Homocysteine in Pregnancy

ADVANCES IN CLINICAL CHEMISTRY, VOL. 53

HOMOCYSTEINE IN PREGNANCY Michelle M. Murphy*,†,1 and Joan D. Fernandez-Ballart*,† *Unit of Preventive Medicine and Public Health, Faculty of Medicine and Health Sciences, IISPV, Universitat Rovira i Virgili, Tarragona, Spain † ´ n (CB06/03), CIBER Fisiopatologı´a de la Obesidad y Nutricio Instituto de Salud Carlos III, Madrid, Spain

1. 2. 3. 4. 5. 6. 7. 8.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Homocysteine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophylactic Folic Acid, Folate Status, and Homocysteine . . . . . . . . . . . . . . . . . . . . . . Choline and Homocysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonnutritional Factors Associated with tHcy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health Benefits of tHcy Lowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Changes in tHcy During Normal Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Animal/In Vitro Studies and Homocysteine and Reproduction . . . . . . . . . . . . 8.3. Prospective Pregnancy Studies in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Elevated tHcy and Subfertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Early Pregnancy Loss/Miscarriage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Embryo, Placental, and Fetal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Gestational Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Other Adverse Pregnancy Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9. DNA-Methylation and Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10. Long-Term Effects of Exposure of Developing Fetus to Homocysteine . . . . 8.11. Future Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract The aim of this review is to evaluate the evidence for and against fasting plasma total homocysteine (tHcy) as a biomarker/risk factor of impaired reproductive function before and during pregnancy. Apart from nutritional 1

Corresponding author: Michelle M. Murphy, e-mail: [email protected] 105

0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53005-7

Copyright 2011, Elsevier Inc. All rights reserved.

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and lifestyle factors, tHcy is also influenced by physiological factors specific to pregnancy such as hemodilution, increased glomerular filtration rate, and endocrinological changes. These lead to a considerable reduction under normal circumstances in tHcy by midpregnancy. Stimulating excess endogenous homocysteine production before and during pregnancy in animal experiments and adding exogenous homocysteine to cell cultures result in the impairment of reproductive and developmental processes from preconception throughout pregnancy and during subsequent development of the offspring. Different studies have confirmed that elevated tHcy is a risk factor for subfertility, congenital developmental defects, preeclampsia, and intrauterine growth retardation. There is conflicting evidence that elevated tHcy is a risk factor for miscarriage, gestational diabetes, premature rupture of the membranes, placental abruption, and offspring with Down syndrome. Prospective, sufficiently powered, studies from preconception/early pregnancy are required to determine whether tHcy is a risk factor for these pregnancy complications.

2. Introduction The investigation of the biological and pharmacological regulation of circulating homocysteine has received considerable interest due to the evidence that associates elevated fasting plasma total homocysteine (tHcy) with increased morbidity and mortality from the earliest stages of life until old age. While this review will focus on pregnancy, regulation of homocysteine metabolism and morbidity associated with alterations in metabolism outside of pregnancy will also be briefly considered because subclinical pathological mechanisms present from before conception may have a considerable influence on pregnancy outcome. Elevated tHcy is often caused by suboptimal status in any of the key micronutrients that regulate it. Thus, it is not easily discerned whether the underlying cause of elevated tHcy or elevated tHcy itself is what contributes to the morbidity and mortality associated with this condition. The evidence for an association between elevated tHcy and pregnancy complications from in vitro, animal, and human studies is reviewed here.

3. Homocysteine Metabolism Homocysteine is formed following transmethylation of the essential sulfurcontaining amino acid, methionine [1]. Homocysteine metabolism is closely regulated by genetic–nutrient interactions and largely dependent on dietary supply of the B-group vitamins: folate, cobalamin, pyridoxine, and riboflavin (to a lesser extent); choline and betaine (Fig. 1) [2]. Suboptimal status or

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HOMOCYSTEINE IN PREGNANCY Glycine THF Serine

Remethylation

5-MTHF Dimethylglycine

BHMT

Choline

Betaine

Transmethylation

MTHFR

Methionine

MTR

MTRR [B2]

B2

5,10-MTHF

B12

Folate cycle

Methionine cycle

S-adenosyl-methionine

S-adenosyl-homocysteine

Homocysteine

Liver and kidney

PO

Serine

N

Transsulphuration

Homocysteine thiolactone

Cystathionine CgS

As

B6

N tR

m

CbS

B6

Cysteine FIG. 1. Simplified diagram of homocysteine metabolism (adapted from Ref. [2]). The roles of key micronutrients (methionine, folate, vitamins B12, B2 and B6, choline, and betaine) are highlighted. THF, tetrahydrofolate; 5,10-MTHF, 5,10-methylenetetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; MTHFR, 5,10-methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; BHMT, betaine–homocysteine methyltransferase; CbS, cystathionine-b-synthase; CgL, cystathionine-g-lysase; PON, paraoxonase; and mtRNAs, methionyl-tRNA-synthase.

deficiency in any of these micronutrients can lead to disturbance of homocysteine metabolism. In healthy humans,
56% of transmethylated methionine is catabolized through the transsulfuration pathway to cysteine and the remainder is remethylated to methionine [3]. The former pathway is initiated when the pyridoxal phosphate-dependent enzyme, cystathionine-b-synthase (CbS), catalyzes the condensation of homocysteine with serine to form cystathionine. Methyl groups for the remethylation route are provided from the folate cycle during the conversion of 5-methyltetrahydrofolate to tetrahydrofolate. Cobalamin is required to bind the methyl group to methionine synthase (MTR) for which it is a cofactor. Functionality of this enzyme is maintained by the flavoprotein, methionine synthase reductase (MTRR) [4]. The transfer of the methyl group to homocysteine results in the remethylation of homocysteine to methionine. An alternative remethylation route occurs in the liver and kidney when betaine (dietary or the product of choline catabolism) is converted to dimethylglycine by betaine–homocysteine methyltransferase. Both low dietary folate and cobalamin intake lead to elevated tHcy [5]. The extent of the

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contribution of the other nutrients involved in remethylation has been shown to vary in situations of different folate status. When folate stores are replete, cobalamin intake becomes a limiting factor for homocysteine remethylation [6]. When daily folate requirements are not met, high choline intake is associated with lower tHcy [7]. Choline and betaine intake were inversely associated with tHcy in the prefortification with folic acid era in the USA. Postfortification, this association is no longer present [8]. Genetic polymorphisms that affect the role of the enzymes implicated in any of the homocysteine metabolic pathways can lead to increased requirements for the micronutrients involved and eventually to increased tHcy if these requirements are not met. The 677 C!T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene [9] is relatively common in Caucasian, Hispanic, and Chinese ethnic groups [10]. MTHFR plays a key role in the folate cycle. Its flavin adenine dinucleotide (FAD)-binding site accepts and transfers the electrons required for the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The reduced affinity of the protein for its FAD-binding site leads to increased susceptibility to FAD dissociation and to reduced enzyme activity in the mutant form compared to the wild type [11]. The resulting reduction in 5-methyltetrahydrofolate production limits the supply of methyl groups from the folate cycle for homocysteine remethylation to methionine and enhances the risk of elevated tHcy in situations of low, although not deficient, folate status [12]. However, tHcy is lower in the presence of optimal folate status in all genotypes compared to their counterparts with suboptimal folate status. The enhanced affinity of MTHFR for FAD in the presence of bound folate to the enzyme [11] may maintain/enhance methyltetrahydrofolate’s role in homocysteine remethylation. However, riboflavin (precursor of FAD) status has also been shown to interact with this polymorphism. tHcy is increased when riboflavin deficiency is induced in pregnant mice with mild Mthfr deficiency [13]. An inverse association between riboflavin status and tHcy has been reported in men, in the presence of the mutant MTHFR 677T allele [14–17], and riboflavin supplementation was shown to lower tHcy in men with the 677TT genotype [18].

4. Prophylactic Folic Acid, Folate Status, and Homocysteine The folate cycle remethylation route has received considerable attention in the field of pregnancy research, given the role of prophylactic folic acid in the prevention of neural tube defects (NTDs) [19–21] and the gradual implementation of mandatory and voluntary fortification of flour/staple foods with folic acid in different countries around the world. This policy was pioneered in the USA and the primary aim was to reduce the incidence of NTDs by

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effectively increasing folic acid intake in the target population (women of fertile age) without harming nontarget groups by exposing them to excessive doses of folic acid [22]. Inevitably, the individual effects of exposure to prophylactic folic acid vary depending on genetic constitution and underlying folate status. As expected, however, global folate status in fortified populations has improved and average tHcy is now lower [23]. NTD rates in the USA and Canada have fallen by about 50% compared to prefortification rates [24]. Moderately elevated tHcy has been associated with NTD-affected pregnancies [25] and other pregnancy complications such as recurrent preeclampsia [26,27], intrauterine growth retardation (IUGR) [28], and preterm birth [29]. Elevated amniotic liquid homocysteine concentration has been associated with NTDs [30] and congenital heart defects [31]. Therefore, the benefit of mandatory fortification with folic acid in reducing the risk of pregnancy complications may extend beyond that of NTD prevention.

5. Choline and Homocysteine Recently, more attention has been given to the role of, and importance of, dietary choline in homocysteine metabolism during pregnancy. Choline contains three methyl groups and is required for phospholipid synthesis and neurotransmitter function. Its metabolism is closely interrelated with that of folate. Rats made folate deficient were reported to have a greater dependence on choline for homocysteine remethylation [32]. It has been suggested that the remethylation of homocysteine may also be more dependent on the choline– betaine pathway in situations of inadequate folate intake in women [7] and MTHFR 677TT Mexican–American men with deficient folate status [16]. Inhibition of choline uptake during pregnancy was associated with growth retardation and NTDs in rats [33]. Feeding diets that only met an eighth of choline requirements to Mthfrþ/ mice before and during pregnancy was associated with ventricular septal defects in the offspring [13]. High periconception dietary intake of methionine, betaine, and choline has been associated with less risk of an NTD-affected pregnancy in a study of Californian women [34]. Dietary data relating to the 3 months before conception was collected retrospectively from the mothers on average 4.6–4.9 months after giving birth. Surprisingly, in another Californian study, high maternal serum choline during midpregnancy was associated with increased risk of cleft lip palate (CLP) in the offspring [35]. However, the authors were unable to provide a biological explanation for this observation. They reported that cases with CLP offspring had lower serum methionine and cobalamin than controls. However, it is not clear whether this concurred with high choline levels in the same patients and whether high choline levels were of dietary or endogenous origin. The latter

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might represent a compensatory mechanism to maintain homocysteine remethylation to methionine. Alternatively, high midpregnancy choline may be marking a separate biological process, unrelated to choline status at the time of neural tube formation. Estradiol injection in rats has been shown to stimulate choline synthesis [36]. Plasma choline gradually increases during pregnancy [37–39], possibly to ensure sufficient polyunsaturated fatty acid supply to the fetus [37]. Thus, endocrinologically driven choline synthesis during midpregnancy might confound the association between dietary choline supply and plasma choline concentrations.

6. Nonnutritional Factors Associated with tHcy The previously mentioned genetic-nutritional control of homocysteine regulation has a major influence on tHcy. However, lifestyle and physiological/ endocrinological factors have also been associated with tHcy in large-scale population studies. Age [40] and sex [40,41] are strong determinants. tHcy is also positively associated with serum creatinine [42–44] and inversely associated with bone mineral density in women [42]. Independent associations between smoking, alcohol consumption and caffeine intake [42,44], exercise [42,45], serum insulin [46], and obesity in women [47] have also been reported. Female hormones have been inversely correlated with tHcy outside of pregnancy in both synthetic and endogenous forms. tHcy was reduced following 17 b-estradiol administration in male rats [48] and men [49]. In a randomized control trial, postmenopausal women on hormone replacement therapy were reported to have lower tHcy following 5–7 years of treatment than controls that had received no substitution treatment [50]. Some of the observations concerning endogenous hormonal fluctuations have not involved a direct measurement of circulating hormone levels. However, the coincidence of key endocrinologically driven changes in the woman’s reproductive cycle with changes in tHcy suggests that they are associated. From puberty, tHcy is lower in girls than in boys and remains lower in women throughout their fertile life than in men [41]. tHcy has also been reported to be lower in the luteal than in the follicular phase of the menstrual cycle [51,52] and in fertile than in menopausal women [41].

7. Health Benefits of tHcy Lowering Elevated tHcy is an independent graded risk factor for vascular disease [53], and even moderately elevated tHcy has been associated with increased risk of cardiovascular disease (CVD), osteoporosis, cognitive decline, and history of

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pregnancy complications [42]. Adult tHcy in the range of 5–15 mmol/L is considered normal [54], although reducing it by 1 mmol/L within this range has been associated with reduced CVD risk [53]. Hyperhomocysteinemia has been classified as moderate (15–30 mmol/L), intermediate (30–100 mmol/L), and severe (> 100 mmol/L) [55] outside of pregnancy. Reference ranges, from large-scale studies, during pregnancy are unavailable to date. Considerable effort in the field of homocysteine research in recent years has focused on the investigation of the health benefits of lowering moderately elevated tHcy. However, secondary prevention of disease progression with tHcy-lowering treatment has proved ineffective in studies of recurrence of cardiovascular events reported so far [56–58]. It remains to be shown whether tHcy lowering is an effective measure in the primary prevention of the lesions that it has been associated with. Preconception supplementation with 400 mg/day of folic acid was not shown to reduce the risk of miscarriage in a Canadian study [59]. However, primary prevention of adverse pregnancy outcome with interventions that lead to lower tHcy has been largely successful. Apart from optimal folate status, elevated cobalamin status has also been shown to offer a considerable degree of protection against NTDs [60]. While this inevitably leads to lowering of tHcy, it is not clear how this contributes, if at all, to the observed protection. Reported regular multivitamin or prenatal supplement use (of unknown composition) from before pregnancy throughout the periconception period was associated with a 45% reduction in risk of preeclampsia [61]. Another prospective cohort study reported a 63% reduction in risk of preeclampsia in women who reported taking high-dose folic acid supplements (1 mg/day) for varying lengths of time from preconception until the beginning of the second trimester of pregnancy [62]. The results from both of these studies need to be confirmed in randomized controlled intervention trials.

8. Pregnancy 8.1. CHANGES IN THCY DURING NORMAL PREGNANCY Pregnancy tHcy is affected by the same underlying nutrient-genetic and lifestyle factors as in the nonpregnant state. However, the considerable variation in tHcy that occurs throughout pregnancy may be as a result of a physiological adaptation to this condition. Longitudinal studies have shown that maternal tHcy is substantially reduced during the first two trimesters of pregnancy compared to preconception concentrations [63,64]. The decrease is only partially explained by folic acid supplement use [63]. Physiological factors such as hemodilution, albumin decline, and enhanced

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renal function all contribute to the decline in tHcy [63–65]. Pregnancy hormones are also likely to have an important influence, given that the decrease in tHcy that occurs between preconception (geometric mean [SD]: 8.2 [1.3] mmol/L) and midpregnancy (5.2 [1.3] mmol/L) cannot be explained by folic acid supplementation or by these physiological effects of pregnancy alone [63]. By 6.5–8 weeks of pregnancy, before the effects of hemodilution and placental hormone production and in the absence of folic acid supplement use, tHcy was already on average 11.5% lower than at preconception. This decrease could be associated with hCG production during early pregnancy, although to the best of our knowledge this has not been investigated. A study of patients receiving ovarian stimulation treatment reported reduced tHcy compared to day 2 (median [min, max]: 9.1 [5.0, 75.3] mmol/L) of the menstrual cycle following hCG administration (8.4 [4.3, 71.6] mmol/L) [66]. However, from day 2 and prior to treatment with hCG, patients had also received follicle-stimulating hormone for a number of days. tHcy has been previously reported to be lower in the luteal than in the follicular phase of the menstrual cycle [51,52]. Subsequently, other pregnancy hormones may also further reduce tHcy until midpregnancy. The decrease that occurs during pregnancy is significantly correlated with the coinciding increase in estradiol [67]. During the last trimester of pregnancy, tHcy returns to similar concentrations observed at preconception in mothers that do not use folic acid containing supplements during the second and third trimesters [68]. This late pregnancy increase in tHcy occurs despite the continuing influence of hemodilution and increased glomerular filtration rate. Other researchers have also observed the increase in tHcy toward the end of pregnancy and attributed it to low folate status [69]. While low folate status will inevitably contribute to the rise in tHcy, late pregnancy tHcy also rises, though not to the same extent, in folic acid supplemented mothers [68]. Either the supplementation regime is not sufficient to maintain adequate folate status or the rise is independent of folate status. The transsulfuration and remethylation pathways have been reported to dominate homocysteine metabolism to different extents depending on the time of pregnancy. A study of methionine kinetics during human pregnancy reported a higher rate of transsulfuration during the first trimester compared to the last and higher remethylation and transmethylation rates in the last trimester compared to the first [70]. These differences were suggested to be due to the dependence of the fetus on maternal cysteine supply in early pregnancy and to the increased methionine requirements of the rapidly developing fetus in late pregnancy. A study of amniotic fluid samples from pregnant mothers collected between 13 and 18 weeks gestation showed that methionine concentrations gradually decreased and that homocysteine concentrations gradually tended to increase during this time frame [71]. Regardless of the cause, tHcy increases within the normal range during late

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pregnancy with normal evolution and outcome. An in vitro study has shown that incubating human myometrium with homocysteine increased its contractility [72]. It remains to be investigated whether the increase in homocysteine during late pregnancy contributes to the onset of uterine contractions in preparation for birth. 8.2. ANIMAL/IN VITRO STUDIES AND HOMOCYSTEINE AND REPRODUCTION There is varied and conflicting evidence in the literature regarding the capacity of moderately elevated tHcy to cause the diseases with which it has been associated. A number of animal and in vitro experiments have been designed to investigate the effects of elevated tHcy from before conception and throughout pregnancy on reproduction and on development in the offspring. These studies have provided valuable information on the biological and clinical effects of provoked endogenous tHcy excess and of exposure to pharmacological doses of homocysteine. However, the challenge remains to interpret the relevance of the evidence provided from acute exposure to supraphysiological homocysteine concentrations in controlled experiments, in the context of chronic exposure to concentrations in the physiological range, and in a free-living environment in humans. The difference in tHcy between the Mthfrþ/ and wild types of the MTHFR knockout mouse is suggested to be analogous to that observed between MTHFR 677TT and 677CC individuals [73]. Inducing folate deficiency and hyperhomocysteinemia (threefold increase in mean tHcy compared to folate replete animals) in mice before mating was associated with fetal resorptions, decreased fetal weight, and developmental delays [74]. Mthfrþ/ female mice fed folic acid-deficient diets before and throughout pregnancy had a lower number of corpora lutea in the ovaries. Pregnancies were affected by different embryonic defects, greater fetal loss, IUGR, and severe placental defects [75,76]. The mean maternal tHcy, during pregnancy, was
70 mmol/L compared to 50 mmol/L in the 677TT model on folic aciddeficient and control diets, respectively [76]. Similarly, inducing choline or riboflavin deficiency in Mthfrþ/ mice was associated with hyperhomocysteinemia during pregnancy (mean tHcy:
40 and
90 mmol/L, respectively, compared to Mthfrþ/ fed on control diets,
38 mmol/L) [13]. There was a greater incidence of embryonic delays and reduced embryonic growth in the offspring of Mthfrþ/ mice that had been fed diets containing a sixth of riboflavin requirements before and during pregnancy. Riboflavin-deficient diets were also associated with reduced left ventricular wall thickness in the offspring. Both riboflavin and choline deficiency (induced by feeding oneeighth of daily choline requirements) were associated with ventricular septal

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defects. Despite being exposed to higher tHcy concentrations in the study of induced choline deficiency than in the study of induced riboflavin deficiency, overall the offspring from the latter experiment had more developmental problems. These results suggest that the combined exposure to the enzyme defect and insufficient riboflavin was more harmful than the elevation in tHcy produced by choline deficiency in animals with the same enzyme defects but adequate riboflavin supply. Nevertheless, the tHcy ranges reported in all of these animal studies would correspond with intermediate hyperhomocysteinemia in nonpregnant humans. While reference ranges have yet to be defined for human pregnancy, such concentrations are considerably higher than those reported in previous pregnancy studies with both normal and pathological outcomes [26,27,39,63,64,68–70]. Although the consequences of the virtual eradication of a single micronutrient from the diet can clearly be attributed to deficiency of that single vitamin under experimental conditions, it is difficult to reproduce free-living conditions in humans in which variation in supply of multiple micronutrients and in lifestyle habits that also affect tHcy may occur at any given moment. The incorporation of homocysteine into protein and tRNA is prevented by the production of the highly reactive thioester, homocysteine thiolactone (HTL) by methionyl-tRNA synthetase during the amino acid editing process of protein synthesis [77,78]. HTL has been reported to be capable of damaging proteins through homocysteinylation of protein lysine residues [79]. High intracellular HTL concentrations (0.25 pmol/105 cells) have been reported in cells cultured in medium containing homocysteine but lacking in cobalamin and folate [80]. However, reported serum HTL concentrations ranged from being undetectable, in half of the healthy adult men studied, to 0.38 nM [81]. Incubation with either HTL or homocysteine during gestational days (GD) 0–2 caused NTDs and congenital heart defects in chick embryos [82]. Later incubation (GD 8.5) caused growth retardation, blisters, and somite development abnormalities in mouse embryos [83]. No NTDs were observed either in this study or in a separate study of mouse embryos in which incubation was with homocysteine (1.3 mM) or HTL (0.65 mM) on GD 8 for 44 h [84]. Incubation of cultured human placental trophoblasts with HTL caused apoptosis [85]. Homozygous MTHFR knockout mice have reduced survival or delayed development and cerebellar abnormalities. Older heterozygous and homozygous knockouts have abnormal aortic lipid deposition and also elevated tHcy and altered S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) levels. These latter abnormalities were associated with DNA hypomethylation in the brain and ovaries [73]. Inducing hyperhomocysteinemia in pregnant rats (26 mM compared to 6 mM in controls by the end of pregnancy) by including 1 g/kg body weight of methionine in their daily drinking water was associated with

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increased lipid peroxidation (indicative of oxidative stress) and apoptosis in the offsprings’ brains [86]. The authors suggested that the apoptosis may have been caused by the oxidative stress and that the fetal brain is more susceptible to oxidative stress due to its less developed antioxidant defense mechanisms. No information is provided regarding the diet composition. On the assumption that daily folate, cobalamin, and other micronutrient requirements were met, this report provides evidence of a potential association between elevated homocysteine and impaired brain development that is not marking folate or cobalamin deficiency. Other studies have shown that fetal exposure to hyperhomocysteinemia during pregnancy was associated with lasting effects in the offspring. Hyperhomocysteinemia induced by feeding pregnant dams methyl-deficient diets was associated with impaired motor and cognitive development in rat pups [87]. Feeding methyl rich diets to the newborn rat pups, as a secondary preventive measure, did not reverse the cognitive impairment associated with regions of the brain in which homocysteine had accumulated during development. Homocysteine was shown to have accumulated in neurons and astrocytes of the hippocampus, cerebellum, striatum, and subventricular zone of the brain. tHcy concentrations in the rat pups were considered to be comparable with mild hyperhomocysteinemia (13  4 mmol/L). Fetal exposure to hyperhomocysteinemia from early pregnancy, reaching a sustained tHcy concentration of 33  3.9 mmol/L, was also associated with impaired cognitive function in the offspring of rats [88]. The hyperhomocysteinemia was induced and maintained by feeding with a very high methionine content diet. The IUGR and impaired postnatal growth observed in the offspring of rats fed methyl donor-deficient diets from preconception throughout pregnancy and lactation were proposed to be due to dysfunction of the gastric ghrelin cell system caused by the remodeling of gastric cell organization [89]. Maternal hyperhomocysteinemia (mean tHcy: 16.4  1.5 mmol/L), induced by feeding rats drinking water containing homocysteine from preconception throughout pregnancy, was associated with a lower number of live births in the intervention than the control group (mean tHcy: 4.7  1.7 mmol/L) and with impaired ossification in the offspring [90]. This effect was despite ensuring that dietary folic acid and pyridoxine supplies were sufficient to meet pregnancy requirements. Whereas the involvement of folate metabolism and homocysteine in agingrelated diseases, several developmental abnormalities, and pregnancy complications has given rise to a large amount of scientific work, the role of these biochemical factors in the earlier stages of mammalian reproduction and the possible preventive effects of folate supplementation on fertility have, until recently, been much less investigated.

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8.3. PROSPECTIVE PREGNANCY STUDIES IN HUMANS The advantage of prospective studies is their enhanced reliability because they are less vulnerable to bias due to the effect of disease on tHcy (‘‘reverse causality’’) and because they control for confounding from established risk factors. Cohorts of women followed from preconception and broadly representative of the population are generally very difficult to collect by virtue of the fact that they require synchronized planning of the pregnancy by the mother and the investigating team. Preconception blood samples were collected from a cohort of 423 Chinese textile industry workers that were planning on becoming pregnant [29,91]. The time between sample collection and the index pregnancy varied by up to 1 year. Information regarding the association between preconception tHcy and gestation length and outcome was obtained from this cohort. Another longitudinal study from preconception throughout pregnancy was carried out in Spain (PREC study, 1992–1996 [63]). Couples planning a pregnancy were invited to do so with the investigating team and blood sample collection was carefully timed to be during the periovulationary phase of the menstrual cycle (conception occurred during this cycle or a maximum of 2 cycles later), at 6–8, 20, and 32 weeks of pregnancy, during labor and from the cord. Although this study was from a relatively small cohort of 93 mothers of medium–high socioeconomic status, its strength is that each woman acted as her own baseline, nonpregnant control and blood samples representative of each phase of pregnancy were collected [63,65,66,92]. Another prospective pregnancy study included the collection of a blood sample during the first trimester, enabling the investigation of early pregnancy tHcy with pregnancy evolution and outcome [69]. The large-scale Child Health and Development nested case–control study collected prenatal blood samples from a cohort of 19,044 pregnant women that received prenatal care in Northern California (1959–1966) and from 12,094 of the offspring at follow-up between 1981 and 1997. This study investigated the association between pregnancy tHcy and schizophrenia in the offspring [93]. The role of homocysteine on pregnancy outcome and development from preconception through to adulthood has been investigated to differing extents throughout the life cycle. Although no study to date has investigated homocysteine and health throughout the life cycle from preconception to old age in the same subjects, different studies have led to evidence covering this topic at all stages of development (Table 1).

8.4. ELEVATED THCY AND SUBFERTILITY In a study of 156 subfertile couples, spermatozoa homocysteine concentration was higher in men with male factor subfertility (58.8 pmol/million cells) and idiopathic subfertility (20.9 pmol/million cells) than in fertile men

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(12.4 pmol/million cells). Follicular fluid homocysteine concentration was higher in women with endometriosis (18.8 nmol/mg protein) compared to patients with unexplained infertility (9.2 nmol/mg protein) and fertile women (12.4 nmol/mg protein) [94]. Both seminal and follicular fluid homocysteine concentrations were reported to be negatively correlated with embryo quality. Increasing tHcy by 1 mmol/L in ejaculate and follicular fluid was associated with 19% and 58% reductions, respectively, in embryo quality. A limitation of this study is the lack of information provided on the suitability of the controls, on lifestyle factors known to be associated with subfertility such as smoking, and on the validity of the regression models used (explicative capacity of the model, significance of the model, number of patients included in the model, nonexistence of colinearity between independent variables, etc.). In a separate study, hyperhomocysteinemic (tHcy > 17 mmol/L) in vitro fertilization (IVF) patients were randomly assigned to tHcy-lowering treated and untreated groups. Increased implantation and pregnancy rates were observed in the treated group in which tHcy was reduced to <17 mmol/L with combined folic acid, cobalamin, and pyridoxine supplementation before attempting IVF, compared to the untreated group of patients [95]. However, the study was not placebo-controlled and it is not stated whether the assessors of IVF success were blind to the supplementation regime. In another study of 181 subfertile couples in which data on BMI, smoking, and folic acid supplementation were considered, neither plasma folate, cobalamin nor tHcy were correlated with embryo quality. However, follicular fluid cobalamin concentration and tHcy were positively and inversely correlated with embryo quality respectively [96]. While follicular fluid cobalamin and tHcy concentrations were not associated with the occurrence of biochemical pregnancy, doubling follicular fluid folate concentration increased the chances of biochemical pregnancy threefold. Follicular fluid tHcy was negatively correlated with follicular diameter in the same study, suggesting a negative effect of elevated tHcy on follicular growth [66]. The risk of subfertility was 12-fold higher in women who had folate receptor blocking autoantibodies during preconception [97]. However, tHcy did not differ between cases and controls in this study. 8.5. EARLY PREGNANCY LOSS/MISCARRIAGE In the majority of studies that have investigated the association between tHcy and miscarriage, biochemical determinations have been carried out after diagnosis of the miscarriage event and no baseline determinations before the event are available. Most studies report elevated tHcy in miscarriage cases. Chorionic villous vascularization was defective (reduced vascular areas and perimeters per measured chorionic area and fewer vascular elements) in the miscarriage tissue collected from 19 recurrent early pregnancy loss

TABLE 1 EVIDENCE FOR ASSOCIATIONS BETWEEN ELEVATED HOMOCYSTEINE, IN DIFFERENT BIOLOGICAL FLUIDS, DURING PRECONCEPTION OR PREGNANCY, AND ADVERSE REPRODUCTIVE OR DEVELOPMENTAL OUTCOMES

Time of sample collection

Preconception

Pregnancy Plasma

Biological fluid Parent Subfertility Endometriosis Miscarriage Preeclampsia Gestational diabetes PROMc Embryo Embryo quality

Follicular fluid

Seminal fluid

[66,94]a [94]

[94]

[94,96]

Fetus Preterm birth NTDs CHDs Birth weight

Maternal

Paternal

[91b]

[103]

Maternal plasma

[29,91]

[68] [29b]

[145] [25,109] [68]

Adult Schizophrenia

[93]

[Reference number of cited study]. No association was found. c Premature rupture of the membranes.

[30,108] [31] [134]

[68]

[94]

[146b]

b

Cord

[98,101b,102] [26–28,102,121,122b] [136–138,139b] [140,141b]

DNA methylation Down syndrome

a

Amniotic fluid

[148]

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patients with elevated tHcy (>18.3mmol/L) and/or postmethionine load hcy > 61.5mmol/L compared to those with normal tHcy (< P95 for each of these measurements based on healthy subjects) in a study in the Netherlands. tHcy was inversely correlated with vascular element perimeter in this study [98]. Pregnancy loss was not attributable to conventional causes such as chromosomal rearrangements, severe uterine anomalies, antiphospholipid antibodies, and thyroid dysfunction in any of these patients. Low folate status at the time of miscarriage diagnosis was associated with an increased risk of miscarriage in a case–control study in Sweden [99]. However, tHcy was not measured in this study. Elevated tHcy was associated with increased risk of miscarriage ( 9.9 mmol/L: twofold;  12.3 mmol/L: fourfold;  15.3 mmol/L: sevenfold) in a case–control study in France that compared 743 women that had miscarried between gestational weeks 8 and 9 with 743 controls that had an elected termination of pregnancy for the first time [100]. Cases and controls were matched for age, number of pregnancies, and time elapsed since miscarriage. In a separate study in Syria, in which pregnancy was confirmed by hCG presence or by ultrasound scan, miscarriage cases (N ¼ 43) were shown to have lower cobalamin status than pregnant controls (N ¼ 32) [101]. Blood samples were collected at the time of miscarriage diagnosis and controls were matched with cases by gestational age. Although tHcy tended to be higher in cases than in controls, it was not significantly so. Despite the observed trends, it is not clear that there were sufficient controls, and although not significantly so, more cases were smokers than controls. Pregnant mothers with tHcy  P90 (based on pregnant control reference concentrations) were twice as likely to have a miscarriage (OR: 2.1 [95% CI: 1.2, 3.6]) in a study of 103 miscarriage cases and 1077 controls [102]. However, although tHcy was determined during the first 20 weeks of pregnancy, it was not known if the miscarriage had occurred before or after the blood sample was collected. Elevated tHcy ( P95) in both mothers (14.0 mmol/L) and fathers (19.6 mmol/L) was associated with fivefold and sevenfold increases in risks of idiopathic recurrent pregnancy loss compared to controls in an Indian study [103]. However, no information was provided regarding the time that had passed between sample collection and the last pregnancy loss. A study of the potential causes of repeated pregnancy loss in data collected from 1020 women in Tennessee at least 6 weeks after a miscarriage event showed that among other possible unconventional causes of miscarriage, tHcy was elevated (>14mmol/L) in 14% of the women [104]. However, the relevance of this finding is not clear, given that there were no controls in this study. Poor pyridoxine status in baseline blood samples (collected before ending contraception with the intention to become pregnant) from 364 women in Anhui, China, was associated with greater risk of miscarriage in a prospective study in which each of conception, pregnancy

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(lasting at least 6 weeks after the onset of the last menstrual period), and early pregnancy loss (pregnancy lasting for less than 6 weeks) were confirmed by urinary hCG detection [91]. However, neither being in the highest tHcy quartile nor having tHcy  12.4 mmol/L was associated with increased risk of pregnancy loss. 8.6. EMBRYO, PLACENTAL, AND FETAL DEVELOPMENT 8.6.1. Neural Tube Defects Apart from the different studies that have associated low maternal folate and/or cobalamin status with risk of an NTD-affected pregnancy, other studies have also reported higher tHcy in mothers with a history of NTDs in their offspring than in control mothers with previous pregnancies unaffected by NTDs [105–107]. Early pregnancy mean tHcy was higher in pregnancies that went on to be affected by NTDs [8.6  2.8 mmol/L] compared to normal pregnancies [7.9  2.5 mmol/L] [25]. Higher amniotic fluid homocysteine concentration was reported in NTD case pregnancies (2.6  1.6 mmol/L) than in controls (1.5  0.4 mmol/L) [30]. Another study reported a higher proportion of amniotic fluid homocysteine concentrations > 1.85 mmol/L (P90 in controls) in pregnancies affected by NTDs compared to unaffected control pregnancies [108]. Analysis of blood samples collected prior to elective termination of an NTD-affected pregnancy showed that tHcy was higher in these pregnancies (mean [min–max] 6.3 [4.8–8.2] mmol/L) than in unaffected pregnancies (mean [min–max] 5.8 [4.6–7.2] mmol/L) [109]. Children with NTDs have also been reported to have higher tHcy (median [range]: 10.0 [4.8–24.3] mmol/L) than control children (4.5 [1.99.6] mmol/L) [110]. 8.6.2. Congenital Heart Defects Elevated maternal tHcy has also been associated with congenital heart defects in the offspring. Median tHcy (3–6 months after giving birth) was higher in mothers (11.9 mmol/L) of children with congenital heart defects than in control mothers (9.4 mmol/L) of unaffected children [111]. Case mothers with tHcy above P90 in controls (13.0 mmol/L) were five times more likely to have a child with a congenital heart defect. This observation was confirmed in subsequent studies in which postpregnancy blood samples were also collected from case and control mothers. Case mothers selected from a birth defects registry and controls (mothers of live births, free of defects) selected from a birth certificate registry were studied [112]. Elevated tHcy (greater than P70 in control mothers) was associated with a greater risk of previously affected pregnancies. Based on tHcy in control mothers, there was a dose–response relationship between increasing tHcy from P70

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(8.59 mmol/L) upward in cases and risk of previously affected pregnancy. Case mothers with tHcy at P70 were four times and those with tHcy at P95 (10.72 mmol/L) were 12 times more likely to have a history of affected pregnancy. This study also reported that more case mothers were smokers than controls. Subsequently, it was reported from the same study that a history of smoking at periconception combined with elevated postpregnancy tHcy (> P75 in control mothers; > 8.59 mmol/L) was associated with a 12-fold increase in risk of history of an affected pregnancy [113]. Another study also confirmed that mothers with hyperhomocysteinemia (tHcy > 14.3 mmol/L), determined
17 months after pregnancy, were almost three times more likely to have had a pregnancy affected by congenital heart defects than normohomocysteinemic mothers [114]. In each of these studies, tHcy was determined after the diagnosis of the complication and, in some cases, a considerable time after the affected pregnancy was over. One study reported that elevated amniotic fluid homocysteine concentration was associated with increased risk of carrying a fetus with an isolated nonsyndromic congenital heart defect [31]. 8.6.3. Placental Development and Function MRNA for both methionine synthase and for 5,10-MTHFR is highly expressed in the human placenta during both early and late pregnancy while expression for CbS and betaine–homocysteine methyltransferase is low and undetectable, respectively [115]. This suggests a high folate requirement for placental homocysteine metabolism. It has recently been shown that homocysteine can cross the human placenta from mother to fetus [116]. The authors propose that this is potentially harmful to placental function and fetal development for various reasons. With regard to placental function, the presence of homocysteine in the syncitiotrophoblast membrane of the placenta could potentially lead to alterations in placental metabolism, vascular function, and induction of apoptosis. Development might be impaired in a fetus deprived of essential amino acids due to competition with homocysteine for transport. However, different studies of uncomplicated pregnancies in healthy women have reported that maternal tHcy at birth is higher than umbilical cord tHcy. Mean maternal tHcy at birth was reported to be 5.4  1.4 mmol/L compared to 4.5  1.8 mmol/L in 35 mother–cord pairs [117]. The authors reported a descending concentration gradient from maternal vein to umbilical vein to umbilical artery. Two subsequent studies confirmed that maternal tHcy at birth is higher than in the cord in larger groups of mother–cord pairs. Mean maternal tHcy at birth was reported to be 8.3  2.9 mmol/L compared to 7.9  2.9 mmol/L in 201 mother–cord pairs in an Irish study [118]. In this study, maternal tHcy was found to be the greatest predictor of cord tHcy. In the PREC study, we observed that maternal tHcy at birth was higher than

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cord in mother–cord pairs that were both supplemented (mean [95% CI]: 6.6 [8.0, 9.1] mmol/L compared to 5.4 [7.7, 9.4] mmol/L in 37 mother–cord pairs) and unsupplemented (8.6 [5.9, 7.3] mmol/L compared to 6.8 [6.0, 7.6] mmol/L in 47 mother–cord pairs) with folic acid during late pregnancy [68]. 8.6.4. Placental Vascularization and Preeclampsia Optimum placental development depends on trophoblast invasion of maternal uterine vessels with resulting changes in their anatomical characteristics from narrow muscular to wide nonmuscular vessels. This enables high flux and low resistance utero-placental blood flow that facilitates the transfer of nutrients across the placenta. Conceivably, in women with underlying undiagnosed pathological vascular conditions, these anatomical adaptations to pregnancy may be impaired. Preeclampsia is a pregnancy-specific disorder that develops after 20 weeks gestation [119]. The placenta appears to be fundamental in the development of preeclampsia, and abnormal vascularization of the placenta is often present [120]. A number of studies have reported that pregnancy tHcy is higher in preeclampsia patients than in controls before the onset of the pregnancy complication and that having elevated tHcy during the first half of pregnancy increases the risk of preeclampsia [26–28,100,121] (Table 2). However, this was not confirmed in a Finnish study in which timing of sample collection was also prior to preeclampsia diagnosis [122]. The number of preeclampsia cases was less in this study. While two of the studies that found an association between elevated early pregnancy tHcy and risk of preeclampsia reported that smoking habits did not differ between cases and controls [28,120], smoking was not reported in the Finnish study. The conclusions regarding the association between tHcy and preeclampsia differ among some studies in which preeclampsia was diagnosed before sample collection for tHcy determination. The main difference between the studies that reported similar tHcy in preeclampsia cases and controls or that found no increased risk associated with elevated tHcy was the timing in blood sample collection. Seven studies in which blood samples were collected after 30 weeks gestation reported elevated tHcy in cases compared to controls [64,123–128]. Three studies that investigated the association between tHcy determined in blood samples collected between 22 and 26 weeks gestation reported no difference between cases and controls [125,129,130]. Interestingly, Hogg et al. [125] reported no difference at 26 weeks but did report a difference at 37 weeks in the same group of patients. Thus, the timing of sample collection appears to be an important factor. The association may be masked when tHcy concentrations are at their lowest during midpregnancy, possibly due to hormonal influences. The weight of the evidence suggests that the association between elevated tHcy and preeclampsia is detectable in early and late pregnancy blood samples. Other studies have also

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reported a dose–response relationship with higher tHcy in cases of severe preeclampsia than mild [126,128]. Some studies have combined the use of tHcy determination with Doppler imaging to investigate their predictive capacity of preeclampsia and IUGR (pregnancy complications associated with impaired placental function). Doppler imaging analysis can be used to obtain information on placental vascularization through the observation of uterine artery waveforms and the registration of the pulsatility index of blood flow (indicative of degree of resistance). No difference in median midpregnancy tHcy (22–24 weeks) was observed between 275 patients with abnormal Dopplers (5.6 mmol/L) and 408 controls (5.4 mmol/L) in a UK study [129]. However, in a prospective study in which tHcy was determined in blood samples collected between 15 and 19 weeks of pregnancy, the combination of abnormal Doppler results with tHcy > P95 (6.3 mmol/L) had higher predictive capacity of a preeclampsia outcome than either elevated tHcy or Doppler alone [28]. The former study provided no information on folic acid supplement use. In the latter, folic acid users after 12 weeks of pregnancy were excluded. However, it is not stated what percentage used supplements during the first trimester. It is not stated in either study whether the blood samples were processed according to the guidelines to prevent artifacts in tHcy determinations [131]. 8.6.5. Fetal Size and Intrauterine Growth Retardation Different studies have investigated the association between elevated maternal tHcy and fetal growth. There is evidence for and against a negative association between maternal tHcy and birth weight. In a large study of 5883 Norwegian women, those with tHcy in the top quartile (10.7–78 mmol/L) were more likely to have previously had a child with very low birth weight compared to those in the lowest quartile (3.6–7.5 mmol/L) [OR: 2.01; 95% CI:1.23, 3.27] [132]. Preconception tHcy  12.4 mmol/L was not associated with increased risk of low birth weight in a study of 423 Chinese mothers [29]. Preconception samples were collected at a routine medical checkup in women that had obtained the necessary governmental approval to become pregnant. A case–control study of 483 mothers that gave birth to children with IUGR (birth weight < P10) and 468 control mothers reported that increasing postpartum (up to 48 h) maternal tHcy by 5 mmol/L was associated with less probability of having had a pregnancy affected by IUGR [133]. tHcy in this study varied from 1.42 to 18.32 mmol/L. In our prospective study in Spanish women, moderately elevated maternal tHcy ( 7.1 mmol/L) at 8 weeks gestation was associated with a threefold increased risk of reduced birth weight in the offspring [68]. In another study, amniotic fluid homocysteine concentrations were higher in pregnancies with a small for gestational age outcome (N ¼ 39; 1.31 [1.64] mM) than in those that resulted in adequate size for gestational age at birth (N ¼ 393; 1.02 [0.55] mM) [134]. In a separate study,

TABLE 2 ASSOCIATION OF PREGNANCY THCY WITH PREECLAMPSIA RISKa Plasma tHcy (mol/L) mean (SD)/P50; [N] Design (year)

Location

Time of sample (gestation week)

Case

Control

OR (95% CI)b

tHcy cutoff (mol/L)

Befored (1999) [120] (2001) [26] (2001) [122] (2003) [27] (2006) [28]

USA Ireland Finland Ireland Turkey

15–22 (16.5  1.5) 15.3  4.0; 14.9  3.4 16 15.9  3.6; 15.6  3.4 16.2  3.3; 16.3  3.2

[52] 9.8 (3.3) [56] 7.0 (1.6) [34] 8.4 (2.4) [71] P50: 7.1 [32]

[56] 8.4 (1.9) [112] 6.9 (1.8) [68] 7.1(1.5)[142] P50: 5.0 [324]

3.2 (1.1–9.2) 2.8 (1.4–5.9) 1.6 (0.6–4.4) 4.1(1.4–12.6) 13.8 (3.1–26.9)

 5.5 > 10 > 7.7  7.8  6.3

(2008) [102]

Canada

4–20 weeks

[65]

[1707]

2.7 (1.4–5.0)i

 7.05–5.71j

Labor Labor 26 37

8.7 (3.1) [20] 9.7 (5.2) [20] 5.2 (1.3) [16] 6.6 (2.1) [46]

5.0 (1.1) [20] 7.2 (2.3) [32] 4.6 (1.4) [409] 5.3 (1.7) [409]

– – – –

(2004) [129] (2004) [64] (2004) [126]

USA USA USA (African American) UK New Mexico Turkey

22–24 30–33
33

P50: 5.5 [586] 3.2 (0.2) [24] 6.1 (1.6) [25]

(2007) [127] (2009) [130] (2009) [128]

Norway Canada China

Term 24–26 ‘‘3rd trimester’’

P50: 5.4 [80] 4.4 (0.6) [15] 10.5 (2.5) [12] 11.2 (3.6) [46] P50: 8.2 [47] 3.4 (0.9) [113] 8.2 (0.8) [24] 10.9 (2.5) [62] 12.6 (1.4) [38]

P50: 6.4 [51] 3.7 (0.9) [443] 6.0 (0.6) [30]

Folate statusc

Smokingc

BMIc

–e NSg – NS Sup users exclh Suppk

Yes – – – NS

Higherf – – – NS

–

–

– – – –

NS NS NS

– – –

NS NS –

– – –

– – –

– – –

– – –

– – –

1.5 (1.0–2.3) 0.7 (0.3–1.3) –

– > 11.0 –

NS Supp ?n

– Lowerm NS

Higher Higher –

d

After (1997) [123]l (1998) [124] (2000) [125]

a Only studies reporting pregnancy tHcy concentrations, gestational age at the time of blood sampling, and with samples collected from complication-free control pregnant patients of similar gestational age at time of sampling as cases are shown. b Risk of preeclampsia in cases compared to controls. c Reported/considered in the study. d Timing of sample collection with respect to diagnosis of preeclampsia. e Information not provided. f Higher in cases. g No significant difference between cases and controls. h Supplement users excluded. i Relative risk. j P90 was calculated in tHcy adjusted for gestational age in weeks for each 2-week interval from 4–5 to 18–20 weeks of pregnancy. tHcy was lower in samples taken later in pregnancy. k Used folic acid supplements. l Time of sample collection: cases ¼ 35  4 weeks, controls ¼ 40  1 week. m Fewer controls were smokers than cases. n States that ‘‘none were known to be supplement users’’; however, neither were patients specifically asked nor was folate status compared between cases and controls.

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maternal tHcy was reported not to differ between 12 pregnancies affected by IUGR (at 30.1  3.5 gestational weeks) and 8 unaffected control pregnancies (at 25.6  6.1 gestational weeks). However, 44.7  14.8% of total albumin was cysteinylated compared to 20.9  6.1% in control pregnancies [135]. 8.7. GESTATIONAL DIABETES tHcy has been shown to vary among different categories of response to glucose loading and tolerance tests performed at 24–28 weeks gestation. In the studies reported to date, tHcy was determined during the same gestational time frame as the glucose metabolism test. tHcy was reported to be higher in women that were diagnosed as glucose intolerant following an oral glucose load of 50 g, compared to women with normal responses [136]. tHcy was also reported to be higher in women that were diagnosed with gestational diabetes following a glucose tolerance test (response to oral load of 100 g of glucose) [137,138]. In the majority of these studies, women with abnormal glucose metabolism were older than the controls that had a normal response to the glucose tolerance tests and in some, their BMI was also higher. Smokers were excluded in some studies, but others do not provide any information regarding this factor. In the normotensive group of control women from a preeclampsia study, glucose intolerant patients had lower tHcy compared to normal controls and there was no difference between tHcy in gestational diabetes patients and controls [139]. Age is not compared between groups and smoking is not considered in this latter study. 8.8. OTHER ADVERSE PREGNANCY OUTCOMES The association between elevated maternal tHcy and presence of or history of a pregnancy affected by premature rupture of membranes (PROM), placental abruption, preterm delivery, and Down syndrome has also been investigated. In a study of PROM, no difference was observed in tHcy measured during weeks 32–35 of pregnancy between cases and controls and having tHcy > P95 (concentration unspecified) did not significantly increase the risk of PROM [140]. It was also reported in this study that cases were more likely to smoke and to have BMI (<19.8) less than controls. The multiple logistic regression analysis was adjusted for these confounding factors. However, the authors recognized that the study may not have been sufficiently powered to detect a difference in risk of PROM between the case and control groups. Another study in which tHcy was measured during gestational weeks 24–32 did not report any difference between cases and controls either [141]. However, no adjustment was made in these studies for smoking or socioeconomic status. More cases were smokers than controls in

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both studies. The former study reported no correlation between smoking and tHcy; however, this does not adjust for the possible effect of smoking on the pregnancy outcome being investigated. Socioeconomic status was lower in cases than in controls in the latter study. Placenta vasculopathy has been investigated primarily in different retrospective studies in which the timing of blood samples varies between 24 h and 48 months after birth. There is evidence for [142,143] and against [144] higher tHcy in cases than in controls. tHcy > 12.4 mmol/L at preconception [91] and above the median or in the highest quartile during the second–third trimester of pregnancy was associated with increased risk of preterm birth [145]. The conclusions regarding the association between elevated maternal tHcy and Down syndrome are conflicting. A prospective case–control study, in which prenatal blood samples were collected, reported no difference in tHcy in 48 cases with Down syndrome affected pregnancies and 192 controls [146]. However, there is considerable diversity in study design, selection of cases and controls, timing of blood sample collection, and adjustment of the analysis for confounding variables among the studies reported to date. The evidence available has been reviewed recently [147]. A number of studies investigating the possible contribution of polymorphisms affecting folate/ cobalamin and homocysteine metabolism to Down syndrome have also been considered in the same review.

8.9. DNA-METHYLATION AND IMPRINTING DNA methylation in the earliest stages of development has a direct influence on gene imprinting in the offspring and is dependent on methyl donor status, of which tHcy is a biomarker. DNA methylation capacity leads to hyper- or hypomethylation of DNA. The balance between each of these situations plays an important role in gene imprinting. An inverse correlation between cord tHcy and LINE1-DNA methylation (indicative of global DNA methylation level) was recently reported in humans [148].

8.10. LONG-TERM EFFECTS OF EXPOSURE OF DEVELOPING FETUS TO HOMOCYSTEINE Apart from congenital defects that are diagnosed in utero or at birth in the offspring of mothers with elevated tHcy during pregnancy, fetal exposure to elevated tHcy may also increase the risk of developing chronic disease in later life. Conceivably, fetuses exposed to elevated tHcy in utero that do not achieve optimal growth may be at increased risk of developing CVD as adults

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[149]. Long-term prospective studies from pregnancy throughout adulthood are required to test this hypothesis. There is conflicting evidence in the literature regarding the association between elevated tHcy in adults and risk of schizophrenia. However, it has been suggested that schizophrenia may actually arise due to anomalies in neurodevelopment rather than neurodegeneration [150]. Results from a nested case–control study in which prenatal samples were collected during the years 1956–1965 and the offspring followed up into adult life suggest that exposure to elevated tHcy in utero may also increase the risk of developing schizophrenia [93]. Fetal exposure to elevated homocysteine (third trimester tHcy  12.1 mmol/L) in utero was associated with a twofold increase in schizophrenia in adult offspring in this study of 63 adult cases and 122 controls. One of the mechanisms proposed was excessive stimulation of the N-methyl-D-aspartate (NMDA) receptor by homocysteine as previously observed in a study of rats [151]. Homocysteine has neuroexcitatory (agonistic at the glutamate binding site) and neuroprotective (antagonistic at the glycine coagonist site) functions at the NMDA receptor. However, in situations of homocysteine excess, overstimulation of the NMDA may occur. Dosedependent neurotoxicity was observed from homocysteine concentrations as low as 10 mM in the presence of 50 mM glycine concentrations. While the in vitro experiments were carried out at glycine concentrations that were double those reported for cerebral spinal fluid (CSF) in healthy newborns [median (95% CI): 12.1 (5.3, 24.4 mmol/L)] [152], the proposed mechanism might be feasible if glycine concentrations were higher in fetuses under unusual circumstances. High cerebral fluid glycine concentrations may occur if the blood–brain barrier is disrupted in cases of stroke or head injury in adults [153,154]. Other studies have reported that perinatal administration of NMDA receptor antagonists to rats lead to apoptopic neurodegeneration in the frontal cortex and long-lasting deficits in behavioral and cognitive function in the offspring [155]. 8.11. FUTURE RESEARCH The advance in knowledge in the field of tHcy and human pregnancy would be greatly enhanced by prospective studies, ideally, from preconception throughout pregnancy and indeed childhood. Such studies are highly time consuming and expensive to run. However, if the collection of preconception blood samples and lifestyle data is not feasible, collection could be started during the first trimester at the latest. Lifestyle habits, especially toxic habits, often change after the first prenatal check up, especially in the case of unplanned pregnancies. There is considerable variation in study designs and quality and quantity of data regarding lifestyle variables. These

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factors may contribute to the conflict in the literature in this field. Further studies are required to establish whether elevated tHcy is associated with increased risk of miscarriage, PROM, placental vasculopathy, gestational diabetes, and Down syndrome. With regard to preeclampsia and IUGR, the mechanism linking elevated tHcy and these complications needs to be elucidated. ACKNOWLEDGMENTS Research grants: MICINN SAF2005-05096; The Spanish Ministry of Health (Instituto de Salud Carlos III, Thematic Network G03/140, and RTIC RD06/0045/0009), FEDER (Fondo Europeo de Desarrollo Regional); Public Health Division, Department of Health, Catalonian Autonomous Government; Centre Catala` de la Nutricio´, Institut d’Estudis Catalans.

REFERENCES [1] G.L. Cantoni, E. Scarano, The formation of S-adenosylhomocysteine in enzymatic transmethylation reactions, J. Am. Chem. Soc. 76 (1954) 4744. [2] J. Liveson, Metabolic diagram, in: R. Carmel, D. Jacobsen (Eds.), Homocysteine in Health and Disease, Cambridge University Press, Cambridge, UK, 2001, p. ix. [3] M.J. MacCoss, N.K. Fukagawa, D.E. Matthews, Measurement of intracellular sulfur amino acid metabolism in humans, Am. J. Physiol. Endocrinol. Metab. 280 (2001) E947–E955. [4] D. Leclerc, A. Wilson, R. Dumas, et al., Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocysteinuria, Proc. Natl. Acad. Sci. USA 95 (1998) 3059–3064. [5] J. Selhub, P.F. Jacques, P.W. Wilson, D. Rush, I.H. Rosenberg, Vitamin status and intake as primary determinants of homocysteinemia in an elderly population, JAMA 270 (1993) 2693–2698. [6] E.P. Quinlivan, J. McPartlin, H. McNulty, et al., Importance of both folic acid and vitamin B12 in reduction of risk of vascular disease, Lancet 359 (2002) 227–228. [7] S.E. Chiuve, E.L. Giovannucci, S.E. Hankinson, et al., The association between betaine and choline intakes and the plasma concentrations of homocysteine in women, Am. J. Clin. Nutr. 86 (2007) 1073–1081. [8] J.E. Lee, P.F. Jacques, L. Dougherty, et al., Are dietary choline and betaine intakes determinants of total homocysteine concentration? Am. J. Clin. Nutr. 91 (2010) 1303–1310. [9] P. Frosst, H.J. Blom, R. Milos, et al., A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase, Nat. Genet. 10 (1995) 111–113. [10] B. Wilcken, F. Bamforth, Z. Li, et al., Geographical and ethnic variation of the 677C>T allele of 5,10-methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas world wide, J. Med. Genet. 40 (2003) 619–625. [11] B.D. Guenther, C.A. Sheppard, P. Tran, R. Rozen, R.G. Matthews, M.L. Ludwig, The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia, Nat. Struct. Biol. 6 (1999) 359–365.

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