Molecular Biology Reports

https://doi.org/10.1007/s11033-019-04776-w

ORIGINAL ARTICLE

Temporal expression of genes involved in folate metabolism

and transport during placental development, preeclampsia and neural
tube defects
Palani Selvam Mohanraj1 · Beenish Rahat2 · Aatish Mahajan2 · Rashmi Bagga3 · Jyotdeep Kaur2

Received: 5 January 2019 / Accepted: 20 March 2019

© Springer Nature B.V. 2019

Abstract
Folate is an essential micronutrient during pregnancy. The differential expression of genes related to folate transport and
metabolism during the advancing gestation and pregnancy complications is not well established. Hence, we studied the
gene expression of folate metabolism and transport proteins in the placenta with advancing gestation, preeclampsia and
neural tube defects (NTD). The expression of folate transporters and enzymes involved in folate metabolism in the placenta
with advancing gestation and pregnancy-related disorders were studied by 2-step RT-PCR. Folate levels were estimated by
microbiological assay using Lactobacillus casei. Significant changes in levels of placental folate metabolizing enzymes were
found in both physiological and pathological pregnancies during advancing gestation. Expression of methyltetrahydrofolate
reductase (MTHFR) (p < 0.001) and cystathionine-β-synthase (CBS) (p < 0.001) was decreased while that of methionine
synthase (MS) (p < 0.001) was increased with advancing gestation. A much-reduced expression of MTHFR (p < 0.01) and
an abnormally high expression of methionine synthase reductase (p < 0.001) were observed in the NTD group. In NTDs,
there was an adaptive up-regulation of folate transporters mainly reduced folate carrier (p < 0.001) and folate receptor alpha
(p < 0.001). MTHFR expression showed a strong positive correlation (r = 0.96, p < 0.01) with folate levels in placenta. Preg-
nant women with preeclampsia had low expression of MS (p < 0.01) in association with low folate levels. Placental folate
metabolizing enzymes exhibited a differential pattern during advancing gestation. Deficient folate status in association with
alteration in expression of enzymes involved in folate metabolism might be associated with pregnancy complications such
as preeclampsia and NTDs.

Keywords  Folate metabolism · Folate transporters · Neural tube defects · Placental development · Preeclampsia

Introduction novo and therefore needs to be supplemented in diet espe-

cially during periods of high demand such as pregnancy and
Folates are a group of water-soluble molecules which belong infancy. Failure of intake of folate in adequate amounts leads
to the family of B9 vitamins. In humans and several other to folate deficiency, which results in several adverse preg-
mammals, unlike bacteria, folate cannot be synthesized de nancy outcomes like neural tube defects and various other
developmental disorders [1, 2].
Folate being negatively charged molecule cannot pass
* Jyotdeep Kaur
jyotdeep2001@yahoo.co.in through the cell membranes and requires specific membrane
transporters for this. Reduced folate carrier (RFC, SLC19A1)
1
Department of Biochemistry, Mahatma Gandhi Medical is the major transporter of folate across biological mem-
College and Research Institute, SBV, Pillaiyarkuppam, branes and is present ubiquitously in various organs [3, 4].
Puducherry, India
2
Folate receptors (FRα and FRβ) are high-affinity folate bind-
Department of Biochemistry, Post Graduate Institute ing proteins that help in transport of folate through receptor-
of Medical Education and Research, Chandigarh 160012,
India mediated endocytosis at neutral to slightly acidic pH [5, 6].
3 Proton-coupled folate transporter (PCFT, SLC46A1) has
Department of Obstetrics and Gynecology, Post Graduate
Institute of Medical Education and Research, Chandigarh, been recently found to play an important role in the absorp-
India tion of folate from intestine at acidic pH [7, 8].

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Folates after transport into the cells are converted to the Post Graduate Institute of Medical Education and Research,
reduced form tetrahydrofolate (THF) which is further con- Chandigarh, India. A total of 75 pregnant women who
verted to 5-MTHF by the enzyme methylenetetrahydrofolate attended the Department of Obstetrics and Gynaecology
reductase (MTHFR). 5-MTHF donates a methyl group to were included in this study. Ethical clearance was obtained
homocysteine to form methionine by the enzyme methio- from the Institute Ethics Committee (intramural), Post
nine synthase (MS), which requires vitamin B ­ 12 as a cofactor. Graduate Institute of Medical Education and Research,
Methionine synthase reductase (MSR) reduces the enzyme Chandigarh (Reference No.-NK/994/MD13643) and writ-
methionine synthase and restores functional form by reductive ten informed consent was obtained from all study subjects
methylation. Methionine generated from methylation of homo- prior to their participation in the study.
cysteine act as a precursor in the production of S-adenosyl
methionine (SAM) which is a primary methyl donor in various Study population
methylation reactions, regenerating homocysteine and methyl-
ated product(s). Homocysteine can be remethylated to methio- The present study was planned to evaluate the expression of
nine or converted to cysteine by entering the transsulfuration folate transporters and enzymes involved in folate metabo-
pathway by the action of cystathionine β synthase (CBS) [9]. lism in placental tissues collected during advancing gesta-
During pregnancy, the requirement of folate increases tion, preeclampsia, and neural tube defect pregnancies. In
several folds. Various studies suggest that folate deficiency this study, placental villous tissues were collected from preg-
during early stages of pregnancy has been associated with nant women with normal pregnancy in first, second and third
neural tube defects [1, 2, 10]. Failure of normal placentation trimesters and pregnant women with a pregnancy compli-
in early gestational period leads to release of various factors cated by preeclampsia or neural tube defect. About 200 mg
from the abnormal placenta which provokes symptoms in of placental tissue was collected from multiple sites within
the mother leading to a maternal syndrome of preeclamp- the placenta and pooled to decrease biological variability
sia [11]. Serum Homocysteine concentration is found to from each subject.
be increased in cases of preeclampsia, which suggests that
folate deficiency may be associated with preeclampsia [12]. Group 1 Pregnant women in the first trimester with a
Very few studies are available in the literature regarding normal pregnancy (n = 15).
the expression of genes related to folate transport during Group 2 Pregnant women in the second trimester with a
advancing gestation in normal pregnancy and pregnancy normal pregnancy (n = 15).
complications [13–15]. However, these studies fail to deter- Group 3 Pregnant women in the third trimester with a
mine the expression of folate pathway enzymes along with normal pregnancy (n = 15).
transporters at various stages of placental development in Group 4 Pregnant women who were diagnosed with
comparison with disease states. The metabolic pathways and preeclampsia (n = 15).
the role of folate transporters and folate pathway enzymes Group 5 Pregnant women diagnosed with a neural tube
in the placental development and pregnancy complications defect in developing fetus (n = 15).
are not well defined and the results are still inconclusive.
The information regarding the expression of folate transport- First trimester group (9–11 weeks of gestation) and sec-
ers along with folate metabolizing enzymes may provide ond trimester group (16–20 weeks) pregnant women, who
insights into the pathophysiology of disease development had undergone medical termination of pregnancy (MTP) by
in pregnancy-related disorders. Therefore, the current study suction evacuation and third trimester (37–40 weeks) preg-
was proposed to determine the expression of folate trans- nant women who had undergone normal vaginal delivery or
porters and key enzymes in folate metabolism during various cesarean section were included in the respective groups 1,
stages of placental development in physiological as well as 2 and 3. The gestational age was calculated from the date
pathological conditions i.e. neural tube defects (NTDs) and of the last menstrual period (LMP) and confirmed by ultra-
preeclampsia in Indian cohort to assess the comparison with sonography. Preeclampsia complicated pregnant women,
previous studies. with clinical symptoms of systolic pressure of 140  mm
Hg or above and diastolic pressure of 90 mm Hg or above
taken 2 times recorded 6 h apart with the patient at bed
Methods rest and proteinuria of greater than 300 mg in 24 h or a 1+
protein on dipstick on or after 34 weeks of gestation (late
Study design onset PE) were included in group 4. Pregnant women with
neural tube defect fetus diagnosed by ultrasonography were
This study was conducted in the Department of Biochem- included in group 5. These women were usually present in
istry and Department of Obstetrics and Gynaecology of the the first or early second trimester. Clinical characteristics

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of the pregnant women included in this study are presented rRNA species on the gel depicted the fine quality of RNA.
in Table 1. Other inclusion criteria include singleton preg- Real-time PCR was performed using SYBR Green Master
nancy with a live fetus, no evident fetal malformations on Mix on Applied Biosystems StepOnePlus™ Real-Time PCR
ultrasonography or diagnosed after the delivery of a baby in System using gene-specific primers (Table 1). Optimum
the current pregnancy and no known genetic disorder in pre- temperature and concentration of primers were determined
vious children. Exclusion criteria include pregnant women using gradient PCR for each gene before performing the real
with anemia, hypertension, diabetes mellitus, thyroid disor- experiments. For each experiment, a non-template reaction
der or pregnancy complications such as intrauterine growth (NTC) and no-amplification control (NAC) were run which
retardation (IUGR), any other cause of hyperplacentosis served as the negative control. Expression of the target gene
such as gestational trophoblastic disease, gestational diabe- was normalized using glyceraldehyde-3-phosphate dehydro-
tes mellitus, and Rh-negative isoimmunised women. genase (GAPDH) as endogenous control. The stability of
GAPDH was verified in all study groups prior to its selec-
Materials tion as the control. The comparative threshold cycle method
(∆∆CT or ­Ct method) [16] was used to quantify the ampli-
TRIzol® Reagent for RNA isolation was purchased from fied transcripts.
Ambion, Life Technologies Corporation, CA, USA, and
cDNA synthesis was done using RevertAidTM M-MuLV- Estimation of folate levels in placental tissue
RT kit (MBI Fermentas, Life Sciences, USA). DyNAmo
Flash SYBR Green qPCR Kit was obtained from Thermo The folate levels were determined by microtiter plate assay
Fisher Scientific Inc. All other routine biochemicals were using Lactobacillus casei as described by Horne and Patter-
of molecular grade and were purchased from Sigma Chemi- son [17]. Readings were taken using microtitre plate reader
cal Co. at 600 nm. All the steps were carried out in aseptic condi-
tions. Intra-assay and inter-assay precision (CV%) were 3.7
Quantitative RT‑PCR analysis and 4.9 respectively.

Total RNA was isolated from placental tissue immediately Statistical analysis
after collection using TRIzol reagent (Ambion, Life Tech-
nologies Corporation, CA, USA) according to the manu- All statistical analyses were performed using GraphPad
facturer’s instructions. After isolation, RNA was quantified Prism software. Between-group comparisons were made
spectrophotometrically and thereafter 1 µg of total RNA using one-way ANOVA; if found significant, the Bonferroni
was reverse transcribed using RevertAidTM MMuLV-RT post hoc test was applied. Pearson’s correlation analysis was
kit (MBI Fermentas, Life Sciences, USA) according to used to estimate the correlations between different param-
manufacturer’s instruction. Before reverse transcription of eters. All statistical tests were two-sided, and differences
RNA to cDNA, its quality was checked on 1.5% formalde- were considered statistically significant at p < 0.05. Unless
hyde agarose gel. Presence of intact bands of 28S and 18S otherwise stated, all data are expressed as mean (SD).

Table 1  Baseline clinical characteristics of pregnancies included in the study

First trimester (n = 15) Second trimester Third trimester Pre-eclampsia (n = 15) Neural tube defect
(n = 15) (n = 15) (n = 15)

Gravidity (range) 1–3 1–3 1–3 1–3 1–3

Parity (range) 1–3 1–3 1–3 1–3 1–3
Gestational age 10.5 ± 1.3 18.3 ± 1.3 38.5 ± 1.2 36.6 ± 1.6 17.7 ± 1.7
(weeks ± SD)
Maternal age 26.0 ± 2.7 25.4 ± 1.8 25.2 ± 2.3 25.7 ± 1.9 25.2 ± 1.4
(years ± SD)
Ethnicity North-Indian North-Indian North-Indian North-Indian North-Indian
Average blood pres- 120/80 120/80 120/80 > 140/90 120/80
sure (systolic/dias-
tolic mmHg)
Delivery mode/sample Suction evacuation for Suction evacuation for Normal vaginal Caesarean section Suction evacuation for
collection medical termination medical termination delivery/caesarean medical termination
of pregnancy of pregnancy section of pregnancy

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Results significantly lower when compared to gestation age-

matched (ST) normal placental tissues.
Table 2 describes the clinical characteristics of the preg-
nancies included in this study. Baseline characteristics Gene expression of folate transporters
were comparable between the study groups. Placental
folate levels and mRNA expression of various genes of Table 3 depicts the average cycle threshold of each gene
the folate pathway in placental tissue were evaluated in under study in order to compare the relative expression of
all study groups. different genes during advancing gestation. The comparison
of each gene as fold change with respect to the first trimester
is presented in Figs. 2 and 3.
Placental folate levels In normal gestation There was no significant change in
RFC expression during advancing gestation. The mRNA
In normal gestation Folic acid levels were found to be levels of PCFT were found to be decreasing with advancing
the maximum and significantly higher (2.04 µg/g tissues gestation though the change was not statistically significant
p < 0.001) in the first trimester as compared to other tri- (Fig. 2).
mester placental tissues (Table  4, Fig.  1). Second and In PE and NTD No significant change in the folate
third-trimester placentas were found to have equal levels transporters was observed in preeclampsia. However, RFC
of folate (884 ng/g tissue and 869 ng/g tissue respectively). expression was found to be significantly higher (24 fold,
In PE and NTD In case of preeclamptic placental tis- p < 0.001) in NTDs as compared to the gestation age-
sues, significantly lower level of folate (around 290 ng/g matched normal second-trimester placenta (Table 3, Fig. 2).
tissue, p < 0.01) was found while the folate levels in neural FRα expression was found to be significantly higher (3.7
tube defect placental tissues were observed to be dras- fold, p < 0.001) in neural tube defects samples as compared
tically reduced to 46 ng/g tissue (p < 0.001) which was to the normal second-trimester placenta (Table 3, Fig. 2).

Gene expression of enzymes of folate metabolism

Table 2  Primer sequences and conditions for real time qRT-PCR In normal gestation Within the normal three-trimester
placentas, MTHFR mRNA expression was the highest in
Gene Primer sequences: 5′–3′ Primer Amp. ­Ta/ first trimester samples and was found to be decreased by
conc. Amp.
nM ­Tm  °C 2- and 4.4-folds in second and third trimester respectively
(Table 4, Fig. 2, p < 0.001 and p < 0.001 respectively). On
RFC F: GAG​CGG​CAG​GAA​GTT​GTA​ 150 64/79.56 estimating the transcript levels of MS, mRNA expression
AG
R: CAG​CTT​GAG​GAG​GAT​GAA​
was the lowest in first trimester samples and increased by
GG
PCFT F: ATG​CAG​CTT​TCT​GCT​TTG​GT 150 64/81.94
R: GGA​GCC​ACA​TAG​AGC​TGG​
AC
FRα F: TGG​CAC​AAG​GGC​TGGAA​ 150 64/82.09
R: CCA​GAT​TTC​ATT​GCA​CAG​
AACAG​
MS F: AGC​CAT​ACT​ACT​GCC​TCT​C 250 62/82.93
R: CGT​CAC​CAT​CAT​CCT​CAT​AG
MSR F: TTG​TGG​TTG​AGC​CGT​GGA​TT 250 64/80.95
R: TAT​CTC​CTC​TTG​TCC​TCT​GCT​
TGA​
MTHFR F: CAG​CAG​TGG​CAG​TGA​GAG​ 150 64/84.44
R: CCT​TGA​GAT​GAG​ATT​GAC​
AGC​
CBS F: AGT​CAT​CTA​CAA​GCA​GTT​C 250 62/86.59 Fig. 1  Folate levels. Folate levels in placental tissues of various
R: AGT​TCA​GCA​AGT​CAA​TGG​ group. FT first trimester, ST second trimester, TT third trimester,
GAPDH F: CGA​CCA​CTT​TGT​CAA​GCT​CA 150 64/86.13 PE preeclampsia, NTD neural tube defect. Values are presented as
R: AGG​GGT​CTA​CAT​GGC​AAC​TG mean ± SD of fold change with respect to first trimester placen-
tal group. ***p < 0.001 versus first trimester samples as control.
Primer conc. primer concentration, Amp. Ta amplicon annealing tem- ###p < 0.001 versus gestational age matched normal placental sam-
perature, Amp. Tm amplicon melting temperature, F forward primer ples (second trimester) as control. $$p < 0.01 versus gestational age
and R reverse primer matched normal placental samples (third trimester) as control

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Table 3  Average CT for mRNA RFC FRα PCFT MS MSR MTHFR CBS

expression of various genes of
folate pathway enzymes and FT 25.93 23.79 29.03 28.87 25.57 23.36 25.69
transports in placental tissues
ST 28.31 25.98 28.98 28.23 27.81 27.20 29.65
from various group
TT 26.21 24.85 28.17 26.43 25.92 26.07 27.79
PE 25.02 23.37 27.09 27.72 26.88 26.86 28.75
NTD 20.90 21.41 27.86 28.02 24.75 28.76 26.44

CT average cycle threshold value, FT first trimester, ST second trimester, TT third trimester, PE preec-
lampsia, NTD neural tube defect, RFC reduced folate carrier, FRα folate receptor alpha, PCFT proton cou-
pled folate transporter, MS methionine synthase, MSR methionine synthase reductase, MTHFR methyl tetra
hydro folate reductase, CBS cystathionine beta synthase

Fig. 2  Expression of folate
transporters. mRNA expres-
sion of RFC, FRα and PCFT in
placental tissues from vari-
ous group. FT first trimester,
ST second trimester, TT third
trimester, PE preeclampsia,
NTD neural tube defect. Values
are presented as mean ± SD
of fold change with respect to
first trimester placental group.
###p < 0.001 versus gestational
age matched normal placental
samples (second trimester) as
control

7.7- and 9.8-folds in second and third trimester respectively age-matched placenta from normal healthy pregnancy
(p < 0.05 and p < 0.001 respectively). On determining the (Fig. 3).
mRNA expression levels of CBS in various placental sam-
ples, a decreasing trend with advancing gestational age was Correlation analysis
observed. It was found to be the highest in first trimester
samples and decreased by 2.5- and 3.3-folds in second and The Pearson’s correlation was used to study the correla-
third trimester respectively (p < 0.001 and p < 0.001 respec- tion between different study parameters among all groups
tively) (Table 4, Fig. 3). including PE and NTD. The analysis revealed that among
In PE and NTD MS was observed in preeclamptic pla- the folate metabolizing enzymes, MTHFR was found to
centas as compared to normal third-trimester placentas have a significantly strong positive correlation(r = 0.96,
(Table 4, Fig. 3). NTD placental samples showed a signifi- p < 0.01) with folate levels (Table 5). There is a strong
cantly decreased mRNA expression of MTHFR (25 fold) as negative correlation (statistically not significant) between
compared to normal second-trimester placentas (p < 0.01). A folate transporters, RFC and FRα with folate levels
3.5-fold (p < 0.01) decreased expression of MSR expression (r = − 0.62 and − 0.67 respectively). We also observed
was found to be significantly higher (7.7-fold, p < 0.001) that RFC has significantly strong positive correlation with
in neural tube defects samples as compared to gestational

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Fig. 3  Expression of folate
metabolizing enzymes. mRNA
expression of MS, MSR,
MTHFR and CBS in placental
tissues from various group.
FT first trimester, ST second
trimester, TT third trimester, PE
preeclampsia, NTD neural tube
defect. Values are presented
as mean ± SD of fold change
with respect to first trimester
placental group. *p < 0.05;
***p < 0.001 versus first
trimester samples as control.
##p < 0.01; ###p < 0.001 versus
gestational age matched normal
placental samples (second
trimester) as control. $$p < 0.01
versus gestational age matched
normal placental samples (third
trimester) as control

Table 4  Folate levels and FT ST TT PE NTD

mRNA expression of various
genes of folate pathway Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
enzymes and transports in
Folate (ng/mg) 2.04 ± 0.71 0.88 ± 0.53*** 0.87 ± 0.53*** 0.29 ± 0.08$$ 0.05 ± 0.02###
placental tissues from various
group RFC (fold change) 1 1.26 ± 0.99 1.25 ± 0.91 2.64 ± 1.92 24.41 ± 16.39###
FRα (fold change) 1 1.14 ± 0.71 0.86 ± 0.70 1.49 ± 0.76 3.75 ± 2.41###
PCFT (fold change) 1 0.85 ± 0.39 0.59 ± 0.53 0.60 ± 0.45 0.56 ± 0.17
MS (fold change) 1 7.67 ± 4.29* 9.83 ± 8.05*** 2.80 ± 1.83$$ 1.36 ± 0.93
MSR (fold change) 1 1.23 ± 0.87 0.34 ± 0.14 0.59 ± 0.35 7.73 ± 7.30###
MTHFR (fold change) 1 0.51 ± 0.42*** 0.23 ± 0.17*** 0.12 ± 0.09 0.02 ± 0.01##
CBS (fold change) 1 0.40 ± 0.30*** 0.30 ± 0.18*** 0.15 ± 0.10 0.49 ± 0.36

Values are presented as mean ± SD of fold change with respect to first trimester placental group
FT first trimester, ST second trimester, TT third trimester, PE preeclampsia, NTD neural tube defect
*p < 0.05; ***p < 0.001 versus first trimester samples as control
##
 p < 0.01; ###p < 0.001 versus gestational age matched normal placental samples (second trimester) as con-
trol
$$
 p < 0.01 versus gestational age matched normal placental samples (third trimester) as control

FRα (r = 0.99, p < 0.001) and MSR (r = 0.99, p < 0.001) Discussion

and weak inverse correlation with other genes, PCFT,
MTHFR, MS, and CBS (r = − 0.39, − 0.54, − 0.41, and In the present study, we aimed to determine the expres-
− 0.36 respectively). Similarly, FRα was also observed sion of folate transporters and enzymes involved in folate
to have a strong positive correlation with MSR (r = 0.97, metabolism in the placenta in different trimesters of
p < 0.01). The correlation of other genes with each other normal pregnancy and pregnancy-related disorders viz.,
and their correlation coefficient (r) values are shown in preeclampsia and NTD. We observed that folate levels
Table 6. were significantly high in the first trimester as compared

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Table 5  Correlation analysis folate level and mRNA expression of patients but a lower level of folates in PE as compared to
various genes of folate pathway enzymes and transporters in placental normal controls [14]. In contrast, we did not observe any
tissue
change in the expression of these transporters in PE. Our
Genes Folate levels versus mRNA p value results showed an increase in RFC and FRα gene expres-
expression sion in NTD. Reza-López et al. have reported increased
Pearson correlation coefficient “r”
levels of PCFT in placentas from birth defect complicated
RFC − 0.62 0.26 pregnancies [15]. In our study, there was no significant
FRα − 0.67 0.22 change in the PCFT expression in the preeclampsia group
PCFT − 0.24 0.69 as well as in NTDs. However, PCFT has been shown to
MS 0.01 0.98 have a role in FRα mediated endocytosis [25]. Our result
MSR − 0.54 0.34 was in accordance with the previous studies that shown
MTHFR 0.96 0.01 PCFT may not be necessary for transplacental transport of
CBS 0.44 0.46 folate because PCFT null subjects who develop hereditary
folate malabsorption do not suffer from any developmental
Statistically significant results with p < 0.05 are highlighted as bold
defects [26, 27]. Prasad et al. [24], have shown that FRα
plays a critical role in the transport of folate across pla-
to second and third-trimester placental groups. This obser- centa. Our study has demonstrated that FRα along with
vation is in agreement with the physiology of the ges- RFC might play an important role in the transport of folate
tation, that in order to meet the high demand for folate in the placenta. Further studies are required with a larger
during the first trimester to provide for cell division and sample size to validate the role of folate transporters in
growth of fetus the placental folate levels are very high complicated pregnancies such as NTD and PE.
[18]. Our observation regarding the significantly lower Mutations in the MTHFR gene and polymorphisms
level of folate in the preeclamptic placental group is con- (C677T and A1298C) are shown to be associated with sev-
sistent with the previous study where a similar finding eral disorders such as cancer and pregnancy complications
of low folate levels in preeclamptic placentas was seen including preeclampsia and neural tube defects [28–30]. In
[19]. This suggests that low folate levels in placenta our study, we observed that the expression of MTHFR was
might play a role in the development of preeclampsia. In the highest in the first trimester and decreased with advanc-
the case of NTD placental group, the folate levels were ing gestation. It was also found to have a significant posi-
found to be drastically reduced. Several studies have ear- tive correlation with folate levels of placental groups. The
lier shown that the deficiency of folate in the diet during expression was significantly lower in preeclampsia and neu-
pregnancy and low maternal serum folate are associated ral tube defects placental group as compared to gestational
with an increased incidence of NTDs [20–22]. However, age-matched placental groups. These findings suggest that
RFC and FRα expression were observed to be significantly folate levels in placenta might regulate the expression of
higher in neural tube defects samples as compared to the MTHFR gene at the mRNA level and may as well contribute
second-trimester group. This suggests that there is an up- significantly in reducing the risk of pregnancy complications
regulation of RFC and FRα genes at the transcript level as such as preeclampsia and neural tube defects.
an adaptive response to low folate levels in placenta. Ear- In our study, we observed that the expression of MS
lier studies observed up-regulation of folate transporters gene was found to be the lowest in the first trimester and
in case of folate deficiency and thus support our findings increases with advancing gestation. The expression was
[23, 24]. Williams et al. observed a reduction in PCFT lower in preeclamptic as well as in neural tube defect sam-
and FRα gene expression in placentas from preeclamptic ples as compared to third and second-trimester placental

Table 6  Correlation analysis Genes FR PCFT MTHFR MTR MTRR​ CBS

between mRNA expression of
various genes of folate pathway r p R p r p r p r p r p
enzymes and transporters in
placental tissue RFC 0.99 < 0.01 − 0.39 0.51 − 0.54 0.34 − 0.41 0.49 0.99 < 0.01 − 0.36 0.55
FR − 0.3 0.62 − 0.56 0.32 − 0.49 0.40 0.97 < 0.01 − 0.35 0.57
PCFT − 0.14 0.82 0.18 0.77 − 0.38 0.53 0.55 0.33
MTHFR − 0.19 0.76 − 0.44 0.46 0.58 0.31
MTR − 0.45 0.45 0.01 0.99
MTRR​ − 0.23 0.70

Statistically significant results with p < 0.05 are highlighted as bold

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group respectively. This suggests that lower expression of Compliance with ethical standards 
MS might result in elevated levels of homocysteine and
hence increase the risk of various pregnancy complica- Conflict of interest  The authors stated that there are no conflicts of in-
tions such as preeclampsia and NTDs [31]. In some pre- terest regarding the publication of this article. Research funding played
no role in the study design; in the collection, analysis, and interpreta-
vious studies the deficiency of this enzyme either due to tion of data; in the writing of the report; or in the decision to submit
deregulation or mutations was shown to be associated with the report for publication.
birth defects, cardiovascular complications and neurologi-
cal impairment [32, 33]. Unexpectedly, lower expression
of MS in NTDs was associated with a higher expression
of MSR gene which might be due to an adaptive response References
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enzyme may lead to inactivation of methionine synthase 57(5):138–141
2. Czeizel AE, Dudas I, Vereczkey A, Banhidy F (2013) Folate
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