folate and
Vitamin B12 levels with abortion, the relation was found to be
insignificant. Thus, folate and Vitamin B12 supplementation
in periconceptional period reduces the risk of NTD, but their
role in abortion needs to be studied on a larger scale. A positive
correlation was seen between serum folate and Vitamin B12
levels. All females planning for conception should be given
folate and Vitamin B12 supplementation.
268 Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 4 ¦ October-December 2018
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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