Methylenetetrahydrofolate Reductase Polymorphisms and Pregnancy Outcome

Abstract

Introduction Aim of the study was to evaluate the effect of methylenetetrahydrofolate reductase (MTHFR) polymorphisms on pregnancy outcome.

Materials and Methods A total of 617 pregnancies of women who were investigated for MTHFR C677T and A1298C polymorphisms prior to pregnancy were included in the study. Cases were classified into “homozygous polymorphisms” (Group I), “heterozygous polymorphisms” (Group II), and patients without polymorphisms who functioned as controls (Group III). Patients with polymorphisms were assigned to a specific protocol at least 3 months before becoming pregnant. Administration of low molecular weight heparin (LMWH) was started very early during pregnancy. The Beksac Obstetrics Index (BOI) was used to estimate the obstetric risk levels for the different groups.

Results We found that the early pregnancy loss (EPL) rate increased as MTHFR polymorphism complexity increased and that the early EPL rate was significantly higher in patients with MTHFR C677T polymorphism compared to patients with MTHFR A1298C polymorphism (p = 0.039). There were significant differences between the previous pregnancies of the patients in the 3 study groups in terms of perinatal complications and EPLs (p = 0.003 and p = 0.019). The BOI decreased as the severity of polymorphisms increased. An association between MTHFR polymorphisms and congenital malformations and chromosomal abnormalities was observed. We could not demonstrate any statistically significant difference between study groups when the 3 groups were compared with regard to the pregnancy outcomes under specific management protocols.

Conclusion MTHFR polymorphisms are potential risk factors for adverse pregnancy outcomes.

Zusammenfassung

Einleitung Ziel dieser Studie war es, die Auswirkungen von Methylentetrahydrofolatreduktase-(MTHFR-)Polymorphismen auf das Schwangerschafts-Outcome zu untersuchen.

Material und Methoden Es wurden insgesamt 617 Schwangerschaften von Frauen, bei denen vor der aktuellen Schwangerschaft eine Untersuchung auf MTHFR-C677T- und -A1298C-Polymorphismen durchgeführt wurde, in die Studie aufgenommen. Die Frauen wurden in 3 Gruppen eingeteilt: „homozygote Polymorphismen“ (Gruppe I), „heterozygote Polymorphismen“ (Gruppe II) sowie Frauen ohne Polymorphismen, die als Kontrolle fungierten (Gruppe III). Patientinnen mit Polymorphismen wurden mindestens 3 Monate vor ihrer Schwangerschaft einem spezifischen Protokoll zugeordnet. Mit der Verabreichung von niedermolekularem Heparin (LMWH) wurde bereits sehr früh während der Schwangerschaft begonnen. Der Beksac Obstetrics Index (BOI) wurde zur Schätzung des geburtshilflichen Risikos der 3 Gruppen verwendet.

Ergebnisse Es zeigte sich, dass der Anstieg der Frühabortrate mit einem Anstieg an MTHFR-Polymorphismus-Komplexität einherging und dass die Frühabortrate bei Frauen mit MTHFR-C677T-Polymorphismen signifikant höher war als bei Frauen mit MTHFR-A1298C-Polymorphismen (p = 0,039). Es gab signifikante Unterschiede zwischen den früheren Schwangerschaften der Patientinnen in den 3 Untersuchungsgruppen in Bezug auf perinatale Komplikationen und Frühaborte (p = 0,003 bzw. p = 0,019). Der BOI nahm mit zunehmendem Schweregrad der Polymorphismen ab. Es wurde auch eine Assoziation zwischen MTHFR-Polymorphismen und angeborenen Fehlbildungen sowie Chromosomenanomalien beobachtet. Beim Vergleich der 3 Studiengruppen in Bezug auf Schwangerschafts-Outcome und Managementprotokoll konnten wir keinen statistisch signifikanten Unterschied feststellen.

Schlussfolgerung MTHFR-Polymorphismen sind ein potenzieller Risikofaktor für einen ungünstigen Schwangerschaftsverlauf.

Key words

pregnancy loss - placenta - preeclampsia - MTHFR

Schlüsselwörter

Abort - Plazenta - Präeklampsie - MTHFR

Introduction

Methylenetetrahydrofolate reductase (MTHFR) polymorphism(s) and adverse “obstetrical/perinatal outcome” relationship has been a matter of interest for a long time [1], [2]. Likewise, the connections between MTHFR polymorphism(s) and repeated miscarriage, intrauterine growth retardation (IUGR), preterm delivery, preeclampsia, congenital abnormalities, fetal aneuploidies and ablation placenta has been reported already by various authors [3], [4], [5], [6], [7]. On the other hand, one must consider the chaotic nature and robustness of these relationship(s) in order to understand the biological rationale behind this “inherited folate metabolism disorder” and to have better management protocols [8], [9], [10], [11].

The MTHFR polymorphisms are relatively common. Several studies reveal that estimated population frequency changes between 5 to 14 percent [2], [9], [11]. MTHFR polymorphisms are more frequent than it has been expected and one should be careful before having epidemiological comments [9], [12]. We believe that MTHFR polymorphisms are genetic risk factors which necessitate some other pathological conditions in order to trigger “chained biological events” that will cause metabolic placental inflammation and intrauterine hypoxia.

MTHFR is an enzyme converting dietary folate (methylenetetrahydrofolate) to its active form (methyltetrahydrofolate) which is the co-enzyme of methionine synthase together with vitamin B₁₂ [13], [14]. Methionine synthase is a critical enzyme taking role in the conversion of homocysteine to methionine which also takes an important role in the DNA methylation process [15]. Homocysteine-Methionine and MTHFR related biochemical pathways are required for various biological processes such as the synthesis of various nucleotides (methylation of cytosine) and DNA methylation [16]. We must also keep “the hazardous structure of dietary folate (methyleneTHF)” in mind and arrange specific dietary protocols not to have impaired pyrimidine (timidilate; uracil → thymine) and DNA synthesis and co-factor deficiencies (vitamin B₁, B₆, B₂, B₃) [15], [17]. At the presence of MTHFR polymorphisms, tetrahydrofolate is converted to dihydrofolate, which in turn is converted to monohydrofolate. This is subsequently reconverted to methyltetrahydrofolate for the clearance of MTHFR. During this process, translational autoregulation of thymidilate synthase and dihydrofolate reductase, and uridine monophosphate conversion to thymidine monophosphate occur, resulting in the formation of tetrameric DNA and related complications.

MTHFR polymorphism(s) is generally accepted as a risk factor for thrombus formation and venous thromboembolism, and has been associated with cardiovascular diseases such as coronary artery disease [18], [19]. Hyperhomocysteinemia and homocysteine related pathway disorders seem to be the critical actors behind these unwanted obstetrical complications and thrombotic events [19], [20], [21]. Balanced homocysteine remethylation and trans-sulfuration are critical metabolic pathways because homocysteine is a vascular toxin which may cause endothelial injury at various organs [22]. And most probably, elevated homocysteine level is responsible from the injury of “intervillous space cellular structures” (endothelial cells of spiral veins, endovascular trophoblasts at the tip of spiral arteries, syncytotrophoblasts, superficial/glandular epithelial cells of decidua, decidual/sertoli cells, etc.) and cause placental inflammation going together with impaired fetal perfusion [23], [24], [25].

The aim of this retrospective study was to evaluate prenatal, obstetrical and perinatal complications in pregnancies with MTHFR C677T and A1298C gene polymorphisms.

Methods

In this study, we have screened “Perinatal Medicine Databank” of the Department of Obstetrics and Gynecology, Hacettepe University between January 2002 and December 2012 and selected “pregnancies” (aged between 18 and 45 years old) who had undergone MTHFR C677T and A1298C polymorphism investigations “before their subsequent pregnancies” due to various reasons (obstetrical, medical, family history etc.) (Hacettepe University Ethical Committee approval number: GO 14/443-34). Polymorphism analyses for MTHFR were performed by a single researcher (FG) with real time polymerase chain reaction (PCR) method over the ten years.

Patients with Factor V Leiden and Prothrombin 20210A mutations, and Antithrombin III, Protein C & S deficiencies, were excluded from the study. Patients with known autoimmune disorders and severe systemic disorders which may affect the study results were also excluded from the study. For the final analysis, a total of 617 patients were recruited for the evaluation in 3 groups. Group I consisted of 227 cases with homozygous or compound heterozygous MTHFR polymorphisms, Group II comprised 257 cases with heterozygous MTHFR polymorphisms (C677T or A1298C), and 133 patients (with various risk factors necessitating MTHFR polymorphism investigation) were recruited for the control group in which MTHFR polymorphisms were negative at all (Group III).

These 617 patients were examined and reevaluated before their subsequent pregnancies. Patients with MTHFR polymorphisms were carefully assigned to a specific treatment/management protocol (methionine restricted diet, 100 mg/day salicylic acid, vitamins B₁, B₂, B₃, B₆, B₉, and B₁₂ daily, and 5 mg folic acid twice a week) at least 3 months before getting pregnant and this protocol was continued during pregnancy. The daily intake of methionine in the general population varies from 1 to 4 g/day. Methionine restricted diet is the diet having less than 1 g/day methionine. Additionally, low dose low molecular weight heparin (LMWH; enoxaparine 1 × 2000 Anti-XA IU/0.2 ml/day) was started very early in their subsequent pregnancies.

The demographic and clinical characteristics of the patients were achieved through patient files and electronic records of the hospital and Division of Perinatal Medicine. Patient demographics (age, parity, gravida, etc.), obstetrics history, maternal serum vitamin and minerals levels, diagnostic laboratory parameters, pregnancy outcome such as gestational weeks at delivery, birth weight (gram), perinatal complications (miscarriage, chromosomal abnormalities, malformations, preeclampsia, intrauterine growth retardation, preterm rupture of membranes, ablation placenta, and stillbirth) were recorded. In this study, we have used “composite perinatal complications” (CPC) in the comparison of the study groups. CPC is consisted of “IUGR, PPROM/preterm delivery, preeclampsia, ablation placenta and stillbirths”. The data about outcomes of the newborns were kindly donated from the records of Neonatology Unit. Necessary consent forms were obtained at each step of the antenatal care program.

Beksac Obstetric Index

Beksac Obstetrics Index (BOI) is used for the evaluation of “risk level” in “pregnancy/patient” groups going together with different types of medical problems and perinatal complications. (BOIp is the calculation of the index during the course of current pregnancy) [26]. This obstetric index was calculated as: (“number of live born children” + “π/10”)/Gravida. In this study, BOIp was calculated and recorded separately during the last pregnancies of the patients in order to compare the study groups in terms of obstetrical history and obstetrical performances.

Statistical analysis

Descriptive statistics were used to describe and compare the baseline characteristics of the groups. Continuous variables were compared using One-Way Anova or Kruskal Wallis tests, and categorical variables were compared using the Pearson Chi-Square test. A p-value of < 0.05 was accepted as statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) v. 20.0 for Windows (SPSS, Inc., Chicago, IL).

Results

Patients demographics

Obstetrical and demographical features of the 3 groups are shown at [Table 1]. There were statistically significant differences between 3 groups in terms of miscarriage and “number of live born children” (p = 0,035 and p = 0,000, respectively) ([Table 1]).

Table 1 Demographic features and maternal serum vitamin and mineral levels.

	Group I (n = 227) (n [%])	Group II (n = 257) (n [%])	Group III (n = 133) (n [%])	p value
Data are given as means ± standard deviation or median (IQR 25 – 75th centiles). a Values calculated by One-Way ANOVA. b Values calculated by Kruskal Wallis test. IQR: Interquartile range.
Maternal age (years)	31.40 ± 5.06	31.49 ± 5.06	31.83 ± 5.02	0.729a
Gravida (n = 1799)	3.0 (2.0 – 4.0) (665 [36.4%])	3.0 (2.0 – 4.0) (779 [43.3%])	2.0 (2.0 – 3.0) (355 [19.7%])	0.095b
Parity (n = 494)	1.0 (0 – 1.0) (167 [33.8%])	1.0 (0 – 1.0) (216 [43.7%])	1.0 (0 – 1.0) (111 [22.5%])	0.057b
Miscarriage (n = 571)	0 (0 – 2.0) (224 [39.2%])	0 (0 – 1.5) (252 [44.1%])	0 (0 – 1.0) (95 [16.7%])	0.035b
Live born (n = 349)	0 (0 – 1.0) (97 [27.8%])	0 (0 – 1.0) (154 [44.1%])	1.0 (0 – 1.0) (98 [28.1%])	0.000b
Blighted ovum (n = 51)	0 (0 – 0) (18 [35.3%])	0 (0 – 0) (27 [52.9%])	0 (0 – 0) (6 [11.8%])	0.428b
Postpartum death (n = 90)	0 (0 – 0) (38 [42.2%])	0 (0 – 0) (39 [43.3%])	0 (0 – 0) (13 [14.5%])	0.472b
Intrauterine death (n = 66)	0 (0 – 0) (30 [45.5%])	0 (0 – 0) (29 [43.9%])	0 (0 – 0) (7 [10.6%])	0.212b
Ectopic pregnancy [n = 10]	0 (0 – 0) (2 [20%])	0 (0 – 0) (7 [70%])	0 (0 – 0) (1 [10%])	0.186b
Molar pregnancy(n = 4)	0 (0 – 0) (0 [0%])	0 (0 – 0) (3 [75%])	0 (0 – 0) (1 [25%])	0.276b
History of fetal anomaly (n = 103)	0 (0 – 0) (33 [32.0%])	0 (0 – 0) (47 [45.6%])	0 (0 – 0) (23 [22.4%])	0.532b
Folic acid (ng/mL)	12.98 ± 5.18	33.03 ± 189.70	13.12 ± 5.20	0.173a
Vitamin B12 (pg/mL)	307.08 ± 158.22	327.41 ± 216.59	344.20 ± 386.29	0.407a
Zinc (pg/mL)	108.20 ± 24.72	106.24 ± 9.13	104.67 ± 32.19	0.814a
Copper (pg/mL)	26.14 ± 17.62	28.86 ± 19.57	25.42 ± 11.82	0.461a

Maternal serum folic acid, vitamin B₁₂, zinc and copper plasma levels were not statistically significantly different in between the 3 groups probably because blood samples were obtained during the beginning of their last pregnancies under medical treatment ([Table 1]).

Previous pregnancies

We have demonstrated that there are statistically significant differences between the previous pregnancies of the multigravid pregnant women of the 3 study groups in terms of the presence of “composite perinatal complications” (p values = Group I vs. III: 0.006, Group II vs. III: 0.001) and composite “early pregnancy complications” (miscarriage/abortion, blighted ovum, ectopic pregnancy and molar pregnancy) (p values = Group I vs. III: 0.005, Group II vs. III: 0.04) ([Table 2]).

Table 2 Comparison of multigravida pregnant women in our three study groups in terms of their previous pregnancy complications.

	Group I (n = 193)	Group II (n = 221)	Group III (n = 114)	p value
				Group I vs. II*	Group I vs. III*	Group II vs. III*
* Pearson Chi-Square test
Early pregnancy complications	118 (61.1%)	125 (56.6%)	51 (44.7%)	0.313	0.005	0.04
Composite perinatal complications	103 (53.4%)	103 (46.6%)	38 (33.3%)	0.169	0.006	0.001
Fetal genetic problems	36 (18.7%)	42 (19.0%)	18 (15.4%)	0.997	0.524	0.467

We have shown that early pregnancy loss rate is statistically significantly increased at patients with MTHFR C677T polymorphism compared to patients with MTHFR A1298C polymorphism (p = 0.039) ([Table 3]). There is no statistically significant difference in between these two polymorphisms in terms of composite perinatal complications (CPC) and genetic disorders. On the other hand, there were no statistical differences between homozygous and heterozygous patients in these three outcomes.

Table 3 Comparison of MTHFR mutation groups in terms of previous pregnancy complications.

	MTHFR 677 homozygous (n = 46) + heterozygous (n = 128) (n = 174)	MTHFR 1298 homozygous (n = 32) + heterozygous (n = 93) (n = 125)	p value
* Pearson Chi-Square test
Early pregnancy complications (homozygous + heterozygous)	(n = 31 + 68) 99 (56.9%)	(n = 16 + 40) 56 (44.8%)	0.039*
Composite perinatal complications (homozygous + heterozygous)	(n = 20 + 66) 86 (49.4%)	(n = 11 + 43) 54 (43.2%)	0.287*
Fetal genetic problems (homozygous + heterozygous)	(n = 6 + 27) 33 (19.0%)	(n = 5 + 15) 20 (16.0%)	0.508*

Previous obstetrical performance (obstetrical backgrounds/histories) of the three groups were compared in terms of a new index, heretofore named BOIp and statistically significant differences were found in between the 3 groups (p < 0.001) (BOIp values were 0.2886 ± 0.2089, 0.3290 ± 0.2102 and 0.4147 ± 0,2274 for Group I, Group II and Group III respectively). Statistically significant differences were detected between Group I versus III and Group II versus III (p < 0.001 and p = 0.001 respectively). Reduced MTHFR activity (Group I > II > III) was associated with lower BOIp values.

Previous pregnancies of 528 multigravid patients (89 primigravid patients were excluded) were also evaluated in terms of the genetic problems of the fetus. One hundred and eleven pregnancies in terms of genetic disorders (chromosomal abnormalities, congenital malformations and gene disorders) were detected in 1193 pregnancies of 528 multigravid patients (9%) (412 pregnancies of 193 patients in Group I, 517 pregnancies of 221 patients in Group II and 264 pregnancies of 114 patients in Group III). It has been demonstrated that 72.1% of these 114 cases were congenital malformations while 11.5% and 7.7% of cases were chromosomal abnormalities and single gene mutations respectively (8.7% of the cases were excluded from statistical analysis due to lack of sufficient information or finding) ([Table 4]). There were no statistical differences between all study groups (Group I vs. II, I vs. III, and II vs. III). Nineteen cases with chromosomal abnormalities were detected and the most common chromosomal anomalies were Turner syndrome (n: 4) and Downʼs syndrome (n: 4). There was no statistically significant difference between study groups in terms of genetic disorders though 18 of 19 “chromosomal abnormality cases” belonged to polymorphism groups.

Table 4 Distribution of congenital malformations, chromosomal anomalies and single gene disorders for the three study groups in previous pregnancies.

	Group I (n = 412)	Group II (n = 517)	Group III (n = 264)	p values
				Group I vs. II*	Group I vs. III*	Group II vs. III*
* Pearson Chi-square test NA = not applicable
Congenital malformations (n = 75)	24 (32%)	33 (44%)	18 (24%)	0.724	0.601	0.815
Chromosomal anomalies (n = 19)	9 (47.3%)	9 (47.3%)	1 (5.4%)	0.626	0.057	0.109
Single gene disorder (n = 8)	2 (25%)	6 (75%)	0	0.268	NA	NA
Unidentified cases (n = 9)	2 (22.2%)	3 (33.3%)	4 (44.4%)	0.844	0.163	0.271
Total (n = 111)	37 (100%)	51 (100%)	23 (100%)	0.647	0.904	0.602

[Table 5] shows the distribution of congenital malformations. The most common malformations were NTD (25,3%) and cardiac anomalies (25,3%). About 90% of NTDs were observed at patients with reduced MTHFR activity (Group I and II). Urinary system anomalies are more frequent in Group I and about 90% of cases concern patients with reduced enzyme activity (Group I and II).

Table 5 Congenital malformations observed in fetuses.

	Group I (n = 24)	Group II (n = 33)	Group III (n = 18)
Neural tube defects (n = 19)	6 (31.5%)	11 (57.8%)	2 (10.5%)
Cardiac anomalies (n = 19)	7 (36.8%)	5 (26.3%)	7 (36.8%)
Urinary tract anomalies (n = 11)	7 (63.6%)	3 (27.2%)	1 (9.0%)
Congenital diaphragm hernia (n = 8)	2 (25%)	4 (50%)	2 (25%)
Multiple anomalies (n = 7)	0	6 (85.7%)	1 (14.3%)
Nonimmune hydrops fetalis (n = 5)	1 (20%)	2 (40%)	2 (40%)
Agenesis of the corpus callosum (n = 3)	0	2 (66.6%)	1 (33.3%)
Omphalocele (n = 2)	1 (50%)	0	1 (50%)
Cleft lip/palate (n = 1)	0	0	1 (100%)

First pregnancies of the study groups were specifically compared in terms of 4 items such as

1. early pregnancy losses,

2. composite perinatal complications (IUGR, PPROM/preterm delivery, preeclampsia, placental abruption and perinatal mortality/stillbirth),

3. term delivery ratio and

4. genetic problems of the newborn (chromosomal abnormalities, congenital malformations and gene defects).

Therefore, 89 of the all patients were removed from the analysis due to primigravidity (whose MTHFR polymorphisms were detected due to various medical problems before getting pregnant). Term delivery rates were 25,3, 26.7 and 41.6% in Group I, Group II and Group III respectively and the differences were statistically significant (p < 0,001) even though Group III itself is a kind of high risk pregnancy group. We have also found increasing “early pregnancy loss” rates going together with MTHFR polymorphism complexity (Group I > II > III).

Pregnancies under treatment

When the 3 groups were compared in terms of their last pregnancies (who were under careful antenatal care), it has been demonstrated that gestational weeks at delivery were 36.67 ± 2.06, 36.91 ± 1.77 and 37.09 ± 2.01 for Group I, II and III respectively with no statistical difference between groups. The birthweights of the neonates in Group I, II and III were found to be 2839.6 ± 519.3 g, 2895.6 ± 475.3 g and 2988.2 ± 566.5 g respectively. We have shown a statistically significant difference only in between Group I and III in terms of birthweight (p = 0.030). The mortality rates were 0.9, 1.2 and 1.1% in Group I, II and III respectively (p = 0.863). We have found low rates of IUGR, oligohydramnios, PPROM, preeclampsia and ablation placenta at the last pregnancies of the study groups probably because last/current pregnancies were under careful antenatal care and specific treatment ([Table 6]). We could not demonstrate any statistical difference between study groups in terms of these complications. On the other hand, early pregnancy loss rate in these patients (patients with MTHFR polymorphisms) was determined as 10.8% which is very much like normal population again probably because last pregnancies of these patients were under intensive care and medical treatment (vitamin B supplementation, low dose low molecular weight heparin and low dose salicylic acid treatment together with methionine restricted diet).

Table 6 Comparison of the outcomes of the last pregnancies of the patients of 3 different groups in terms of obstetrical complications and perinatal outcomes including genetic problems and congenital malformations.

	Group I (n = 227)	Group II (n = 257)	Group III (n = 133)	p value
PPROM: Preterm premature rupture of membranes IUGR: Intrauterine growth restriction
Preeclampsia (n = 20 [%])	5 (2.2%)	10 (3.9%)	5 (3.8%)	0.538
PPROM (n = 18 [%])	7 (3.1%)	7 (2.7%)	4 (3.0%)	0.970
IUGR (n = 11 [%])	1 (0.4%)	8 (3.2%)	2 (1.5%)	0.820
Ablatio placenta (n = 1 [%])	1 (0%)	0 (0%)	0 (0%)	0.423
Oligohydramnios (n = 11 [%])	5 (2.2%)	6 (2.3%)	0 (0%)	0.213
Stillbirth (n = 6 [%])	2 (0.9%)	3 (1.2%)	1 (0.8%)	0.863

Discussion

The question is the explanation of the mechanisms involved in increased perinatal morbidity and mortality in pregnant women with MTHFR gene polymorphisms (C677T and A1298C). It has been reported that several biological processes are disturbed due to the decreased MTHFR enzyme activity related to these mutations [20], [21], [25], [27]. Thus, increased homocysteine (injury of the cellular components of intervillous space of the placenta which results in impaired implantation and decreased fetal perfusion) and decreased methionine (impaired DNA methylation) levels may be responsible from various perinatal and obstetrical complications as well as the unwanted complications due to the accumulation of inactive dietary folic acid (activation of tetrameric DNA, timidilate) [16], [20], [24].

MTHFR enzyme polymorphisms are associated with increased risk for chromosomal abnormalities [28]. These polymorphisms can even cause consequent trisomic fetuses in the same patient [28], [29]. Association of MTHFR polymorphism(s) and congenital malformations were also reported by several authors (neural tube defects (NTD), cleft lip/palate, congenital heart defects etc.) [4], [30], [31], [32], [33]. We have demonstrated similar findings in our series, 18 of the 19 (94.7%) pregnancies with chromosomal anomalies were in the MTHFR homozygous or heterozygous group. We have also demonstrated that the most common malformations were NTD (25.3%) and cardiac anomalies (25.3%), and about 90% of NTDs affected patients with reduced MTHFR activity.

Additionally, recurrent miscarriages and adverse perinatal outcomes such as intrauterine growth restriction, preeclampsia, preterm labor, preterm premature rupture of membranes (PPROM), ablation placenta and stillbirth were reported in pregnancies with MTHFR polymorphisms [5], [6], [34], [35]. We have found increasing “early pregnancy loss” rates going together with MTHFR polymorphism complexity in terms of enzyme activity (Group I > II > III). We have also demonstrated that there are statistically significant differences in between the previous pregnancies of the multigravid pregnant women of the 3 study groups in terms of the presence of “composite perinatal complications” and “early pregnancy complications” (miscarriage/abortion, blighted ovum, ectopic pregnancy and molar pregnancy) (p = 0.003 and p = 0.019). However, there are conflicting publications in this field due to the complexity of the problem [27], [36], [37].

The association between MTHFR polymorphisms and recurrent pregnancy loss has already been known for a long time, and it has been reported that low-dose acetylsalicylic acid (ASA) and low molecular weight heparin (LMWH) have a positive impact on pregnancy outcome [36], [38]. Our findings are consistent with the literature. We have also demonstrated that “MTHFR homozygous group” (Group I) had the highest early pregnancy loss rate in their first pregnancies (untreated pregnancies) when compared to the others.

Our study group consisted of patients that were preconceptionally evaluated and prepared for their subsequent pregnancies. Low dose LMWH, low dose ASA, folic acid, pyridoxine and cobalamin treatments and diet containing lower methionine levels were started as early as possible when they got pregnant under intensive antenatal care program. We believe that this treatment modality improved the pregnancy outcomes.

In clinical practice, we are dealing with a wide spectrum of patients with various health disorders causing different perinatal complications. The critical issue is the existence of multiple variables behind the maternal health disorders and perinatal complications [1], [34], [39]. This reality makes life difficult for physicians in the categorization and comparison of risk levels for different patient groups. Therefore, in this study we have used a new obstetric index which is named “Beksac Obstetrics Index” (BOI) which is defined as (number of living children + [Π/10])/gravida [26]. We believe that BOI is especially profitable during pregnancy and critical for the planning/envisioning of the management of risk factors. In this perspective, when comparing all of the groups in terms of BOI, increasing MTHFR polymorphism severity (Group I > II > III) was associated with lower BOI values and these results were statistically significant.

In conclusion, MTHFR polymorphisms and impaired “homocysteine/methionine” metabolism disorders are potential risk factors for “placental inflammation and impaired fetal perfusion” going together with obstetrical/perinatal complications. The same mechanisms are also responsible for the “impaired nucleotide and DNA synthesis” and most probably the reason of increased incidence of congenital abnormalities and fetal aneuploidies.

We believe that pregnancies with poor obstetrical history concerning repeated miscarriages and obstetrical complications might be screened for MTHFR polymorphisms, and we recommend methionine restricted diet, 100 mg/day salicylic acid, vitamins B₁, B₂, B₃, B₆, B₉, and B₁₂ daily, and 5 mg folic acid twice a week together with enoxaparine (1 × 2000 Anti-XA IU/0.2 ml/day) for pregnant women with these polymorphisms.

Conflict of Interest

The authors declare that they have no conflict of interest.

• References

• 1 Lykke J, Bare L, Olsen J. et al. Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort. J Thromb Haemost 2012; 10: 1320-1325
• 2 Nurk E, Tell GS, Refsum H. et al. Associations between maternal methylenetetrahydrofolate reductase polymorphisms and adverse outcomes of pregnancy: the Hordaland Homocysteine Study. Am J Med 2004; 117: 26-31
• 3 Pitkin RM. Folate and neural tube defects. Am J Clin Nutr 2007; 85: 285S-288S
• 4 Pan X, Wang P, Yin X. et al. Association between maternal MTHFR polymorphisms and nonsyndromic cleft lip with or without cleft palate in offspring, a meta-analysis based on 15 case-control studies. Inter J Fertil Steril 2015; 8: 463
• 5 Burke G, Robinson K, Refsum H. et al. Intrauterine growth retardation, perinatal death, and maternal homocysteine levels. N Engl J Med 1992; 326: 69-70
• 6 Goddijn-Wessel TA, Wouters MG, van de Molen EF. et al. Hyperhomocysteinemia: a risk factor for placental abruption or infarction. Eur J Obstet Gynecol Reprod Biol 1996; 66: 23-29
• 7 Tiwari D, Bose PD, Das S. et al. MTHFR (C677T) polymorphism and PR (PROGINS) mutation as genetic factors for preterm delivery, fetal death and low birth weight: A Northeast Indian population based study. Meta Gene 2015; 3: 31-42
• 8 Rady PL, Szucs S, Grady J. et al. Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A. Am J Med Genet 2002; 107: 162-168
• 9 Sazci A, Ergul E, Kaya G. et al. Genotype and allele frequencies of the polymorphic methylenetetrahydrofolate reductase gene in Turkey. Cell Biochem Funct 2005; 23: 51-54
• 10 Angeline T, Jeyaraj N, Granito S. et al. Prevalence of MTHFR gene polymorphisms (C677T and A1298C) among Tamilians. Exp Mol Pathol 2004; 77: 85-88
• 11 Perez ABA, DʼAlmeida V, Vergani N. et al. Methylenetetrahydrofolate reductase (MTHFR): incidence of mutations C677T and A1298C in Brazilian population and its correlation with plasma homocysteine levels in spina bifida. Am J Med Genet 2003; 119: 20-25
• 12 Esfahani ST, Cogger EA, Caudill MA. Heterogeneity in the prevalence of methylenetetrahydrofolate reductase gene polymorphisms in women of different ethnic groups. J Am Diet Assoc 2003; 103: 200-207
• 13 Nazki FH, Sameer AS, Ganaie BA. Folate: metabolism, genes, polymorphisms and the associated diseases. Gene 2014; 533: 11-20
• 14 Bhargava S, Tyagi S. Nutriepigenetic regulation by folate–homocysteine–methionine axis: a review. Mol Cell Biochem 2014; 387: 55-61
• 15 Şahin TG, Sayal B, Coşgun E. et al. Methylenetetrahydrofolate Reductase Enzyme Mutations and Relationship of Homocysteine Vitamin B12 and Folate Blood Levels. Gynecol Obstet Reprod Med 2016; 19: 1-6
• 16 Stover PJ. One-carbon metabolism–genome interactions in folate-associated pathologies. J Nutr 2009; 139: 2402-2405
• 17 Aydın E, Ceylan AC, Beksaç MS. The Relationship Between Methylation Defects and Different Genetic Disorders: Two Case Reports. Gynecol Obstet Reprod Med 2016; 22: 110-112 doi:10.21613/GORM.2016.484
• 18 Frederiksen J, Juul K, Grande P. et al. Methylenetetrahydrofolate reductase polymorphism (C677T), hyperhomocysteinemia, and risk of ischemic cardiovascular disease and venous thromboembolism: prospective and case-control studies from the Copenhagen City Heart Study. Blood 2004; 104: 3046-3051
• 19 Harpel PC, Zhang X, Borth W. Homocysteine and hemostasis: pathogenetic mechanisms predisposing to thrombosis. J Nutr 1996; 126 (Suppl. 04) 1285S
• 20 Tsitsiou E, Sibley CP, DʼSouza SW. et al. Homocysteine transport by systems L, A and y+L across the microvillous plasma membrane of human placenta. J Physiol 2009; 587: 4001-4013
• 21 Jansson T. Novel mechanism causing restricted fetal growth: does maternal homocysteine impair placental amino acid transport?. J Physiol 2009; 587: 4123
• 22 Hoffer LJ. Homocysteine remethylation and trans-sulfuration. Metabolism 2004; 53: 1480-1483
• 23 Solanky N, Jimenez AR, DʼSouza S. et al. Expression of folate transporters in human placenta and implications for homocysteine metabolism. Placenta 2010; 31: 134-143
• 24 Beksaç K, Örgül G, Çagan M. et al. Retrospective evaluation of pregnant women with celiac disease. J Turk Ger Gynecol Assoc 2017; 18: 56
• 25 Kamudhamas A, Pang L, Smith SD. et al. Homocysteine thiolactone induces apoptosis in cultured human trophoblasts: a mechanism for homocysteine-mediated placental dysfunction?. Am J Obstet Gynecol 2004; 191: 563-571
• 26 Beksaç MS, Aydın E, Turgal M. et al. An Obstetrics Index for the Assessment of Risk Levels of “High Risk Pregnancy” Groups. Gynecol Obstet Reprod Med 2016; 21: 10-13
• 27 Alfirevic Z, Roberts D, Martlew V. How strong is the association between maternal thrombophilia and adverse pregnancy outcome?: A systematic review. Eur J Obstet Gynecol Reprod Bio 2002; 101: 6-14
• 28 da Silva LR, Vergani N, Galdieri Lde C. et al. Relationship between polymorphisms in genes involved in homocysteine metabolism and maternal risk for Down syndrome in Brazil. Am J Med Genet A 2005; 135: 263-267
• 29 Turgal M, Yazicioglu A, Ozyuncu O. et al. Impaired DNA methylation leading to heterotrisomy. J Obstet Gynaecol 2013; 33: 904-905
• 30 Wenstrom KD, Johanning GL, Owen J. et al. Amniotic fluid homocysteine levels, 5, 10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites. Am J Med Genet 2000; 90: 6-11
• 31 van Beynum IM, Kapusta L, den Heijer M. et al. Maternal MTHFR 677C> T is a risk factor for congenital heart defects: effect modification by periconceptional folate supplementation. Eur Heart J 2006; 27: 981-987
• 32 Amorim MR, Lima MA, Castilla EE. et al. Non-Latin European descent could be a requirement for association of NTDs and MTHFR variant 677C> T. Am J Med Genet 2007; 143: 1726-1732
• 33 Botto LD, Yang Q. 5, 10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol 2000; 151: 862-877
• 34 Wu X, Zhao L, Zhu H. et al. Association between the MTHFR C677T polymorphism and recurrent pregnancy loss: a meta-analysis. Genet Test Mol Biomarkers 2012; 16: 806-811
• 35 Nair RR, Khanna A, Singh R. et al. Association of maternal and fetal MTHFR A1298C polymorphism with the risk of pregnancy loss: a study of an Indian population and a meta-analysis. Fertil Steril 2013; 99: 1311-1318.e4
• 36 Kosar A, Kasapoglu B, Kalyoncu S. et al. Treatment of adverse perinatal outcome in inherited thrombophilias: a clinical study. Blood Coagul Fibrinolysis 2011; 22: 14-18
• 37 Laskin CA, Spitzer KA, Clark CA. et al. Low molecular weight heparin and aspirin for recurrent pregnancy loss: results from the randomized, controlled HepASA Trial. J Rheumatol 2009; 36: 279-287
• 38 Aracic N, Roje D, Drmic Hofman I. et al. Low molecular weight heparin treatment and impact of inherited thrombophilia type in pregnancies with previous adverse outcome. J Matern Fetal Neonatal Med 2015; 28: 306-310
• 39 Wang X, Fu J, Li Q. et al. Geographical and Ethnic Distributions of the MTHFR C677T, A1298C and MTRR A66G Gene Polymorphisms in Chinese Populations: A Meta-Analysis. PLoS One 2016; 11: e0152414