European Journal of Obstetrics & Gynecology and Reproductive Biology 188 (2015) 113–117

The importance of MTHFR, MTR, MTRR and CSE expression levels inCaucasian women with preeclampsia

Agnieszka Seremak-Mrozikiewicz a,b,c, Anna Bogacz b,d,*, Joanna Bartkowiak-Wieczorek b,d,Hubert Wolski a,e, Boguslaw Czerny f,g, Malgorzata Gorska-Paukszta b, Krzysztof Drews a,c

a Division of Perinatology and Women’s Diseases, Poznan University of Medical Sciences, Poznan, Polandb Department of Pharmacology and Phytochemistry, Institute of Natural Fibres and Medicinal Plants, Poznan, Polandc Laboratory of Molecular Biology in Division of Perinatology and Women’s Diseases, Poznan University of Medical Sciences, Poznan, Polandd Laboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences, Poznan, Polande Division of Gynecology and Obstetrics, Podhale Multidisciplinary Hospital, Nowy Targ, Polandf Department of General Pharmacology and Pharmacoeconomics, Pomeranian Medical University, Szczecin, Polandg Department of Stem Cells and Regenerative Medicine, Institute of Natural Fibres and Medicinal Plants, Poznan, Poland

A R T I C L E I N F O

Article history:

Received 2 January 2015

Received in revised form 26 February 2015

Accepted 2 March 2015

Keywords:

Preeclampsia

Gene expression

Methionine and homocysteine metabolism

A B S T R A C T

Objective: Preeclampsia (PE) is a major cause of maternal and perinatal mortality and morbidity. The

studies suggest that both polymorphisms and changes of expression in genes encoding enzymes

involved in the methionine and homocysteine metabolism (MHM), such as methylenetetrahydrofolate,

reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR) and

cystathionine gamma-lyase (CSE), could play a role in the development of hypertension during

pregnancy. The aim of the study was to determine the expression level of MTHFR, MTR, MTRR and CSE

genes in the development of PE in Caucasian women.

Study design: The control group consisted of 74 healthy pregnant women and 90 patients with diagnosed

pre-eclampsia. Total RNA was isolated from placenta and the mRNA level of examined genes was to

determine using real-time PCR.

Results: The expression level of MTHFR gene showed no statistically significant difference in the study

group as compared to the control group. An increase of mRNA levels for MTR and CTH was observed by

124.7% (p < 0.0001) and 26.6% (p > 0.05), respectively. However, a decrease of placental expression was

noted for MTRR by 50% in preeclamptic women as compared to control group (p < 0.0001).

Conclusions: Our findings suggest that the elevated RNA expression of MTR in placenta of preeclamptic

patients is probably results of a potential compensation mechanism of the MHM while elevated CSE

expression indicates that homocysteine may be eliminated through the alternate transsulfuration

pathway.

� 2015 Published by Elsevier Ireland Ltd.

Contents lists available at ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /e jo g rb

Introduction

Preeclampsia (PE) present in around 5–10% of all pregnantwomen worldwide is associated with an increased risk of maternaland fetal morbidity and mortality. The aetiology of both pre-eclampsia and gestational hypertension remains essentiallyunknown. There is evidence suggesting that these disorders are

* Corresponding author at: Poznan University of Medical Sciences, Department

of Clinical Pharmacy and Biopharmacy, Marii Magdaleny 14, Poznan, Poland.

Tel.: +48 0616687837.

E-mail address: aniabogacz23@o2.pl (A. Bogacz).

http://dx.doi.org/10.1016/j.ejogrb.2015.03.009

0301-2115/� 2015 Published by Elsevier Ireland Ltd.

partly genetic in origin. Numerous studies search for genesprimarily responsible for PE development [1,2]. In recent years,it is claimed that vasodilator factors play an important role ingenetic background of PE because participate in the regulation ofblood pressure. The H2S synthesized by cystathionine gamma-lyase (CSE) acts as vasodilator factor and could be a potentvasodilator of the placental vasculature. This compound alsoexhibits antioxidant effects and stimulates angiogenesis. Addi-tionally, the H2S plays an important role in regulation of thebalance between growth and death of cells by inhibition of theCSE/H2S pathway in the excessive apoptosis of vascular smoothmuscle cells [3,4]. In 2008, Yang et al. pointed to the directvasodilator effect of H2S in the blood pressure regulation [4]. Thus,

A. Seremak-Mrozikiewicz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 188 (2015) 113–117114

abnormal function of the CSE/H2S pathway is associated withpathomechanism of cardiovascular diseases, including atheroscle-rosis and hypertension because CSE enzyme also participates inhomocysteine metabolism [5–7].

Furthermore, both polymorphisms and changes of expressionin genes encoding enzymes involved in the methionine andhomocysteine metabolism (MHM), such as methylenetetrahydro-folate, reductase (MTHFR), methionine synthase (MTR), methio-nine synthase reductase (MTRR) and CSE, could play a role in PEdevelopment. It has been shown that normal pregnant women hadlow plasma concentrations of homocysteine during gestationwhereas hyperhomocysteinemia occurred in women with PE [8–11]. The MTHFR enzyme converts dietary folates to 5-methylte-trahydrofolate which donates the methyl group for the remethyla-tion of homocysteine (Hcy) to methionine catalyzed by methioninesynthase (MTR). Methionine adenosyltransferase acts on thismethionine to produce S-adenosylmethionine (SAM) that isconverted to S-adenosylhomocysteine (SAH) and further hydro-lyzed to homocysteine. Hcy is either remethylated to methionineor is utilized for cysteine synthesis catalyzed by cystathionine b-synthase. Besides, methionine synthase for its functional regener-ation requires the participation of methionine synthase reductasewhich is encoded by the MTRR gene [12–14]. Recently, it has beenshown that changes in the methionine and homocysteinemetabolism may be related to the systemic damage that leadsto the occurrence of PE [15,16]. The study conducted by Hill et al.showed that reduced MTHFR activity leads to a decrease in SAMproduction [17].

We hypothesize that changes in the expression levels ofenzymes involved in the MHM cycle may be related to develop-ment of PE. The aim of the study was to determine the mRNA levelof MTHFR, MTR, MTRR and CSE genes that may contribute todevelopment of PE in Caucasian women.

Materials and methods

Patients

The patients were Caucasian of Polish origin recruited inDivision of Perinatology and Women’s Diseases, Poznan Universityof Medical Sciences. The Bioethical Committee of PoznanUniversity of Medical Sciences approved the study. All patientswere informed about the aim of the study and gave the writtenconsent.

To the study 90 pregnant women with PE (29 women with mildPE and 61 women with severe PE) were enrolled (age 28.89 � 5.33years, systolic blood pressure 161.21 � 19.89 mmHg, diastolicblood pressure 101.82 � 11.90 mmHg, gestational age at delivery33.22 � 3.64 weeks). The PE was recognized according to ACOG

Table 1Sequences of primers used for the RT-PCR analysis and PCR conditions.

Gene Primer sequence (50 ! 30) PCR con

Cycle

GAPDH F

GAPDH R

GAA GGT GAA GGT CGG AGT C

GAA GAT GGT GAT GGG ATT TC

35

MTHFR F

MTHFR R

GAA GTA CGA GCT CCG GGT TCC

AGA ACA TGA AGC TGA CG

40

MTR F

MTR R

GGA TGA TGG CAT GCT AGA TG

CAC CAT AGC AGC TCC ATA CT

40

MTRR F

MTRR R

GTG AGC AAG CTG TGG TAC AT

GTG TAT TCT GAA TCA CCG AGA

40

CSE F

CSE R

CAG GAT CCA GAG CAA TGG AC

GGT GTA ATT GCT GCC TCT AGT

40

F—forward.

R–reverse.

The sequences for used primers were designed with the use of the Oligo 6.0 program

(American College of Obstetricians and Gynecologists, 2011) criteria[18]. In each woman the blood pressure was measured sitting or lyingwith the sphygmomanometer (twice with 6 h interval). The urinesample was analyzed and the proteinuria was recognized by presenceof protein �30 mg/dl in sample (or in test strip as result 1+).

The control group included 74 healthy pregnant women (age28.60 � 4.25 years, systolic blood pressure 107.35 � 11.23 mmHg,diastolic blood pressure 66.24 � 9.09 mmHg, gestational age atdelivery 39.18 � 1.18 weeks). The exclusion criteria were as follow:diabetes mellitus, obesity, cardiovascular diseases, chronic hyperten-sion, renal and endocrinal diseases, thromboembolism, age lowerthan 18 years, multifetal pregnancy.

From each preeclamptic and healthy pregnant patient imme-diately after birth the fragment from the central part of placentawas collected. The fragments were sampled in maternal side inorder to obtain villous and decidua. These fragments of tissueswere placed in liquid nitrogen and immediately transported tolaboratory, where in few minutes they were stored in �80 8C to themoment of analysis.

RNA extraction and cDNA synthesis

Total cellular RNA was isolated from the placenta tissue usingTriPure Isolation Reagent (Roche, Germany) according to themanufacturer’s protocol. The concentrations and the purity of RNAwere determined by measuring the absorbance at 260 and 280 nmin a spectrophotometer (BioPhotometer Eppendorf, USA). RNAsamples were stored at �80 8C. Complementary DNA wassynthesized from 2 mg of total RNA in a total volume of 20 mlusing the Transcriptor First Strand cDNA Synthesis Kit (Roche,Germany). The obtained transcripts were stored at �20 8C or useddirectly for the real-time quantitative PCR (RT-PCR).

Real-time PCR

The level of mRNA expression was analyzed by using RT-PCR.The primers and RT-PCR conditions used for MTHFR, MTR, MTRR,CSE and GAPDH amplifications were described in Table 1. Alloligonucleotide sequences were synthesized by TIB Molbiol(Poland). Amplicon size and reaction specificity were confirmedby agarose gel electrophoresis and melting curve analysis. RT-PCRwas carried out using a LightCycler1 480 Instrument (Roche,Germany) and a LightCycler1 480 SYBR Green I Master (Roche,Germany) according to the manufacturer’s protocol. GAPDH wasused as a housekeeping gene for normalization (endogenousinternal standard). The PCR program was initiated with anactivation at 95 8C for 10 min. Each PCR cycle comprised adenaturation step at 95 8C, an annealing step at a specifictemperature and an extension step at 72 8C. The quantitative

ditions

Denaturation Annealing Extension

95 8C, 20 s 57 8C, 15 s 72 8C, 15 s

95 8C, 20 s 57 8C, 15 s 72 8C, 15 s

95 8C, 20 s 58 8C, 10 s 72 8C, 15 s

95 8C, 20 s 63 8C, 10 s 72 8C, 15 s

95 8C, 20 s 61 8C, 15 s 72 8C, 15 s

(National Biosciences, USA).

A. Seremak-Mrozikiewicz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 188 (2015) 113–117 115

PCR was monitored by measuring the increase in fluorescence bythe binding of SYBR Green I dye to the generated double-strandedcDNA. All samples were run in duplicate using the LightCycler1

480 Instrument and the melting curves were analyzed using theLightCycler1 480 Basic Software.

Statistical analysis

The mRNA content for studied genes was expressed asmean � SEM. The experimental data were analyzed using the SPSS17.0 for Windows software. Mean values were compared by one-wayANOVA test. The value of p < 0.05 was considered as statisticallysignificant.

Results

In this study, an analysis of selected clinical and biochemicalparameters in the preeclamptic women and the control group wasperformed. A shorter gestational age at delivery (33.22 � 3.64 vs.39.18 � 1.18 weeks, p < 0.0001) as well as higher systolic

Table 2Comparison of selected clinical parameters in the pregnant women with PE and contro

Parameter Control group n = 74 Women with PE (

Age (years)

range

p

28.60 � 4.25

18–42

28.89 � 5.33

18–40

ns

Gestational age at delivery (weeks)

range

p

39.18 � 1.18

37–42

33.22 � 3.64

25–40

<0.0001

Systolic blood pressure (mmHg)

range

p

107.35 � 11.23

90–140

161.21 � 19.89

110–220

<0.0001

Diastolic blood pressure (mmHg)

range

p

66.24 � 9.09

50–90

101.82 � 11.90

70–130

<0.0001

Height (cm)

range

p

165.38 � 4.18

150–180

164.23 � 5.43

149–176

ns

Body weight before pregnancy (kg)

range

p

60.13 � 8.38

40–97

63.26 � 15.64

42–128

0.009

Body weight during pregnancy (kg)

range

p

74.34 � 9.12

52–106

81.19 � 13.83

55–139

0.026

Infant birth weight (g)

range

p

3454.43 � 403.12

2560–4640

1963 � 887.49

620–3940

<0.0001

Number of pregnancies

range

p

1.45 � 0.69

1–6

1.43 � 0.53

1–4

ns

Delivery mode

Number of spontaneous birth (%)

Number of cesarean section (%)

p

(46,67%)

(53.33%)

(11.67%)

(88.33%)

<0.0001

Hemoglobin (mmol/L)

range

p

6.62 � 0.69

4.5–8.8

8.36 � 2.14

3.90–13.30

<0.0001

Erythrocytes (10^6/mL)

range

p

3.78 � 0.35

2.5–4.81

4.18 � 0.85

3.24–9.92

<0.0001

Leukocytes (10^3/mL)

range

p

18.39 � 7.20

8.27–56.12

10.95 � 4.17

4.14–25.29

ns

Platelets (g/L)

range

p

200.77 � 61.84

9.1–314.6

209.85 � 65.19

88.00–398.00

ns

Hematocrit (L/L)

range

p

0.34 � 3.16

0.219–0.36

0.37 � 0.05

0.20–0.45

ns

Protein in urine (mg/dL)

range

p

10.12 � 11.34

0–25

357.74 � 253.11

165.8–1334.2

<0.0001

* p < 0.05 as compared between the control group and study group (one-way ANOVA

(161.21 � 19.89 vs. 107.35 � 11.23 mmHg, p < 0.0001) and diastolic(101.82 � 11.90 vs. 66.24 � 9.09 mmHg, p < 0.0001) blood pressurewere observed for women with preeclampsia. These women werecharacterized by a higher body weight both before (p < 0.009) andduring the pregnancy (p < 0.026) compared to the control group. Thestudy group also had higher values of hemoglobin (p < 0.0001),erythrocytes (p < 0.0001) and platelets. In contrast to healthywomen, the study group had the presence of protein in the urine(165.8–1334.2 mg/dl, p < 0.0001). Moreover, the control group hadhigher levels of leukocytes in the blood compared to women withhypertension (Table 2). Correlation was also noted with respect to theinfant birth weight (1963 � 887.49 vs. 3454.43 � 403.12 g;p < 0.0001). Furthermore, in women with severe PE the infant birthweight was 1508.27 � 656.07 g (Table 2). As a confirmation of thecorrect selection of the women in the examined groups may indicatethat there were no statistically significant differences in relation tothe age of women. The lack of correlation was also found in relation tothe number of pregnancies.

Analysis of expression level of MTHFR gene showed nostatistically significant difference in the study group as compared

l group.

total) n = 90 Women with mild PE n = 29 Women with severe PE n = 61

30.22 � 5.46

18–38

ns

28.82 � 4.82

21–40

ns

35.18 � 3.41

28–40

0.002

32.24 � 3.65

25–40

<0.0001

174.81 � 14.02

110–210

<0.0001

182.57 � 16.54

155–220

p < 0.0001

106.29 � 13.42

70–130

<0.0001

110.00 � 10.31

100–130

<0.0001

165.92 � 7.16

149–176

ns

164.09 � 5.64

155–175

ns

64.74 � 17.11

42–128

0.008

64.45 � 14.32

45–100

0.006

80.62 � 14.89

56–139

0.031

80.54 � 14.91

55–115

0.035

2491.20 � 834.70

910–3940

<0.0001

1508.27 � 656.07

620–3140

<0.0001

1.66 � 0.92

1–4

ns

1.42 � 0.75

1–4

ns

(11.96%)

(88.04%)

<0.0001

(10.87%)

(89.13%)

<0.0001

8.55 � 2.01

5.29–12.50

<0.0001

8.35 � 2.75

3.90–13.30

<0.0001

4.10 � 0.53

3.24–5.61

<0.0001

4.40 � 1.12

3.50 � 9.92

<0.0001

10.43 � 1.97

7.10–15.90

ns

11.21 � 4.94

4.14–25.29

ns

202.78 � 70.09

88.00–398.00

ns

209.85 � 57.58

103.00–339.00

ns

0.36 � 0.04

0.28–0.44

ns

0.36 � 0.05

0.20–0.45

ns

75.00 � 0.00

75.00–75.00

<0.0001

410.86 � 147.54

150.00–500.00

<0.0001

test).

Fig. 1. The placental expression level of MTHFR, MTR, MTRR, and CTH in pregnant

women with PE. Control group was defined as 100%. Data were presented as

mean � SEM. *** p < 0.0001 as compared to the control group (one-way ANOVA test).

A. Seremak-Mrozikiewicz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 188 (2015) 113–117116

to the control group. An increase of mRNA levels in PE group forMTR and CTH was observed by 124.7% (p < 0.0001) and 26.6%(p > 0.05), respectively. However, a decrease of placental expres-sion was noted for MTRR by 50% in preeclamptic women ascompared to control group (p < 0.0001) (Fig. 1). Analyzing thedistribution of women with mild and severe preeclampsia weshowed a decrease of MTRR mRNA level by 55% (p < 0.0001) inwomen with severe PE. A significant increase of expression wasobserved for CTH by 58% (p < 0.001) in the same group ascompared to the control. No significant changes in the CTHexpression has not been demonstrated in women with mild PE. Inaddition, there were no significant differences in the MTHFR genein women with severe and mild PE. Furthermore, an increase ofmRNA MTR levels was observed by 122% (mild PE) and 128%(severe PE), respectively (Fig. 2).

Comment

In this study we determined if changes in the expression levelsof MTHFR, MTR, MTRR and CSE genes involved in MHM areconnected with PE occurrence. The data obtained in our studyindicated that MTHFR expression did not alter in women with PEin comparison to other study genes. In addition, we showed asignificant decrease of mRNA level for MTRR in study group inrelated to control group. However, an increase of placentalexpression in preeclamptic women was observed for MTR andCTH genes. These results suggested that the elevated RNAexpression of MTR in placenta of preeclamptic patients is probably

Fig. 2. The placental expression level of MTHFR, MTR, MTRR, and CTH in pregnant

women with mild and severe PE. Control group was defined as 100%. Data were

presented as mean � SEM. * p < 0.05, ** p < 0.001, *** p < 0.0001 as compared to the

control group (one-way ANOVA test).

results of a potential compensation mechanism of the methionineand homocysteine metabolism in the physiopathology of thisdisease.

Similar effect to our experiment was observed by Perez-Sepulveda et al. [11]. They showed also an increase of mRNA levelof MTR in placental tissue obtained from patients with PE. Thisincrease of MTR expression was associated with decrease 2-methoxyestradiol (2-ME) synthesized by cathechol-O-methyl-transferase (COMT) that induces the differentiation of theendovascular cytotrophoblast cells in the presence of hypoxia[19]. Additionally, in another study they showed low levels ofcirculating 2-ME at the first trimester of pregnancy in patients wholater developed preeclampsia [20]. According to Kanasaki et al.similar effect on low levels of 2-ME in women with PE wasobserved during the third trimester of pregnancy [21]. It seemsthat not only a decrease of COMT expression but alterations in themethionine and homocysteine metabolism could be involved inthe pathogenesis of PE. The SAM plays an important role becauseacts as the principal methyl donor within cells. These methylgroups are used for DNA methylation as well as production of 2-MEthrough COMT. Low levels of SAM may be caused by a decreasedactivity of MTHFR and/or MTR [22]. However, in the conductedstudies it was observed a lack of differences in the concentrationsof SAM or SAH between the study group and the control group[11]. This fact can be explained by supplementation of folic acidin the diet providing appropriate amounts of methionine forconversion into SAM. Moreover, authors suggest that other processmay lead to reduction of 2-ME even though the concentration ofSAM is not reduced. They claimed that the aromatase pathwayresponsible for the conversion of androgens into estrogens maycontribute to reduced production of 2-ME being a naturalmetabolite of 17-b-estradiol [23,24]. However, further studieswill be needed to confirm thishypothesis. In addition, it should beemphasized that there are a few studies which examine changes ofMTHFR, MTR and MTRR enzymes in women with PE. So far, there islack of data on the placental MTRR and CSE expression amongpreeclamptic women and hence the implications of these findingsmay not currently be very clear.

We postulate that decrease of MTRR mRNA level may be causedby many factors such as diet rich in folic acid and vitamin B12,administration of the drugs (methyldopa). A slight increase of theplacental CSE expression level between preeclamptic women andcontrol group indicates that homocysteine may be eliminatedthrough the alternate transsulfuration pathway which is sup-ported by the increased mRNA levels of CSE and/or CBS.

Furthermore, analysis of expression of MTHFR and MTR wasconducted in pregnant female Wistar rats using animal model. Itwas shown that the MTHFR mRNA levels were lower in both thevitamin B12 deficient groups at normal and excess levels of folicacid as compared to the control. Analyzing of MTR expression levelthe authors also showed that in the group with vitamin B12deficiency in the presence of excess folic acid levels was areduction of MTR mRNA level. They explain this fact that a decreaseof the MTHFR and the MTR expression levels may signify thealtered remethylation of homocysteine in the placenta. Thesechanges may also have the influence on the epigenetic program-ming of the developing fetus [12]. However, further studies arehelpful for validation of these results.

In conclusion, our findings suggest that the elevated RNAexpression of MTR in placenta of preeclamptic patients is probablyresults of a potential compensation mechanism of MHM in thephysiopathology of this disorder. In addition, the elevated CSEexpression indicates that homocysteine may be eliminatedthrough the alternate transsulfuration pathway which is sup-ported by the increased level of CSE and/or CBS enzymes. We alsopostulate that many factors such as diet rich in folic acid and

A. Seremak-Mrozikiewicz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 188 (2015) 113–117 117

vitamin B12, drugs may influence the decrease of MTRR level andmodulate MHM cycle.

Condensation

This study shows the changes in the expression level of genesinvolved in the methionine and homocysteine metabolism inwomen with PE.

Acknowledgement

The study was supported by statutory project from the PoznanUniversity of Medical Sciences (Poland).

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