Increased MMA concentration and body mass index are associated with spontaneous abortion in Brazilian women

Clinica Chimica Acta 411 (2010) 423–427

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n c h i m

Increased MMA concentration and body mass index are associated with spontaneous abortion in Brazilian women A pilot study Elvira M. Guerra-Shinohara a,⁎, Perla M. Pereira a, Ananka M. Kubota a, Thaiomara A. Silva a, Jucilene L. Reis a, Gerson S. Miyashita a, Vânia D'Almeida b, Robert H. Allen c, Sally P. Stabler c a b c

Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Science, University of Sao Paulo, Brazil Department of Biosciences, Universidade Federal de São Paulo, Brazil Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, United States

a r t i c l e

i n f o

Article history: Received 8 September 2009 Received in revised form 16 December 2009 Accepted 17 December 2009 Available online 28 December 2009 Keywords: Cobalamin Folate Homocysteine Methylmalonic acid Polymorphisms Body mass index Spontaneous abortion

a b s t r a c t Background: The pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors. We evaluated the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age, body mass index (BMI), cigarette smoking, alcohol ingestion, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin B6) and vitamin-dependent metabolites were analyzed. Methods: Study population included 100 healthy women that attended pre-natal care in 2 health centers of Sao Paulo, Brazil, and in whom pregnancy outcome was known. Folate and cobalamin status was measured in blood specimens collected between 4 and 16 weeks. The genotypes for 8 gene polymorphisms were evaluated by PCRRFLP. Results: Eighty-eight women had normal pregnancy outcome (Group 1), while 12 experienced a miscarriage after blood collection (Group 2). Increased methylmalonic acid (MMA) concentrations were found in Group 2 (median [25th–75th percentile]=274 [149–425] nmol/l) relative to Group 1 (138 [98–185]) (Pb 0.01). No differences between the groups were observed for serum cobalamin, serum or red cell folate, and serum total homocysteine or allele frequencies for 8 polymorphisms. In a conditional logistic regression analysis including age, gestational age, serum creatinine, MMA, cystathionine, body mass index (BMI), cigarette smoking, alcohol ingestion and use of multivitamins the risk of abortion was significantly associated with MMA (OR [95% CI]=3.80 [1.36, 10.62] per quartile increase in MMA), BMI (OR [95% CI]=5.49 [1.29, 23.39] per quartile) and gestational age (OR [95% CI]=0.10 [0.01, 0.77] per increase of interval in gestational age). Conclusions: Increased serum MMA and BMI concentrations are associated with spontaneous abortion in Brazilian women. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Many pregnancies are lost during early gestation and the pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors [1,2]. Low folate status has been associated with adverse pregnancy outcomes (placental abruption, recurrent pregnancy loss and preeclampsia) and congenital malformations [3–9]. In addition, increased total homocysteine (tHcy) concentration has been considered a marker of pregnancy complications and adverse pregnancy outcomes [10–12]. Few studies have

⁎ Corresponding author. Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Science, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, CEP 05508-900, Sao Paulo, SP, Brazil. Tel.: +55 11 3091 3785; fax: +55 11 3813 2197. E-mail address: [email protected] (E.M. Guerra-Shinohara). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.12.014

measured folate and tHcy concentrations in prospective studies prior to the adverse pregnancy outcome [9,13,14]. Although some case–control studies have shown decreased cobalamin concentrations in women with unexplained recurrent abortions [15–18], none of them has measured methylmalonic acid (MMA) and other metabolic markers of cobalamin or folate deficiencies before pregnancy loss. Obesity predisposes to poor pregnancy outcome, including sudden and unexplained intrauterine death [19]. It has also been reported that the risks of early miscarriage and recurrent early miscarriages (N3 successive miscarriages b12 weeks) were significantly higher among obese women [20]. Alcohol ingestion and cigarette smoking have been related as an independent, positive association with adverse pregnancy outcome [21,22]. It was shown that smoking during pregnancy not only affects placental function, thus causing obstetrical complications, but nicotine also crosses the placenta and acts as neuroteratogen [22].

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Genetic variants related to folate, cobalamin and homocysteine metabolism are associated with the risk of experiencing a miscarriage [2,11,12,23,24]. Furthermore, the combination between genotypes 677CT/1298CC and 677TT/1298CC in the methylenetetrahydrofolate reductase gene (MTHFR) was found in spontaneous aborted embryos but not in the healthy neonatal group, suggesting that these mutations are related to decreased fetal viability [25]. The objective of this study was to evaluate the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age, body mass index (BMI), cigarette smoking, alcohol consumption, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin B6) and vitamin-dependent metabolites were analyzed. 2. Materials and methods 2.1. Subjects Participants were recruited from pregnant women who attended 2 public health centers in Sao Paulo City, Brazil after admission for prenatal care from February 2004 to December 2005. In the current study, a total of 138 healthy women were recruited between 4 and 16 weeks of pregnancy, and they were followed by new examinations in the centers at 16, 28 and 36 weeks of pregnancy. Blood was collected in the fasting state at their first visit. The pregnancy outcome was known for 100 women, 88 women had labour at term with healthy newborns (Group 1) and 12 women miscarried (Group 2). We did not obtain data on pregnancy outcome for 38 women because they moved from Sao Paulo City to another city or state, or were unable to attend the health center on appointment day. The study protocol was approved by the Investigational Review Board of both the University of São Paulo (Brazil) and the University of Colorado (Denver CO), and written informed consent was obtained from all participants prior to entering the study. 2.2. Data collection Pregnancies were confirmed by a positive serum human chorionic gonadotrophin (hCG) test in urine by the investigating team. All the pregnancies were achieved by natural conception. Maternal data such as socioeconomic data (monthly per capita income of family, educational concentration and occupation), ethnic group, reproductive history, cigarette smoking, alcohol ingestion and vitamin supplement intake were obtained through an interview. Height and weight were measured while subjects were wearing light clothing, and BMI was calculated. The gestational age at the time of blood sampling was calculated from the date of the last menstrual period. The women were classified as Caucasian and African according to ethnic morphological criteria as previously published [26,27]. There was no Asian-descent woman in this sample. After delivery, we asked the women about the pregnancy outcome and infant birth weight, gender and outcome. 2.3. Blood sampling and biochemical measurements Two blood samples were drawn using the BD Vacutainers System®, one with EDTA for measuring red blood cell folate (RBC folate) and for genetic analysis, and the other without anticoagulant for all other measurements. The tube without anticoagulant was centrifuged within 15 min, and serum was frozen at −80 °C until further use. The determination of metabolites (tHcy, MMA, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine, cystathionine, 2methylcitric acid, cysteine, alpha-aminobutyrate, glycine, serine, N,Ndimethylglycine (DMG) and N-methylglycine (MG)) concentrations was performed at the University of Colorado Health Sciences Center by stable isotope dilution capillary gas chromatography–mass spectrometry and by liquid chromatography–mass spectrometry methods

[28,29]. Serum folate, red blood cell folate and cobalamin concentrations were determined by a chemiluminescent method (Immulite 2000®, Siemens Medical Solutions, Los Angeles, CA). Serum iron (SI), total iron binding capacity (TIBC), creatinine, AST and ALT were analyzed using commercial kits for Advia 1650® Chemistry Systems (Siemens New York NY). Ferritin concentration was determined by the Axsym System® (Abbott Laboratories, Abbott Park, IL). The value of transferrin saturation (TS) was obtained by ratio between the concentrations of SI and TIBC and it was expressed as a percentage. Total serum vitamin B6 (pyridoxal 5′-phosphate, pyridoxal, pyridoxine, pyridoxamine and 4pyridoxic acid) was quantified using HPLC with ultraviolet detection and isocratic elution [30]. 2.3.1. Genetic analysis Genomic DNA was isolated from whole blood by a salting-out method described by Salazar et al. [31]. Genotyping was performed using polymerase chain reaction followed by restriction digests (PCRRFLP) as described previously for methylenetetrahydrofolate reductase (MTHFR) 677CNT [32], MTHFR 1298ANC [33], reduced folate carrier (RFC1) 80ANG [34], methionine synthase (MTR) 2756ANG [27], methionine synthase reductase (MTRR) 66ANG [27], transcobalamin 2 (TC2) 776CNG [27], TC2 67ANG [27] and glutamate carboxypeptidase II (GCP2) 1561CNT [35]. 2.4. Statistical analysis The women with normal pregnancy outcome and those who had spontaneous abortion were classified as Group 1 and Group 2, respectively. The Kolmogorov–Smirnov test was used to test the normality of the continuous variable distributions. The distributions of per capita income, schooling, number of pregnancies, BMI, gestational age, creatinine, cobalamin, serum folate, ferritin, transferrin saturation, MMA, tHcy, 2-methylcitric acid, alpha-aminobutyrate, glycine, DMG, MG, SAH and SAM/SAH were skewed, and were log-transformed before analyses. The following criteria were adopted: anemia was defined by hemoglobin values b11.0 g/dl and iron deficiency by ferritin values b20 µg/l. Student's t-test, Mann–Whitney test, χ2 and Fisher's Exact test were used to compare independent groups. Correlations between vitamins and metabolites were examined by Spearman p test. The comparisons between vitamins and vitamin-dependent metabolites were repeated after adjusting for confounding variables (maternal age, gestational age, body mass index (BMI) and use of multivitamins). A conditional multiple logistic regression analysis was performed for evaluating the predictors for spontaneous abortion. The independent variables included were: quartiles of creatinine, quartiles of MMA, quartiles of cystathionine and quartiles of BMI. The maternal age and the gestational age were also included as categorical variables (maternal age: b20, 20 to 29 and ≥30 years, gestational age: b9, 9 to 12 and ≥13 weeks of pregnancy) as well as cigarette smoking, alcohol ingestion and use of multivitamins. The cut-off values of concentrations in each quartile were: MMA (103, 145 and 204 nmol/l); cystathionine (74, 88 and 115 nmol/l); BMI (21.3, 23.4 and 26.3 kg/m2) and creatinine (0.7, 0.7 and 0.8 mg/dl). All statistical analyses were performed by using SPSS for WINDOWS software (version 16.0; SPPS, Chicago, IL) and differences were determined at P b 0.05. 3. Results 3.1. Characteristics of population Of the 100 included subjects, 88 women had normal pregnancy outcome (Group 1), 12 had a miscarriage after the blood had been drawn (Group 2). In Group 2, 11 miscarriages occurred in the first trimester of pregnancy and one in the beginning of the second trimester

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(20th week). The frequencies of Caucasians in Groups 1 and 2 were respectively, 56.8 and 41.7%. The frequency of Africans was 43.2% in Group 1 (17.0% black and 26.2% mulattoes); and 58.3% in Group 2 (25.0% black and 33.3% mulattoes). The gestational age at the time of recruitment, and hence, blood sampling varied from 4 to 16 weeks. No difference was observed between the median (25th–75th percentile) of ages in Group 1: 25.5 (21.3–30.0) years and Group 2: 28.0 (24.3–34.5) years, P = NS (Table 1). None of the women included in the present study took folic acid supplementation in the preconceptional time. The frequencies of cigarette smokers and alcohol consumers were similar in the two groups (Table 1). No difference between the allele frequency in the 2 groups for MTHFR 677CNT, MTHFR 1298ANC, MTR 2756ANG, MTRR 66ANG, TC2 776CNG, TC2 67ANG, RFC1 80ANG and GCP2 1561CNT polymorphisms was observed (Table 1). The frequencies of anemia and iron deficiency were similar in both groups (Table 1).

3.2. Vitamin and metabolite status Increased MMA concentrations were found in Group 2 (median [25th–75th percentile] = 274 [149–425] nmol/l) relative to Group 1 (138 [98–185]) (P b 0.01). The concentrations of other variables were

increased in Group 2 (P b 0.05): serum cystathionine (128 [88–165] versus 90 [73–114] nmol/l); serum total cysteine (247 [224–268] versus 224 [202–241] µmol/l); and serum glycine (176 [159–240] versus 159 [137–191] µmol/l). The differences of MMA and cystathionine between the groups remained significant even after adjustment in maternal age, gestational age, use of multivitamins, creatinine and BMI. No differences between the groups were observed for serum cobalamin, serum or red cell folate, vitamin B6, ferritin, transferrin saturation, tHcy, 2-methylcitric acid, methionine, alpha-aminobutyrate, serine DMG, MG, SAH, SAM and SAM/SAH ratio (Table 2). Cobalamin was inversely related to MMA in both groups while the association between cobalamin and tHcy was found only in Group 1. No correlation was found between MMA and creatinine in both groups. Folate (serum and RBC folate) was inversely associated with tHcy in both groups. No association was found between vitamin B6 and MMA or vitamin B6 and tHcy, in both groups. A direct association was found between creatinine and tHcy concentrations in both groups (Table 3). No association was found between BMI and MMA in Group 1 (r = −0.15, P =0.198) and Group 2 (r= −0.04, P =0.907). Table 2 Vitamin and metabolite concentrations in women that had normal pregnancy outcome (Group 1) and those who had spontaneous abortion (Group 2). Variables

Table 1 Characteristics of women with (Group 2) and without (Group 1) spontaneous abortion. Cobalamin (pmol/l)

Variables

Group 1 N = 88

Group 2 N = 12

P value

Age (year) Per capita income (US$)

25.5 (21.3, 30.0) 50.00 (46.67, 125.00) 9.0 (5.0, 11.0)

28.0 (24.3, 34.5) 70.83 (30.83, 125.00) 8.0 (5.5, 10.8)

NSa NSa

Red blood cell folate (nmol/l)

NSa

Vitamin B6 (nmol/l)

Education level (number of years) Number of pregnancies (including the actual pregnancy) Body mass index (in kg/m2) Gestational age at recruitment (weeks) Hemoglobin (g/dl) Ethnic groups Caucasian African Cigarette smoking No Yes Alcohol consumption No Yes Use supplements (in the present pregnancy) No Yes Allele frequency (polymorphism) Allele T (MTHFR 677CNT) Allele C (MTHFR 1298ANC) Allele G (MTR 2756ANG) Allele G (MTRR 66ANG) Allele G (TC2 776CNG) Allele G (TC2 67ANG) Allele G (RFC1 80ANG) Allele T (GCP2 1561CNT) Anemia Iron deficiency

2.0 (1.0, 3.0)

2.0 (1.0, 5.3)

NS

Serum folate (nmol/l)

a

22.8 (21.1, 26.1) 11.0 (9.0, 13.0)

27.0 (24.4, 28.1) 9.5 (7.5, 11.0)

0.005 NSa

12.5 (11.9, 13.1)

12.6 (11.9, 13.3)

NSb

50 (56.8%) 38 (43.2%)

5 (41.7%) 7 (58.3%)

NSc

76 (86.4%) 12 (13.6%)

10 (90.9%) 1 (11.1%)

NSd

72 (81.8%) 16 (18.2%)

9 (75.0%) 3 (25.0%)

NSd

60 (68.2%) 28 (31.8%)

11 (91.7%) 1 (8.3%)

NSd

25.6% 24.4% 21.0% 43.7% 33.5% 14.2% 56.2% 1.7% 5 (5.7%) 21 (24.4%)

16.7% 20.8% 20.8% 58.3% 25.0% 16.7% 37.5% 4.2% 1 (8.3%) 3 (25.0%)

NSd NSd NSd NSd NSd NSd NSd NSd NSd NSd

Ferritin (μg/l) a

Transferrin saturation (%) Serum creatinine (mg/dl) MMA (nmol/l) tHcy (μmol/l) Cystathionine (nmol/l) 2-methylcitric acid (nmol/l) Cysteine (μmol/l) Methionine (μmol/l) Alpha-aminobutyrate (μmol/l)

Group 1: women with normal pregnancy outcome; Group 2: women who had spontaneous abortion. Median (25th, 75th percentile) and frequency (absolute and relative) were shown, respectively, for continuous and categorical variables. Criterion adopted: anemia (Hb b 11 g/dl); iron deficiency (ferritin b20 µg/l). a Mann–Whitney test. b Student's t-test. c χ2. d Fisher's Exact test.

425

Glycine (μmol/l) Serine (μmol/l) N.N-dimethylglycine (μmol/l) N-methylglycine (μmol/l) SAH (nmol/l) SAM (nmol/l) SAM/SAH ratio

Median (25th, 75th percentile) N Group 1

Group 2

269 (206, 334) 86 27.4 (19.6, 39.3) 88 1213 (917, 1396) 82 13.9 (11.0, 16.5) 76 40.4 (20.6, 63.3) 86 29.6 (22.5, 37.3) 87 0.70 (0.70, 0.80) 87 138 (98, 185) 76 4.6 (4.1, 5.5) 76 90 (73, 114) 76 108 (99, 122) 76 224 (202, 241) 76 24.2 (21.5, 26.4) 76 9.3 (7.9, 11.8) 76 159 (137, 191) 76 100 (89, 113) 76 3.0 (2.4, 3.6) 76 0.8 (0.7, 1.0) 76 10.9 (8.7, 12.6) 76 60.5 (54.9, 67.1) 76 5.7 (4.8, 7.2) 76

271 (214, 368) 12 26.3 (20.9, 40.7) 12 1145 (911, 1392) 12 13.6 (11.2, 17.2) 10 46.1 (15.5, 93.3) 12 27.3 (19.9, 43.4) 12 0.80 (0.70, 0.88) 12 274 (149, 425) 10 5.1 (4.8, 6.8) 10 128 (88, 165) 10 124 (102, 182) 10 247 (224, 268) 10 26.6 (22.6, 30.2) 10 10.8 (7.9, 16.3) 10 176 (159, 240) 10 111 (97, 125) 10 3.1 (2.6, 3.9) 10 1.0 (0.8, 1.3) 10 10.3 (7.7, 11.8) 10 64.2 (58.3, 72.1) 10 6.6 (5.6, 8.4) 10

P

NS NS NS NS NS NS ND 0.008 NS 0.006 NS 0.017 NS NS 0.041 NS NS 0.033 NS NS NS

Group 1: women that had normal pregnancy and babies, Group 2: women who had spontaneous abortion. The biochemical determinations were measured before the miscarriage. MMA: methylmalonic acid; tHcy: total homocysteine; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.

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Table 3 Spearman correlations between vitamin and metabolite levels in two groups of women (Groups 1 and 2). MMA

Cobalamin

Serum folate

RBC folate

Vitamin B6

Creatinine

tHcy

Group 1

Group 2

Group 1

Group 2

r = − 0.43 P b 0.001 N = 76 r = − 0.04 P = NS N = 76 r = − 0.04 P = NS N = 71 r = − 0.16 P = NS N = 76 r = − 0.02 P = NS N = 76

r = − 0.64 P = 0.048 N = 10 r = − 0.41 P = NS N = 10 r = 0.10 P = NS N = 10 r = 0.03 P = NS N = 10 r = − 0.28 P = NS N = 10

r = − 0.29 P = 0.012 N = 76 r = − 0.33 P = 0.003 N = 76 r = − 0.28 P = 0.018 N = 71 r = 0.18 P = NS N = 76 r = 0.34 P = 0.003 N = 76

r = 0.37 P = NS N = 10 r = − 0.78 P = 0.008 N = 10 r = − 0.71 P = 0.021 N = 10 r = 0.06 P = NS N = 10 r = 0.76 P = 0.011 N = 10

Group 1: women that had normal pregnancy and babies, Group 2: women who had spontaneous abortion. MMA: methylmalonic acid; tHcy: total homocysteine.

3.3. Multiple logistic regression model In a conditional logistic regression analysis including age, gestational age, serum creatinine, MMA, cystathionine, BMI, cigarette smoking, alcohol ingestion and use of multivitamins the risk of abortion was significantly associated with MMA (OR [95% CI] = 3.80 [1.36, 10.62] per quartile increase in MMA), BMI (OR [95% CI] = 5.49 [1.29, 23.39] per quartile) and gestational age (OR [95% CI] = 0.10 [0.01, 0.77] per increase of interval in gestational age). 4. Discussion Impaired cobalamin and folate status may affect tHcy remethylation to methionine, other methylation reactions, and DNA synthesis, and therefore cell proliferation and normal growth. Aberrations in folate pathways such as maternal folate deficiency, maternal hyperhomocysteinemia and the 677TT genotype of methylenetetrahydrofolate reductase (MTHFR) 677CNT polymorphism have been associated with spontaneous abortion [15]. Moreover, some experimental studies showed that homocysteine itself can be embryotoxic [36], or it can interact with hemostatic genetic determinants [37] or it can block methylation reactions by increasing the SAH concentrations, and consequently reduce the SAM/SAH ratio [38]. In humans, the excess homocysteine may cause alterations in chorionic and decidual vessels leading to defective implantation of the embryo. A direct relationship between high tHcy concentrations and alterations in vascularisation of chorionic villosities (reduced vascular areas, perimeters and diameters) was shown by Nelen et al. [39]. Although several studies show that tHcy is an independent risk factor for spontaneous abortion, the present study does not confirm this finding because the women from Group 2 did not have increased tHcy concentrations as compared to those with normal pregnancy outcome, and tHcy did not predict spontaneous abortion in the logistic regression model. However, we were able to show inverse correlations between folate (serum and erythrocyte) and tHcy concentrations in both groups studied. It has been proposed that cobalamin deficiency might explain infertility due to failure of ovulation, abnormal cell division in the fertilized ovum and/or failure of implantation due to megaloblastoid epithelial changes in the endometrium [40]; moreover, this deficiency could be responsible for the early cessation of the embryo's development [41]. Although MMA is considered a marker

of functional cobalamin deficiency and renal dysfunction [29], in the present study, there was no difference in cobalamin and creatinine concentrations between the two groups. In addition, significant inverse correlations were found between cobalamin and MMA concentrations and no significant correlations were observed between creatinine and MMA in both groups. On the other hand, increased MMA and BMI, and low gestational age were selected as predictors of spontaneous abortion in the conditional logistic regression which included some confounding variables such as creatinine, age, cigarette smoking, alcohol ingestion and the use of multivitamins. The increased MMA concentrations found in Group 2 could be related to the mechanisms of miscarriage. We hypothesize that another associated metabolic alteration could explain how with the same background (vitamin intake and genetics) and the same cobalamin and creatinine concentrations, those in Group 2 would have higher concentrations of MMA. Exposure to tobacco and alcohol consumption are considered risk factors for miscarriages [21], however we are unable to show the effects of these two factors in the present study. In the present study, anemia and iron deficiency frequencies were similar in the two groups and folate status was not associated with spontaneous abortion. Folate deficiency was not found in a previous study with Brazilian women of childbearing-age [42]. The absence of a difference between folate (serum and RBC) concentrations in the 2 groups of women, in the present study, might be a result of the food folate enrichment program present in Brazil or it could be due to the very short time use of supplementation by the women. Although folic acid supplementation is recommended for pregnant women and women planning to become pregnant in some countries [43], it is an unusual practice in public health centers in Brazil from 2004 to 2005 as was confirmed by findings that none of the women took folic acid supplementation in the preconceptional time. The allele frequencies of polymorphisms in the RFC1 and GCP2 genes (related to the absorption of folate), TC2 gene (transport of cobalamin into the cells), MTHFR, MTR and MTRR genes (related to homocysteine metabolic pathways) were similar in the two groups. The analysis of influence of the polymorphisms alone or in combination with vitamin or vitamin-dependent metabolites was not possible because of the small sample size. In summary, no differences in the studied polymorphisms related to impaired folate and cobalamin metabolism were detected between groups. Homocysteine concentrations were also similar, however, increased MMA, cystathionine, cysteine and glycine concentrations were found in Group 2. We propose that another associated metabolic alteration could explain higher concentrations of MMA. In this study, other causes of miscarriage different from vitamin deficiency, such as chromosomal, endocrine, thrombophilic, autoimmune, etc. causes, were not evaluated. Although pregnancies were confirmed by a positive hCG test, the presence of a viable fetus by ultrasound scan before the blood collection was not confirmed in this study. However, the strength of this study includes the fact that the specimens were collected in the fasting state. Although the number of cases is small, it shows that increased MMA and BMI concentrations could be considered as risk factors for miscarriage. Future prospective larger studies are necessary to demonstrate the role of increased MMA as a possible cause of the miscarriage or the presence of other metabolic alteration related with this condition. Acknowledgments This study was supported financially by Fundação de Amparo à Pesquisa do Estado de São Paulo — FAPESP, Brazil (Proc. 03/09660-1 and 06//06406-5) (EMGS) and NIA-AG09834 (SPS). EMGS, TAS, GSM and VDA were granted with fellowships by CNPq. PMP and AMK were granted with fellowships by FAPESP. We thank all the women who participated in this study.

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