TY - JOUR
AU - Guéant,, J.L.
AB - Abstract Folates belong to the vitamin B group and are involved in a large number of biochemical processes, particularly in the metabolism of homocysteine. Dietary or genetically determined folate deficiency leads to mild hyperhomocysteinemia, which has been associated with various pathologies. Molecular mechanisms of homocysteine-induced cellular dysfunction include increased inflammatory cytokine expression, altered nitric oxide bioavailability, induction of oxidative stress, activation of apoptosis and defective methylation. Whereas the involvement of folate metabolism and homocysteine in ageing-related diseases, in several developmental abnormalities and in 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. In the present article, the possible roles of folates and homocysteine in male and female subfertility and related diseases are systematically reviewed, with regard to the epidemiological, pathological, pharmacological and experimental data of the literature from the last 25 years. fertility, folates, homocysteine, MTHFR polymorphism, human reproduction Introduction Folates are a group of inter-convertible co-enzymes, differing by their oxidation state, number of glutamic acid moieties and one-carbon substitutions. They are involved in amino acid metabolism, purine and pyrimidine synthesis and methylation of a large number of nucleic acids, proteins and lipids. Of particular interest is the interface between folate metabolism and the homocysteine/methionine cycle. Homocysteine, a sulfhydryl-containing amino acid that is not used in protein synthesis, originates exclusively from the one-carbon-donating metabolism of methionine, and it is remethylated into methionine with folates acting as methyl donors ( Lucock, 2000 ). Over the past decade, there has been a growing body of evidence that even a moderately elevated serum homocysteine concentration is associated with an increased risk of ageing-related diseases, such as atherosclerotic, thromboembolic and neurodegenerative disorders, and also with early pathological events of life ( Herrmann, 2001 ; Gueant et al. , 2003 ). The latter category includes a number of developmental abnormalities, particularly neural tube defects, as well as late pregnancy complications, such as pre-eclampsia, abruptio placentae , intrauterine growth retardation, preterm birth and intrauterine fetal death ( Eskes, 2000 ; Nelen, 2001 ; Hague, 2003 ; Steegers-Theunissen et al. , 2004 ; Tamura and Picciano, 2006 ). Whereas a large amount of scientific work has investigated the roles of folate metabolism and hyperhomocysteinemia in malformations and pathologies of the ongoing pregnancy, there is only little information on a possible involvement of these biochemical phenomena in the earlier stages of reproductive physiology and in related diseases. In the present article, current data on the influence of folate metabolism on male and female reproductive tracts and fertility are reviewed by means of a systematic review of the Medline database-indexed literature since 1980. Biochemical background In most mammalian cells, accumulating homocysteine is removed either by remethylation into methionine or by trans-sulfuration into cysteine (Scott and Weir, 1998; Fowler, 2005 ). In the trans-sulfuration pathway (Figure 1 ), homocysteine is condensed with serine in an irreversible reaction catalyzed by cystathionine-beta-synthase (CBS) to form cystathionine, which in turn is reduced to cysteine and alpha-ketobutyrate by cystathionine lyase. Both of these enzymes depend on pyridoxal 5-phosphate, an active from of vitamin B6. Figure 1. Open in new tabDownload slide Biochemistry of folate and homocysteine ( A ) Pathways of folate and homocysteine metabolism (the folate and methionine cycles are highlighted). Hcy is remethylated into methionine (Met) by MTR with 5-methylTHF as a methyl donor and cobalamin (B12) as a co-enzyme. 5-MethylTHF is produced by the FAD-dependent enzyme 5,10-MTHFR. 5,10-MethyleneTHF is also a one-carbon donor in the synthesis of thymidylate and after conversion into 5,10-methenyltetrahydrofolate (MethenylTHF) and further into 10-formyltetrahydrofolate (FormylTHF), in the synthesis of purines. After the release of their one-carbon unit, all of these substituted folates are converted to THF which is finally recycled into MethyleneTHF during the conversion of serine to glycine by the enzyme serine hydroxymethyltransferase (SHMT). Met is further transformed into SAM, the universal methyl donor for methylation of nucleic acids, proteins, polysaccharides, phospholipids, etc. After release of the methyl group, SAM is converted into SAH, which is reversibly hydrolyzed into Hcy. The alternative pathway by which Hcy is remethylated into Met takes place in the liver and uses betaine as a methyl donor; this reaction is catalyzed by BHMT. Hcy is also metabolized into cystathionine by the vitamin B6-dependent CBS which is further cleaved into cysteine and alpha-ketobutyrate by cystathionine lyase (CyL). Cysteine is further incorporated into peptides, glutathione and other molecules. Abbreviations: MAT, methionine adenosyltransferase; MT, methyltransferases; X, substrate to be methylated; SAHH, S -adenosylhomocysteine hydrolase; TS, thymidylate synthase; MTHFD, 5,10-methylenetetrahydrofolate dehydrogenase. ( B ) Chemical formulae of the key reaction, i.e. the remethylation of homocysteine, at the interface between the methionine and folate cycle. Methyltetrahydrofolate (MethylTHF) consists of a pteridine ring, para-aminobenzoic acid and glutamic acid (the three constituents of all biologically active folates), with the methyl group at position 5. In the present reaction, catalyzed by MTR, this methyl group is transferred to homocysteine, with cobalamin (B12) acting as an intermediate carrier. Figure 1. Open in new tabDownload slide Biochemistry of folate and homocysteine ( A ) Pathways of folate and homocysteine metabolism (the folate and methionine cycles are highlighted). Hcy is remethylated into methionine (Met) by MTR with 5-methylTHF as a methyl donor and cobalamin (B12) as a co-enzyme. 5-MethylTHF is produced by the FAD-dependent enzyme 5,10-MTHFR. 5,10-MethyleneTHF is also a one-carbon donor in the synthesis of thymidylate and after conversion into 5,10-methenyltetrahydrofolate (MethenylTHF) and further into 10-formyltetrahydrofolate (FormylTHF), in the synthesis of purines. After the release of their one-carbon unit, all of these substituted folates are converted to THF which is finally recycled into MethyleneTHF during the conversion of serine to glycine by the enzyme serine hydroxymethyltransferase (SHMT). Met is further transformed into SAM, the universal methyl donor for methylation of nucleic acids, proteins, polysaccharides, phospholipids, etc. After release of the methyl group, SAM is converted into SAH, which is reversibly hydrolyzed into Hcy. The alternative pathway by which Hcy is remethylated into Met takes place in the liver and uses betaine as a methyl donor; this reaction is catalyzed by BHMT. Hcy is also metabolized into cystathionine by the vitamin B6-dependent CBS which is further cleaved into cysteine and alpha-ketobutyrate by cystathionine lyase (CyL). Cysteine is further incorporated into peptides, glutathione and other molecules. Abbreviations: MAT, methionine adenosyltransferase; MT, methyltransferases; X, substrate to be methylated; SAHH, S -adenosylhomocysteine hydrolase; TS, thymidylate synthase; MTHFD, 5,10-methylenetetrahydrofolate dehydrogenase. ( B ) Chemical formulae of the key reaction, i.e. the remethylation of homocysteine, at the interface between the methionine and folate cycle. Methyltetrahydrofolate (MethylTHF) consists of a pteridine ring, para-aminobenzoic acid and glutamic acid (the three constituents of all biologically active folates), with the methyl group at position 5. In the present reaction, catalyzed by MTR, this methyl group is transferred to homocysteine, with cobalamin (B12) acting as an intermediate carrier. During the remethylation into methionine (Figure 1 ), a methyl group provided by 5-methyltetrahydrofolate (5-methylTHF) is transferred to homocysteine by methionine synthase (MTR). In this ubiquitous reaction, cobalamin (vitamin B12) is involved as an intermediate carrier of the methyl group. Alternatively, homocysteine can also be remethylated into methionine by betaine–homocysteine methyltransferase (BHMT), in which the methyl group is provided by betaine that is transformed into dimethyl-glycine. However, in contrast with the MTR reaction, this alternative pathway seems to be limited to the liver; in particular, the possibility of a BHMT-expression in human gonads has not yet been investigated ( Chadwick et al. , 2000 ; Delgado-Reyes et al. , 2001 ). Whatever the remethylation pathway, the resulting methionine will be either incorporated into various peptides or transformed into S -adenosylmethionine (SAM) by methionine adenosyltransferase, which transfers an adenosyl group from ATP to methionine. SAM is the universal methyl donor in a large number of methylation reactions, and thus plays a key role in cellular function. During these reactions, which are catalyzed by specific methyltransferases, SAM has a methyl group removed to form S -adenosylhomocysteine (SAH). Finally, SAH is hydrolyzed in a reversible reaction into homocysteine. The total sequence of the preceding reactions is called the homocysteine/methionine cycle. This cycle could not turn accurately without the normal functioning of a second cycle, the folate cycle. The methyl-donating 5-methylTHF originates from 5,10-methyleneTHF by the flavine adenine dinucleotide-dependent 5,10-methyleneTHF reductase (MTHFR). After the remethylation of homocysteine to methionine, demethylated THF will be converted again into 5,10-methyleneTHF during the conversion of serine to glycine. MTHFR has a pivotal regulatory function in the folate cycle, as it directs the folate pool toward the remethylation of homocysteine at the expense of DNA and RNA synthesis ( Fowler, 2001 ); besides its conversion to 5-methylTHF by MTHFR, 5,10-methyleneTHF is further metabolized in several one-carbon transfer reactions during the synthesis of thymidylate (methylation of deoxyuridine 5′-monophosphate (dUMP) to deoxythymidine 5′-monophosphate dTMP), as well as during the synthesis of purines (Figure 1 ). An impaired function of these metabolic pathways leads to accumulation of homocysteine, either by insufficient trans-sulfuration (through CBS mutations or vitamin B6 deficiency) or by a blockage of remethylation. In the latter case, folate or cobalamin deficiency may be involved; importantly, a single nucleotide polymorphism of the MTHFR gene, C677T, encodes a thermolabile variant of the enzyme, characterized by an alanine-to-valine substitution at position 222 and a 50% reduction in enzyme activity ( Frosst et al. , 1995 ). As a consequence, the MTHFR C677T polymorphism has been associated with moderately elevated serum homocysteine concentration, particularly in patients with insufficient folate supply ( Harmon et al. , 1996 ; Jacques et al. , 1996 ). The prevalence of the homozygous TT allele is ∼10% in Caucasians, Australians and White Americans, but it seems to be influenced by ethnicity as well as by folate status ( Botto and Yang, 2000 ). Consistently, our group found an association of T allele frequency and the rate of cases with folate deficiency among seven population samples: the prevalence of homozygotes was highest in Mexicans and Italians who also have the highest plasma folate concentrations, whereas it was lowest in West Africans who have the lowest folate status ( Gueant-Rodriguez et al. , 2006 ). A second polymorphism of the same gene (A1298C) does not seem to be associated with hyperhomocysteinemia ( van der Put et al. , 1998 ). Clinical implications of the MTHFR C677T polymorphism include increased risk for several diseases, such as the pregnancy complications mentioned earlier, particularly in subjects with low folate status, but also for some other pathologies, such as colorectal neoplasias, in which the risk might be reduced in mutated subjects with sufficient folate supply ( Ueland et al. , 2001 ; Peyrin-Biroulet et al. , 2004 ). The cellular and molecular mechanisms underlying these effects have been investigated principally in the field of atherosclerosis, using either animal models or in vitro culture of endothelial cells or other components of the vascular wall; these studies have been recently reviewed in detail elsewhere ( Lawrence de Koning et al. , 2003 ; Austin et al. , 2004 ) and will be briefly recalled next (Figure 2 ). Figure 2. Open in new tabDownload slide Cellular and molecular mechanisms of hyperhomocysteinemia-induced cell dysfunction. Figure 2. Open in new tabDownload slide Cellular and molecular mechanisms of hyperhomocysteinemia-induced cell dysfunction. First, homocysteine has been shown to induce vascular inflammation by enhancing the expression of pro-inflammatory cytokines, such as monocyte chemoattractant protein 1 (MCP-1), which regulates migration and activation of monocytes/macrophages, and interleukin 8 (IL-8), which is an important chemoattractant for neutrophils and T-lymphocytes ( Poddar et al. , 2001 ). Second, homocysteine decreases the bioavailability of nitric oxide (NO), one of the major endothelium-dependent vasodilators that is produced by the endothelial isoform of nitric oxide synthase (eNOS). This effect is caused either by an accelerated oxidative inactivation of NO and/or eNOS ( Zhang et al. , 2000 ; Romerio et al. , 2004 ) or by an increase in serum assymetric dimethylarginine, an endogenous inhibitor of eNOS ( Stuhlinger et al. , 2003 ). Third, there is a large body of evidence that hyperhomocysteinemia is associated with the production of reactive oxygen species (ROS) in endothelial and smooth muscle cells. The mechanism of this oxidative stress relies either on auto-oxidation of the highly reactive thiol group of homocysteines ( Starkebaum and Harlan, 1986 ) or on the formation of intracellular superoxide and peroxyl radicals with concomitant inhibition of cellular antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase ( Weiss, 2005 ). Fourth, a more recent concept concerns activation of the unfolded protein response (UPR) that is triggered when unfolded or misfolded proteins accumulate in the endoplasmic reticulum (ER) ( Kaufman, 2002 ). This ER stress induces the expression of several molecular chaperones and other stress response proteins, which are aimed at restoring correct protein folding or retranslocating defective proteins back to the cytosol for degradation in the proteasomes. In case of a prolonged ER stress, the UPR extends the to activation of apoptosis by various signaling pathways ( Xu et al. , 2005 ). This is precisely what happens in human endothelial cells after exposure to homocysteine in vitro : while inducing misfolding in the ER by altering the local redox potential and interfering with disulfide bond formation, homocysteine activates UPR and, subsequently, growth arrest ( Outinen et al. , 1999 ) and apoptosis ( Zhang et al. , 2001 ). Homocysteine-induced endothelial apoptosis probably also involves other mechanisms such as the classical p53 pathway ( Lee et al. , 2005 ). Furthermore, folate deficiency and genetically determined low MTHFR activity lead to an insufficient remethylation of homocysteine to methionine and a decreased SAM production and SAM/SAH ratio. Insufficient availability of SAM then results in impaired methylation reactions, with multiple consequences, especially as far as DNA methylation is concerned. In homozygous MTHFR 677 TT patients, the resulting deficit in 5-methylTHF has been associated with DNA hypomethylation in peripheral blood mononuclear cells ( Stern et al. , 2000 ; Ueland et al. , 2001 ; Friso et al. , 2002 ). Thus, defective methylation may lead to aberrant gene expression resulting in abnormal fetal development and malignant diseases ( Reik and Walter, 2001 ). Finally, dietary folate deficiency and the resulting decreased cellular synthesis of 5,10-methyleneTHF, as well as reduced MTHFR activity lead to an accumulation of dUMP and thus to an excessive incorporation of uracil into DNA, with the subsequent repair mechanisms increasing the risk of chromosome breakage ( Blount et al. , 1997 ; Fenech, 2001 ). Whether all or some of these pathogenetic mechanisms of endothelial dysfunction are also involved in folate deficiency-induced alterations of male and female reproductive functions is currently unknown. However, several experimental data are in favor of such similarities, as will be discussed next. Folates and male fertility Epidemiological data Several authors investigated the prevalence of the different MTHFR 677 genotypes among infertile male patients (Table I ). In a German study, 255 patients seeking fertility counseling and 200 fertile controls were included, but precise inclusion criteria were not provided (Bezold et al. , 2001). The frequency of wild-type CC and heterozygous CT carriers was not significantly different in patients and controls. However, the prevalence of mutant TT homozygotes was significantly higher among patients when compared with controls (18.8% vs. 9.5%). The authors thus concluded a possible role of MTHFR polymorphism in the pathogenesis of male infertility. In contrast, this conclusion was not shared by a Dutch group who did not find any significant difference in the prevalence of MTHFR polymorphism between 113 healthy fertile men and 77 subfertile patients with moderate oligospermia and conception failure in female partners for at least 1 year: TT homozygosity was found in 9.1% of patients and in 13.3% of controls ( Ebisch et al. , 2003 ). In Italian men, the prevalence of TT homozygosity was even higher, between 20.4% and 27.6%, and yet not significantly different in fertile and infertile patients ( Stuppia et al. , 2003 ). Table I. MTHFR polymorphism and male infertility Reference . Geographic origin . Patients . Results . Bezold et al. (2001) Germany 255 subfertile men Higher prevalence of 677TT vs. fertile controls Ebisch et al. (2003) Netherlands 77 subfertile men Prevalence of 677TT not different vs. fertile controls Stuppia et al. (2003) Italy 93 infertile men Prevalence of 677TT not different vs. fertile controls Singh et al. (2005) India 151 infertile men (severe oligoastheonteratozoospermia) Higher prevalence of 677TT vs. fertile controls Park et al. (2005) South Korea 373 infertile men (unexplained infertility) Prevalence of 677TT higher; prevalence of 1298CC not different vs. fertile controls Paracchini et al. (2006) Italy 105 infertile men Higher prevalence of 677TT vs. fertile controls Reference . Geographic origin . Patients . Results . Bezold et al. (2001) Germany 255 subfertile men Higher prevalence of 677TT vs. fertile controls Ebisch et al. (2003) Netherlands 77 subfertile men Prevalence of 677TT not different vs. fertile controls Stuppia et al. (2003) Italy 93 infertile men Prevalence of 677TT not different vs. fertile controls Singh et al. (2005) India 151 infertile men (severe oligoastheonteratozoospermia) Higher prevalence of 677TT vs. fertile controls Park et al. (2005) South Korea 373 infertile men (unexplained infertility) Prevalence of 677TT higher; prevalence of 1298CC not different vs. fertile controls Paracchini et al. (2006) Italy 105 infertile men Higher prevalence of 677TT vs. fertile controls Open in new tab Table I. MTHFR polymorphism and male infertility Reference . Geographic origin . Patients . Results . Bezold et al. (2001) Germany 255 subfertile men Higher prevalence of 677TT vs. fertile controls Ebisch et al. (2003) Netherlands 77 subfertile men Prevalence of 677TT not different vs. fertile controls Stuppia et al. (2003) Italy 93 infertile men Prevalence of 677TT not different vs. fertile controls Singh et al. (2005) India 151 infertile men (severe oligoastheonteratozoospermia) Higher prevalence of 677TT vs. fertile controls Park et al. (2005) South Korea 373 infertile men (unexplained infertility) Prevalence of 677TT higher; prevalence of 1298CC not different vs. fertile controls Paracchini et al. (2006) Italy 105 infertile men Higher prevalence of 677TT vs. fertile controls Reference . Geographic origin . Patients . Results . Bezold et al. (2001) Germany 255 subfertile men Higher prevalence of 677TT vs. fertile controls Ebisch et al. (2003) Netherlands 77 subfertile men Prevalence of 677TT not different vs. fertile controls Stuppia et al. (2003) Italy 93 infertile men Prevalence of 677TT not different vs. fertile controls Singh et al. (2005) India 151 infertile men (severe oligoastheonteratozoospermia) Higher prevalence of 677TT vs. fertile controls Park et al. (2005) South Korea 373 infertile men (unexplained infertility) Prevalence of 677TT higher; prevalence of 1298CC not different vs. fertile controls Paracchini et al. (2006) Italy 105 infertile men Higher prevalence of 677TT vs. fertile controls Open in new tab A further study included 151 Indian patients with severe male infertility ( Singh et al. , 2005 ). These patients presented either with severe oligozoospermia or with azoospermia; those with known genetic causes (chromosomal abnormalities or Y chromosome microdeletions) had been excluded. In this population, there were significantly more homozygous TT carriers (4% vs. 0%), as well as heterozygous CT carriers (26.5% vs. 18.5%), compared with 200 fertile control subjects. The authors emphasized the importance of the general prevalence of MTHFR polymorphism and the nutritional status of the population, when comparing the results of different studies: in general, Western European people have rather high serum folate and low homocysteine concentrations that do not vary consistently among the different MTHFR 677 genotypes. Thus it is possible that despite a higher prevalence of TT homozygotes, the clinical or biological effects of this polymorphism might be masked because of a higher nutritional vitamin intake. In contrast, Indian people generally have low serum folate and high homocysteine concentrations because of a poorer nutritional status; thus, despite a very low frequency of the mutated T allele, an abnormal reproductive phenotype depending on the MTHFR polymorphism is easily detectable. Therefore the authors suggested that similar observations could probably be made in African and Asian subjects, and that MTHFR polymorphism-related infertility could be prevented by nutritional improvement in these populations. Actually, these conclusions are consistent with our study mentioned earlier, analysing folate metabolism and MTHFR polymorphism in Western Europeans, Mexicans and West Africans ( Gueant-Rodriguez et al. , 2006 ). Furthermore, such an interaction between nutritional status and genotype has also been suggested in a Spanish population where the prevalence of MTHFR 677-mutated subjects doubled since the mid-1970s, which coincided with the development of folic acid supplementation programs for pregnant women ( Munoz-Moran et al. , 1998 ). Thus, a systematic supplementation could have rescued mutated fetuses whose viability may otherwise be reduced: when both MTHFR polymorphisms, C677T and A1298C, were determined in another population sample from South Europe, the presence of three or four mutated alleles could only be detected in aborted fetuses, but not in living neonates ( Isotalo et al. , 2000 ). In contrast, the genotype analysis of 1777 individuals, divided into four age groups, showed a progressive increase of genotypes with two mutated alleles in the younger subjects when compared with the older, reflecting an unblocking of the usual genetic selection by changes in the diet and folate intake ( Reyes-Engel et al. , 2002 ). In accordance with the Indian study ( Singh et al. , 2005 ), Korean authors also found a higher prevalence of the MTHFR C677T mutant homozygotes only among men with unexplained infertility, normal karyotype and no chromosome Y microdeletions (Park et al. , 2005). These differences remained significant when subgroups of infertile patients with either azoospermia or severe oligoasthenoteratozoospermia, but not with moderately altered sperm parameters, were investigated. The authors also determined the frequency of the A1298C mutation that was, however, equally distributed among infertile patients and fertile controls. Finally, in a prospective study based on the follow-up of 105 Italians, the authors confirmed the MTHFR C677T homozygous variant to be a significant risk factor for male infertility ( Paracchini et al. , 2006 ). However, this was no longer the case in a subgroup of patients who, in addition to the MTHFR polymorphism, also presented with a common deletion of GSTM1 , a gene encoding one of the glutathione transferases. This effect could be explained by the GSTM1 deletion causing an increase in glutathione, which in turn stimulates SAM synthase activity, thus counteracting the deleterious effects of MTHFR polymorphism on the subsequent methylation reactions. The authors therefore emphasized that these types of gene interactions have to be taken into account when interpreting the results of case–control studies that focus only on one genetic parameter. Pharmacological and experimental data A few therapeutic trials investigating the possible effect of folate supplementation on male fertility have been reported. In the first of them, 40 either normozoospermic or oligozoospermic patients received 10 mg folic acid per day for a period of 30 days ( Landau et al. , 1978 ). The authors did not notice any correlation between serum or seminal fluid folate concentrations and total sperm count, and there was no beneficial effect of folic acid on sperm parameters. However, the treatment period may have been too short, as spermatogenesis is generally thought to extend > 3 months in the human. Therefore, another group administered a daily dose of 15 mg folinic acid for 3 months to 65 infertile men with an excessive round cell count in their ejaculate ( Bentivoglio et al. , 1993 ). Semen analysis was performed twice: before as well as at the end of the treatment. All female partners had normal fertility evaluation. In contrast with the preceding study, the authors noted statistically significant modifications of all analysed sperm parameters at the end of the treatment period, particularly an increase in sperm density (from 15 to 22.6 × 10 6 /ml) and motility (from 17.7% to 27.8%) as well as a decrease in round cell count (from 9.7 to 6.4 × 10 6 /ml). Moreover, among these 65 couples presenting with infertility for 3.2 years on average, 24 conceived within 6 months following the folinic acid treatment and 17 women delivered. The concomitant improvement of sperm parameters and reduction in round cells led to the conclusion that folate supplementation had a beneficial effect on spermatogenesis, possibly by increasing cellular cohesion within the seminiferous epithelium, thus preventing abnormal release of immature germ cells into the lumen. Further therapeutic intervention studies were performed using a randomized, placebo-controlled protocol to compare subfertile patients with fertile controls. Subfertility was defined as the absence of pregnancy within 1 year of unprotected intercourse and moderate oligozoospermia, i.e. a sperm concentration between 5 and 20 × 10 6 /ml. In a first study, the authors included 94 patients and 99 controls who were divided into four groups, receiving either folic acid (5 mg/day) and a placebo, or zinc sulfate (66 mg/day) and a placebo, or both drugs (folic acid and zinc sulfate) or two placebos for 6 months ( Wong et al. , 2002 ). Serum folate and zinc concentrations were not different in subfertile and fertile subjects before the onset of treatment. Only concomitant administration of folic acid and zinc led to a significant 74% increase in sperm density and total count in subfertile patients, whereas in fertile controls no significant improvement was detected. Folic acid alone increased the sperm density by 40% in subfertile patients, but this did not reach statistical significance. Thus in this study, folate supplementation alone was not sufficient to significantly improve sperm parameters in subfertile patients, but it showed a synergistic effect with zinc sulfate. The importance of this oligo-element in spermatogenesis has been investigated by several authors and recently reviewed ( Wong et al. , 2000 ). Interactions between folate and zinc metabolism have also been emphasized: zinc deficiency impairs folate intestinal absorption, as zinc plays a role in the conversion of pteroylpolyglutamates to the monoglutamate form ( Tamura and Kaiser, 1991 ; Favier et al. , 1993 ). The same Dutch group subsequently investigated whether the effect of folate and zinc supplementation depended on the MTHFR 677 genotype ( Ebisch et al. , 2003 ). Unexpectedly, it was shown that a significant increase in sperm count was limited to wild-type MTHFR 677 CC carriers, whereas no effect was observed in CT heterozygotes and in homozygous TT mutants. The authors hypothesized that residual MTHFR activity in heterozygous and homozygous mutant patients was insufficient to produce the expected effect following folic acid administration, compared with wild-type enzyme activity, although this is not consistent with the above-mentioned assumption that a high folate status may overcome the biological effects of MTHFR mutants. Polymorphism of other enzymes of the folate metabolism might also interfere. However, it has to be mentioned also that in this study, results from subfertile and fertile men had been pooled. Recently, the same authors used an identical study design to confirm the beneficial effect of the combined folic acid and zinc sulfate supplementation in subfertile patients, although to a lesser extent, as the increase in sperm count only reached 18% ( Ebisch et al. , 2006b ). In this study they showed subfertile patients to have lower serum inhibin B and higher FSH baseline concentrations, as well as an identical testosterone concentration, compared with fertile controls, but none of these parameters was modified by the treatment, thus the observed beneficial effect was not involving endocrine function of the testis. Unlike supplementation, folate deprivation is conceivable only in animal models. Rats fed a folic acid-deficient diet showed a major reduction in sperm count ( Mayr et al. , 1999 ), whereas various dihydrofolate reductase (DHFR) inhibitors, such as etopride ( Malik et al. , 1995 ) and pyrimethamine ( Cosentino et al. , 1990 ; Kalla et al. , 1997 ) proved to be efficient male contraceptives in mice and rats, by significantly reducing sperm count as well as motility. Given the key role of MTHFR in folate metabolism, the most interesting animal model is the MTHFR knock out mouse, whose MTHFR activity has been abolished by gene targeting ( Chen et al. , 2001b ). These animals had a seriously compromised survival, and males presented with high homocysteine concentration, abnormal spermatogenesis and severe infertility ( Kelly et al. , 2005 ). In the neonatal testis of MTHFR-deficient animals, the number of germ cells was extremely reduced, because of a lack of post-natal proliferation, as well as an abnormal activation of apoptosis. In the adult testis, seminiferous tubules appeared either devoid of germ cells or with a maturation blockage. Both the survival and the reproductive phenotype could be significantly improved by betaine supplementation. This further illustrates the need for an intact folate cycle to maintain normal spermatogenesis, and suggests that the alternative homocysteine remethylation pathway is also operating in the testis. To summarize the data from the above-mentioned pharmacological interventions, folic acid supplementation seems to have a positive effect on spermatogenesis and sperm parameters, either alone or in combination with other nutritional factors, but the subgroup of patients who might benefit from such therapy, as well as the optimal doses, still have to be determined. Furthermore, it has to be emphasized that no controlled, randomized study investigating the impact of folate supplementation on the pregnancy and live birth rate in the female partners of the treated male patients is currently available. Presence of folates in the male reproductive tract Seminal plasma folate concentration and biochemical forms have been determined in 48 men with low habitual fruit and vegetable consumption, using a microbiological assay (Wallock et al. , 2001). Results showed that total folate concentration was 1.5 times higher in seminal than in blood plasma. No polyglutamates were detected, as the results were identical before and after treatment of the samples with folate conjugase. Furthermore, seminal plasma contained a significant proportion (26%) of non-methyltetrahydrofolates, whereas in the blood plasma, 5-methylTHF is largely predominant. Blood and seminal plasma folate concentrations were positively correlated, except for the non-methyltetrahydrofolates. Sperm density and total count in the ejaculate showed a significant positive correlation with non-methyltetrahydrofolate concentration, but not with total folate or 5-methylTHF, suggesting a role for non-methyltetrahydrofolates in spermatogenesis or sperm maturation. Furthermore, seminal plasma has been shown to contain a high affinity folate-binding protein, which shares immunoreactivity with the human milk folate-binding protein ( Holm et al. , 1991 ). Immunohistochemical and ultrastructural studies suggested this binding protein to be secreted by the epididymal and vas deferens epithelium, but not by the seminiferous epithelium, prostate or seminal vesicles. Moreover, part of this binding protein was associated with prostasome-like vesicles that adhere to the spermatozoa in the epididymal duct, whereas testicular spermatozoa are devoid of immunoreactive folate-binding protein ( Malm et al. , 2005 ). This observation further underlines a possible physiological role of folate metabolism in male gamete maturation: the authors hypothesize either a bacteriostatic function on folate-requiring micro-organisms or a mechanism for folate uptake into spermatozoa, possibly via megalin-mediated endocytosis ( Birn et al. , 2005 ). These observations clearly need further investigation, especially with regard to possible differences between fertile and infertile men. A recent Chinese study did not find any difference in seminal plasma folate and cobalamin concentrations in 44 infertile and 176 fertile men; instead, the concentration of seminal ROS was significantly higher in the infertile group and negatively correlated with seminal folate and cobalamin ( Chen et al. , 2001a ). Finally, high affinity folate-binding sites have also been detected in human testicular tissue ( Holm et al. , 1999 ), as well as MTHFR activity, which proved to be five times higher in the mouse testis than in other tissues ( Chen et al. , 2001b ), but the cellular origin of this activity has not been determined. Pathophysiology of folate deficiency-induced male infertility Besides a direct effect of folate deficiency on the nucleic acid synthesis and thus on the proliferation of rapidly dividing cells such as male germ cell precursors, most of the pathophysiological mechanisms that have been described in endothelial cells, in case of folate deficiency and subsequent accumulation of homocysteine, also operate in the male reproductive tract. However, in many aspects of male infertility, the causal relationship between these cellular and molecular events and altered folate metabolism has not yet been investigated. Inflammatory cytokines, such as those induced by hyperhomocysteinemia, have been associated with impaired sperm parameters and male infertility: MCP-1 could play a role in testicular inflammation ( Aubry et al. , 2000 ), whereas seminal IL-8 concentration was significantly higher in men with genital tract inflammation when compared with that in healthy controls ( Sanocka et al. , 2003 ). The NO signaling pathways are involved in penile erection, spermatogenesis, dynamics of the blood–testis barrier, sperm motility, capacitation, acrosome reaction and fertilization ( Rosselli et al. , 1998 ; Herrero et al. , 2003 ; Lee and Cheng, 2004 ). Thus, any alteration of NO bioavailability, e.g. by hyperhomocysteinemia, may have direct consequences on male reproductive functions. A considerable amount of literature has demonstrated that although a small quantity of ROS is necessary for normal sperm function and sperm–oocyte fusion, excessive oxidative stress will induce sperm DNA damage and adversely influence sperm function, fertilization and early embryo development; these mechanisms have been recently reviewed (Lewis and Aitken, 2005; Weiss, 2005 ; Agarwal et al. , 2006 ). As mentioned above, another pathophysiological impact of homocysteine metabolism involves UPR-induced apoptosis. There is currently no information available on this phenomenon in male reproduction. However, it is well established that other cell death pathways such as Fas ligand-induced apoptosis play an important role in testicular physiology, by regulating the clonal expansion of germ cells, and abnormal apoptosis has been implicated in various andrological pathologies, such as impaired spermatogenesis, reduced sperm motility or increased sperm DNA fragmentation ( Said et al. , 2004 ). To our knowledge, sperm DNA methylation has not yet been investigated in patients with MTHFR polymorphism. It is clear, however, that methylation and the related gene imprinting play an important role during spermatogenesis. This is illustrated by the fact that in primordial germ cells, inherited imprinted genes have their methylation imprints erased ( Mann, 2001 ). Subsequently, a paternal-specific remethylation occurs during spermatogonial and spermatocytic differentiation ( Rousseaux et al. , 2005 ). The relationship between defective DNA methylation and impaired spermatogenesis has been demonstrated by bisufhite genomic sequencing of sperm DNA, with regard to H19, a paternally de novo methylated gene that is imprinted during the premeiotic stages of spermatogenesis ( Marques et al. , 2004 ). In 15% of moderate oligozoospermia and in 30% of severe oligozoospermia, H19 was shown to be incompletely methylated, whereas all of the normozoospermic controls showed normal methylation. In another study, sperm DNA methylation has been proposed as a prognostic factor in in vitro fertilization (IVF): these authors used a 5-methylcytosine immunoassay with flow cytometry detection to quantify DNA methylation, and observed a pregnancy rate of 33% per cycle in the normal methylation group vs. 8.3% in the low methylation group ( Benchaib et al. , 2005 ). These observations are in accordance with previous animal studies using 5-aza-2-deoxycytidine, a hypomethylating agent, in neonatal mice whose testes contain only premeiotic germ cells. This treatment completely blocked differentiation to the spermatocyte stage ( Raman and Narayan, 1995 ). When administered to adult mice or rats, the resulting effect was a severe impairment of spermatogenesis with reduced pregnancy rates in females ( Doerksen et al. , 2000 ; Kelly et al. , 2003 ). Methylation defects also affect other molecules than DNA. Particularly, phospholipid methylation has been shown to be highly active during the synthesis of phosphatidylcholine in rat Leydig cells ( Moger, 1985 ). The major methyl donor, SAM, stimulated hCG-mediated testosterone synthesis in purified rat Leydig cells in vitro , whereas SAH had opposite effects ( Papadopoulos et al. , 1987 ). Thus folate deficiency-induced hypomethylation may impair not only the exocrine, but also the endocrine, functions of the male gonad. Finally, it has also been suggested that the adverse reproductive outcome in patients with homozygous MTHFR polymorphism may be related to homocysteine-induced precocious atherosclerotic vascular alterations, impairing the blood flow in the testicular arteries ( Rossato, 2004 ). Folates and Female Fertility Folates and homocysteine in the female reproductive tract Folates, methionine, homocysteine and vitamins B6 and B12 have been measured in the follicular fluid of 14 patients undergoing oocyte retrieval for IVF ( Steegers-Theunissen et al. , 1993 ). None of these women had a previous history of recurrent spontaneous absorption or malformations. When compared with serum concentrations, follicular fluid concentrations of folates and homocysteine were not different, but methionine as well as vitamin B6 and B12 concentrations were significantly lower. Follicular fluid results from passive blood plasma diffusion through the basement membrane between the theca interna and the granulosa layer, as well as from active secretion by granulosa cells. Actually, prior to ovulation, granulosa cells, and the oocyte they enclose, are avascular and therefore depend on the diffusion of blood plasma nutrients supplied by the thecal capillary network. Thus the question was raised whether disturbances of this micro-environment around the oocyte may have deleterious consequences on the reproductive competence of the female gamete. A Polish group measured homocysteine concentration in follicular fluid of 40 patients undergoing IVF, 20 of whom received folic acid supplementation ( Szymanski and Kazdepka-Zieminska, 2003 ). These authors found follicular fluid homocysteine concentration to be significantly lower in folate-supplemented patients; moreover, there was a negative correlation between follicular fluid homocysteine concentration and the degree of maturity of the retrieved oocytes. The clinical significance of this observation remains to be determined, since in another study, follicular fluid homocysteine concentration was not predictive of the success of the IVF procedure; however, there were only 15 patients included in that study, with heterogeneous etiologies ( Jerzak et al. , 2003 ). Recently, homocysteine and other thiol concentrations have been determined in the ejaculate and follicular fluid of 156 couples undergoing IVF ( Ebisch et al. , 2006a ). Follicular fluid homocysteine concentration was significantly higher in women with endometriosis when compared with patients having unexplained infertility. Moreover, follicular fluid as well as seminal plasma homocysteine concentrations showed a significant negative correlation with embryo quality on day 3 after IVF, but whether there was any further correlation with the chances of pregnancy has not been investigated in that study. The question of a possible effect of folic acid on ovarian function was raised in the late 1960s, when it has been shown that in immature superovulated rats, either excess or deficiency of folates partially inhibited ovulation ( Willmott et al. , 1968 ). In rhesus monkeys, a folate-restricted diet led to irregular menstrual cycles, whereas pre-ovulatory serum estradiol as well as mid-luteal progesterone concentrations decreased progressively when compared with animals under normal diet. Ovarian biopsies of folate-deprived monkeys demonstrated degeneration of graafian follicles, with an increase in atretic and cystic follicles, as well as a depletion of granulosa cells and a reduction or even an absence of corpora lutea ( Mohanty and Das, 1982 ). Thus, a sufficient folate intake seems to be necessary to maintain normal ovarian histology and endocrine function in this primate model. Unfortunately, fertility had not been assessed in these animals, but another group studied the probability of pregnancy in golden hamsters according to the diet ( Mooij et al. , 1992 ). In animals fed with a folic acid-free diet for 2 weeks before mating, the number of pregnancies as well as the red blood cell folate concentration were not different when compared with control animals. However, when the folate-free diet was prolonged up to 16 weeks before mating, none of the females was fertile, while their folate concentrations had decreased significantly. Recently, we reported on a low vitamin B2 and B12, folate and choline diet fed to female rats 1 month before mating (Blaise et al. , 2005). These animals showed normal fertility, but pups exposed to this diet in utero and during the suckling period showed significant growth retardation and a 25% perinatal mortality. Gonadal histology has not yet been assessed in that model. Folate metabolism and estrogens The hypothesis of a relationship between folate metabolism and estrogens has been investigated in a large number of studies since the early 1970s, focussing on the possible involvement of oral contraceptives in a reduction of folate intestinal uptake and serum concentration ( Lindenbaum et al. , 1975 ). Thirty years later, the interactions of sex steroids and folate metabolism still remain controversial, but there seems to be convergent, though not unanimous, evidence of an estrogen-induced decrease in serum homocysteine concentration. Serum homocysteine concentration is actually lower in females than in males ( Boers et al. , 1983 ), lower in premenopausal than in post-menopausal women ( Wouters et al. , 1995 ; Hak et al. , 2000 ) as well as throughout the follicular phase of the menstrual cycle ( Tallova et al. , 1999 ). Homocysteine concentration has also been shown to increase after bilateral oophorectomy in a series of 30 patients ( Kapral et al. , 2002 ) and to decrease again after the onset of hormonal replacement therapy (HRT). The impact of HRT on homocysteine has been investigated by a number of authors, and most of them agree with the inhibitory effect of estrogens on homocysteine production. These studies have been recently reviewed ( Mijatovic and van der Mooren, 2001 ). In several other studies, however, no significant effect of HRT on homocysteine concentration could be detected ( Berger et al. , 2000 ; Farag et al. , 2003 ). Similarly, the impact of oral contraceptives on homocysteine metabolism needs to be clarified ( Lindenbaum et al. , 1975 ; Brattstrom et al. , 1992 ; Steegers-Theunissen et al. , 1992b ; Merki-Feld et al. , 2002 ; Lussana et al. , 2003 ), concerning the role of estrogens as well as that of the progestin component of these products. Finally, two groups have monitored serum homocysteine concentration throughout long protocol stimulation for IVF (Bettahar-Lebugle et al. , 2002; Roopnarinesingh et al. , 2006 ). These protocols include first a pituitary down-regulation by a GnRH agonist, followed by ovarian stimulation with hMG or FSH. Thus serum estrogen concentration varies between two extremes in these patients during the IVF protocol; nevertheless, in both studies, homocysteine concentration did not show any variations that could have been expected from the preceding results. Folate metabolism and Polycystic ovary syndrome According to the Rotterdam criteria, polycystic ovary syndrome (PCOS) is defined by at least two of the following abnormalities: (i) oligo-or anovulation, (ii) clinical or biological hyperandrogenism and (iii) polycystic ovaries on pelvic ultrasound ( Azziz, 2004 ). PCOS is classically related to an increased cardiovascular risk, which may be accounted for by the associated insulin resistance, hyperandrogenism, or possibly, hyperhomocysteinemia. Thus a number of studies have investigated homocysteine and folate metabolism and confirmed the presence of increased serum homocysteine concentration in obese as well as in non-obese PCOS patients ( Yilmaz et al. , 2005 ). MTHFR polymorphism does not seem to be more frequent in these patients than in healthy controls ( Tsanadis et al. , 2002 ), and the possible determinants of elevated homocysteine concentration are still debated among authors who found significant correlations between homocysteine and insulin resistance or hyperandrogenism ( Yarali et al. , 2001 ; Schachter et al. , 2003 ; Vrbikova et al. , 2003 ; Bayraktar et al. , 2004 ; Wijeyaratne et al. , 2004 ) and those who did not ( Loverro et al. , 2002 ; Kilic-Okman et al. , 2004 ; Yilmaz et al. , 2005 ). Interestingly, administration of insulin sensitizers, such as metformin, as it is proposed in PCOS patients to improve ovulation induction and other parameters ( Stadtmauer and Oehninger, 2005 ), has led to a further increase of serum homocysteine in these patients, despite the decrease in insulin resistance ( Vrbikova et al. , 2002 ; Kilicdag et al. , 2005b ). This effect may be explained by a metformin-induced folate depletion ( Wulffele et al. , 2003 ), and may be prevented by concomitant folate supplementation ( Kilicdag et al. , 2005a ). An adequate folate supplementation will also prevent an increase in plasma homocysteine during weight loss, which is the first therapeutic measure to be taken in obese PCOS patients ( Henning et al. , 1998 ; Volek et al. , 2002 ; Ortega et al. , 2006 ). Finally, only two studies did not report elevated homocysteine concentration among patients with polycystic ovaries: one study including patients with polycystic-appearing ovaries but possibly not all had PCOS ( Sills et al. , 2001 ), and an Italian study including 70 PCOS patients with low folate intake and a very high prevalence of the mutated 677T allele, as is usually observed in that population ( Orio et al. , 2003 ). Folate metabolism and outcome of assisted reproduction techniques To investigate the question whether folate metabolism had an impact on the ovarian response to ovulation induction treatments for IVF (Table II ), 105 IVF patients were included in a prospective study and had their MTHFR 677 genotype determined ( Thaler et al. , 2006 ). A total of 269 IVF cycles were started and 245 led to oocyte retrieval. The analysis of these cycles showed that patients with a MTHFR 677 CT or TT genotype required significantly higher FSH doses for ovulation induction than homozygous wild-type patients, whereas they nevertheless had a lower ovarian response. Similarly, the number of oocytes collected and the maximal serum estradiol concentration were significantly lower in patients carrying the mutated T allele, whether they were homozygous or heterozygous. Fertilization and implantation rates were not affected by the MTHFR polymorphism. The preceding differences remained significant when the subgroup of patients over > years old was analysed; however in younger patients, no significant differences were observed. Thus there may be an accelerated depletion of the ovarian reserve in patients with MTHFR polymorphism, which becomes clinically evident beyond the age of 35. MTHFR polymorphism had no impact on fertilization and implantation rates, which was consistent with the findings of an Italian group who showed there was no difference in the prevalence of MTHFR polymorphism between 234 women who conceived spontaneously and 162 patients with implantation failure after IVF, most of whom were undergoing their first cycle (Martinel Li et al. , 2003 ). In contrast, an Israeli group reported a significantly higher prevalence of MTHFR C677T homozygotes as well as other inherited thrombophilia mutations in patients with post-IVF implantation failure ( Azem et al. , 2004 ). These authors however selected only patients with at least four consecutive IVF implantation failures despite the transfer of at least three good quality embryos, thus MTHFR polymorphism could be involved in such repeated and otherwise unexplained failures. In a recent study including 602 IVF patients, women with a heterozygous MTHFR 677 CT rather than a homozygous CC genotype had a slightly, but significantly increased chance to have a viable pregnancy, whereas for the MTHFR 1298 polymorphism, the homozygous AA genotype was significantly linked to a better IVF outcome ( Haggarty et al. , 2006 ). Finally, in another recent study on 197 IVF couples, neither A1298C nor C677T polymorphism, in any of the partners, was associated with embryo quality, the chance of pregnancy and the risk of early pregnancy loss ( Dobson et al. , 2007 ). However, in the two latter studies all of the female patients had been taking folate supplementation prior to their IVF attempt, thus a possible effect of these polymorphisms could have been masked. Table II. MTHFR metabolism and IVF outcome Reference . Patients . Impact . Martinelli et al. (2003) 162 IVF patients Prevalence of 677TT not different in case of post-IVF implantation failure (1st attempt) vs. fertile controls Azem et al. (2004) 45 IVF patients Higher prevalence of 677TT in case of at least four post-IVF implantation failures vs. fertile controls Haggarty et al. (2006) 602 IVF patients Higher chance of live birth in 1298AA vs. 1298CC and in 677CT vs. 677CC patients (all patients supplemented with folate) Dobson et al. 197 IVF couples No impact of MTHFR polymorphism on embryo quality, chance of pregnancy and risk of early pregnancy loss (all patients supplemented with folate) Thaler et al. (2006) 105 IVF patients Higher FSH doses, lower ovarian response in 677CT/TT vs. 677CC patients when > 35 years; no differences < 35 years Reference . Patients . Impact . Martinelli et al. (2003) 162 IVF patients Prevalence of 677TT not different in case of post-IVF implantation failure (1st attempt) vs. fertile controls Azem et al. (2004) 45 IVF patients Higher prevalence of 677TT in case of at least four post-IVF implantation failures vs. fertile controls Haggarty et al. (2006) 602 IVF patients Higher chance of live birth in 1298AA vs. 1298CC and in 677CT vs. 677CC patients (all patients supplemented with folate) Dobson et al. 197 IVF couples No impact of MTHFR polymorphism on embryo quality, chance of pregnancy and risk of early pregnancy loss (all patients supplemented with folate) Thaler et al. (2006) 105 IVF patients Higher FSH doses, lower ovarian response in 677CT/TT vs. 677CC patients when > 35 years; no differences < 35 years Open in new tab Table II. MTHFR metabolism and IVF outcome Reference . Patients . Impact . Martinelli et al. (2003) 162 IVF patients Prevalence of 677TT not different in case of post-IVF implantation failure (1st attempt) vs. fertile controls Azem et al. (2004) 45 IVF patients Higher prevalence of 677TT in case of at least four post-IVF implantation failures vs. fertile controls Haggarty et al. (2006) 602 IVF patients Higher chance of live birth in 1298AA vs. 1298CC and in 677CT vs. 677CC patients (all patients supplemented with folate) Dobson et al. 197 IVF couples No impact of MTHFR polymorphism on embryo quality, chance of pregnancy and risk of early pregnancy loss (all patients supplemented with folate) Thaler et al. (2006) 105 IVF patients Higher FSH doses, lower ovarian response in 677CT/TT vs. 677CC patients when > 35 years; no differences < 35 years Reference . Patients . Impact . Martinelli et al. (2003) 162 IVF patients Prevalence of 677TT not different in case of post-IVF implantation failure (1st attempt) vs. fertile controls Azem et al. (2004) 45 IVF patients Higher prevalence of 677TT in case of at least four post-IVF implantation failures vs. fertile controls Haggarty et al. (2006) 602 IVF patients Higher chance of live birth in 1298AA vs. 1298CC and in 677CT vs. 677CC patients (all patients supplemented with folate) Dobson et al. 197 IVF couples No impact of MTHFR polymorphism on embryo quality, chance of pregnancy and risk of early pregnancy loss (all patients supplemented with folate) Thaler et al. (2006) 105 IVF patients Higher FSH doses, lower ovarian response in 677CT/TT vs. 677CC patients when > 35 years; no differences < 35 years Open in new tab Nonetheless, the above mentioned results raise the question of whether folate supplementation could improve IVF outcome, particularly in patients beyond 35 years and presenting with MTHFR polymorphism, and, if so, at which doses and for which treatment duration. Folate metabolism and the risk of multiple pregnancies A negative impact of the MTHFR C677T polymorphism on follicular growth and maturation may also explain an earlier observation of a significantly lower prevalence of the MTHFR 677T allele in women who spontaneously conceived dichorionic twins when compared with those having spontaneous singleton pregnancies ( Hasbargen et al. , 2000 ). The frequency of homozygous wild-type, heterozygous and homozygous mutation carriers was 48.7, 41.7 and 9.6%, respectively, in 156 mothers with singleton pregnancies, whereas in 40 women with dichorionic twin pregnancies, these frequencies were 72.5, 22.5 and 5.0%, respectively. Thus a reduced MTHFR activity and the ensuing reduced SAM availability or hyperhomocysteinemia could inhibit polyovulation, which is a condition for spontaneous dichorionic pregnancies ( Hall, 2003 ). On the other hand, it may also be hypothesized that low folate availability and hypomethylation, either due to poor dietary folate intake or genetic polymorphism, could lead to undetected loss of a dichorionic co-twin, a relatively common phenomenon called the vanishing twin ( Landy and Keith, 1998 ). Whatever the exact mechanism, the influence of folate metabolism on the probability of dizygotic twinning is further illustrated by the observation that the rate of twin births is low in populations with a high prevalence of mutated MTHFR alleles, whereas it is rather high in ethnic groups with a low mutation frequency ( Hasbargen et al. , 2000 ). However, other authors did not find any association between the prevalence of dizygotic twinning and the MTHFR genotype in large samples of Australian and Dutch populations ( Montgomery et al. , 2003 ). Nevertheless, there has been considerable controversy about whether or not periconceptional folic acid supplementation to prevent neural tube defects, would expose women to a higher risk of spontaneous multiple pregnancy and birth. The question was first raised in a Hungarian placebo-controlled study including > 5500 pregnant women and showing a 40% increase of the multiple birth rate in women who took vitamin supplements when compared with those who did not ( Czeizel et al. , 1994 ). Other authors reported similar findings ( Werler et al. , 1997 ; Ericson et al. , 2001 ; Vollset et al. , 2005 ). However, different confounding elements could have biased these results, such as the inclusion of pregnancies after infertility treatments that are known to induce up to 25% multiple pregnancies ( Berry et al. , 2005 ), or the use of different kinds of supplements (folic acid alone at very low or very high doses, multivitamin preparations, and sometimes supplements of unknown composition), or the retrospective study design with self-reported supplementation or an extremely low proportion of women who used supple meats. In contrast, several studies investigating the prevalence of multiple births before and after the onset of systematic food fortification programs in countries where this was the case did not find any increase that could have been attributable to folic acid intake in the general population ( Waller et al. , 2003 ; Kucik and Correa, 2004 ; Lawrence et al. , 2004 ; Signore et al. , 2005 ). Moreover, the data from a community intervention program of folic acid supplementation in a homogeneous Chinese rural population including > 242 000 women did not find any difference in multiple birth rate of women under periconceptional supplementation with 0.4 mg folic acid when compared with those who were not supplemented ( Li et al. , 2003 ). The Hungarian authors extended their study and included over 38 000 pregnant women; when only folic acid supplementation (at a high dose of 6 mg/day) was considered, the frequency of twinning was still higher than in untreated women, but this did not reach statistical significance ( Czeizel and Vargha, 2004 ). In a recent Swedish study, possible confounding factors, such as infertility treatments, immigration, maternal age, parity and smoking, have been taken into account; nevertheless, the authors conclude folic acid supplementation to be a relatively weak but significant risk factor for dizygotic twinning ( Kallen, 2004 ). Finally, in a recent study including 602 patients undergoing IVF and 932 women who conceived naturally, the authors also demonstrated a small but significant increase in the risk of dizygotic twinning among the infertile population ( Haggarty et al. , 2006 ). Thus, this question is still a matter of debate. Folate metabolism and early pregnancy loss The question of the involvement of folate metabolism in embryonic viability has also been raised with regard to the relationship among folates, homocysteine and early pregnancy loss. Such a relationship has been suggested first in CBS-deficient patients with homocystinuria who presented with severe hyperhomocysteinemia and a spontaneous abortion rate of almost 50% ( Mudd, 1985 ), although currently, a careful monitoring of these patients has allowed a better pregnancy outcome to be achieved ( Levy et al. , 2002 ). In a population-based case–control study, women with low plasma folate levels had a higher risk of early pregnancy loss than women with normal or high levels ( George et al. , 2002 ). Moderate hyperhomocysteinemia has also been found to be a risk factor for recurrent early pregnancy loss (REPL) ( Steegers-Theunissen et al. , 1992a ; Wouters et al. , 1993 ; Coumans et al. , 1999 ; Del Bianco et al. , 2004 ) and even for first early pregnancy loss ( Gris et al. , 2003 ). A recent meta-analysis confirmed an increased risk of hyperhomocysteinemia for REPL, defined as two or more spontaneous abortions before 16 weeks of menstrual age ( Nelen et al. , 2000 ). As to the possible impact of MTHFR polymorphism, 677 TT homozygosity has been shown to increase the risk in some studies, whereas in others, no such effect could be detected; these studies have been reviewed elsewhere ( Zetterberg, 2004 ). In a recent meta-analysis by Rey et al. (2003) , this polymorphism was not found to increase the risk for REPL. There are several reasons to explain the inconsistency of these results. First, there is no homogeneous definition of REPL (at least two or three consecutive spontaneous abortions), as well as of hyperhomocysteinemia (fasting or afterload concentrations). Second, the possible impact of the fetal MTHFR genotype on the risk of REPL has not been investigated in most of the studies. Recently, Zetterberg et al. (2002) emphasized the importance of this parameter, as they observed an OR of 14.2 (95% confidence interval: 1.78–113) in spontaneously aborted embryos presenting with one or more MTHFR 677T and 1298C alleles when compared with the wild-type (677CC and 1298AA) genotype. Thus the risk could even be higher when both the mother and her fetus are homozygous, as has been shown for the risk of neural tube defects ( Christensen et al. , 1999 ). Third, possible gene–gene interactions have to be taken into account, as has already been mentioned in the context of male infertility. As an example, the interference of a transcobalamin (TC) polymorphism, TC C776G , which influences homocysteine metabolism has been investigated ( Zetterberg et al. , 2003 ). TC is a vitamin B12-binding protein and plays a role in the transport and bioavailability of this vitamin. Patients with a heterozygous or homozygous TC 776 mutation have lower serum TC concentration and a tendency toward a higher homocysteine concentration ( Namour et al. , 2001 ). Genotype analysis in fetal tissues from 76 spontaneous abortions, most of them occurring earlier than week 12, showed that embryos presenting with a combined MTHFR 677TT and TC 776CG or TC 776GG genotype had an increased risk for REPL when compared with embryos that had only one of these mutated genotypes ( Zetterberg et al. , 2003 ). Fourth, as suggested by the preceding observation, etiologies other than folate deficiency or MTHFR polymorphism may lead to hyperhomocysteinemia and subsequent pregnancy loss. The association between REPL and vitamin B12 has been illustrated by two case reports concerning a 38 year-old woman with four episodes of early spontaneous abortion vitamin B12 deficiency and bone marrow megaloblastosis ( Candito et al. , 2003 ), and a 36 year-old patient with documented familial and personal history of Addison–Biermer disease, who had experienced 12 episodes of spontaneous abortion in the absence of any other known causes of REPL ( Gueant et al. , 2004 ). Despite several pathophysiological hypotheses including impaired cell proliferation, increased oxidative stress, apoptosis, reduced extra-embryonic vascular development and hypomethylation ( Zetterberg, 2004 ; Latacha and Rosenquist, 2005 ), it is not clear whether hyperhomocysteinemia is causally related to REPL or whether it is only a marker of the increased risk of REPL. Actually, lowering homocysteine concentration by B-vitamin supplementation has been shown to have a positive effect in several case reports and in small series, with spontaneous pregnancies occurring after a few months of treatment in patients who had previously experienced between 4 and 12 early spontaneous abortions ( Quere et al. , 1998, 2001 ; Candito et al. , 2003 ; Gueant et al. , 2004 ). Pathophysiology of folate deficiency-induced female infertility Possible mechanisms of the deleterious effects of folate deficiency and homocysteine accumulation on female fertility include, as in the male, reduced cell division (e.g. of oogonia during oogenesis or of granulosa cells during folliculogenesis), inflammatory cytokine production, altered NO metabolism, oxidative stress, apoptosis and defective methylation reactions. Excessive MCP-1 and IL-8 production has been associated with endometriosis-related infertility ( Calhaz-Jorge et al. , 2003 ; Gmyrek et al. , 2005 ), whereas NO is involved in nearly all steps of female reproduction, i.e. in ovulation, early embryonic cleavage, implantation, regulation of arterial pressure, uterine quiescence and, finally, labor contractions and cervical ripening. Importantly, physiological NO concentrations are within a narrow range and either excess or lack of NO will induce an adverse reproductive outcome ( Maul et al. , 2003 ; Thaler and Epel, 2003 ). Similarly, oxidative stress and apoptosis play a role in physiological events, such as follicular development and cyclic endometrial changes, as well as in various pathological situations, such as infertility, endometriosis, spontaneous abortions, pre-eclampsia, gestational diabetes, fetal embryopathies and preterm labor ( Agarwal et al. , 2005 ; Hussein, 2005 ). Thus, MTHFR polymorphism-related hyperhomocysteinemia may activate apoptosis, leading to follicular atresia, as suggested by the above-mentioned rhesus model. Whereas homocysteine-induced apoptosis remains to be investigated in granulosa cells and other follicular constituents, it has already been demonstrated in other tissues, such as the endothelium and trophoblast ( Steegers-Theunissen et al. , 2000 ; Di Simone et al. , 2003 ; Suhara et al. , 2004 ). Finally, insufficient availability of methyl groups for DNA, protein and lipid methylation could impair proliferation and differentiation of the granulosa layer, thus inhibiting follicular maturation as well as steroidogenesis. In fact, it has recently been shown that DNA methylation is involved in the regulation of differential aromatase expression in bovine granulosa and corpus luteum cells ( Vanselow et al. , 2005 ). Conclusion Folate metabolism is involved in a large number of physiological and pathophysiological processes in the field of andrology and gynecology. There is a growing body of evidence demonstrating a relationship between folate and other B vitamin deficiencies, hyperhomocysteinemia and gonadal abnormalities, such as altered spermatogenesis and impaired ovarian reserve, as well as male and female infertility. However, the exact mechanisms underlying these phenomena still need to be further investigated. Whereas the benefit of a periconceptional folate supplementation in women has been generally accepted as far as the risk of neural tube defects is concerned, the effects of this preventive intervention on other developmental abnormalities of the growing fetus, as well as on the success rate of assisted reproduction techniques are still unclear. Similarly, in the male, there is a considerable lack of information on the exact role of folate metabolism and folate supplementation in normal reproductive functions as well as in the treatment of infertility. Finally, it has to be emphasized that further studies need to take into account gene–gene as well as gene–environment interactions, which have often biased previous work and led to contradictory conclusions. References Agarwal A , Gupta S , Sharma RK . Role of oxidative stress in female reproduction , Reprod Biol Endocrinol , 2005 , vol. 3 (pg. 28 - 47 ) Google Scholar Crossref Search ADS PubMed WorldCat Agarwal A , Prabakaran S , Allamaneni S . What an andrologist/urologist should know about free radicals and why , Urology , 2006 , vol. 67 (pg. 2 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Aubry F , Habasque C , Satie AP , et al. Expression and regulation of the CC-chemokine monocyte chemoattractant protein-1 in rat testicular cells in primary culture , Biol Reprod , 2000 , vol. 62 (pg. 1427 - 35 ) Google Scholar Crossref Search ADS PubMed WorldCat Austin RC , Lentz SR , Werstuck GH . Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease , Cell Death Differ , 2004 , vol. 11 Suppl 1 (pg. S56 - 64 ) Google Scholar Crossref Search ADS PubMed WorldCat Azem F , Many A , Ben Ami I , et al. Increased rates of thrombophilia in women with repeated IVF failures , Hum Reprod , 2004 , vol. 19 (pg. 368 - 70 ) Google Scholar Crossref Search ADS PubMed WorldCat Azziz R . PCOS: a diagnostic challenge , Reprod Biomed Online , 2004 , vol. 8 (pg. 644 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Bayraktar F , Dereli D , Ozgen AG , et al. Plasma homocysteine levels in polycystic ovary syndrome and congenital adrenal hyperplasia , Endocr J , 2004 , vol. 51 (pg. 601 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Benchaib M , Braun V , Ressnikof D , et al. Influence of global sperm DNA methylation on IVF results , Hum Reprod , 2005 , vol. 20 (pg. 768 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Bentivoglio G , Melica F , Cristoforoni P . Folinic acid in the treatment of human male infertility , Fertil Steril , 1993 , vol. 60 (pg. 698 - 701 ) Google Scholar Crossref Search ADS PubMed WorldCat Berger PB , Herrmann RR , Dumesic DA . The effect of estrogen replacement therapy on total plasma homocysteine in healthy postmenopausal women , Mayo Clin Proc , 2000 , vol. 75 (pg. 18 - 23 ) Google Scholar Crossref Search ADS PubMed WorldCat Berry RJ , Kihlberg R , Devine O . Impact of misclassification of in vitro fertilisation in studies of folic acid and twinning: modelling using population based Swedish vital records , BMJ , 2005 , vol. 330 (pg. 815 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Bettahar-Lebugle K , Feugeas O , Wittemer C . Evolution of homocysteine during ovarian stimulation for IVF or ICSI , Gynecol Obstet Fertil , 2000 , vol. 30 (pg. 121 - 8 ) Google Scholar Crossref Search ADS WorldCat Bezold G , Lange M , Peter RU . Homozygous methylenetetrahydrofolate reductase C677T mutation and male infertility , New Engl J Med , 2001 , vol. 344 (pg. 1172 - 3 ) Google Scholar Crossref Search ADS PubMed WorldCat Birn H , Zhai X , Holm J , et al. Megalin binds and mediates cellular internalization of folate binding protein , FEBS J , 2005 , vol. 272 (pg. 4423 - 30 ) Google Scholar Crossref Search ADS PubMed WorldCat Blaise S , Alberto JM , Nedelec E , et al. Mild neonatal hypoxia exacerbates the effects of vitamin-deficient diet on homocysteine metabolism in rats , Pediatr Res , 2005 , vol. 57 (pg. 777 - 82 ) Google Scholar Crossref Search ADS PubMed WorldCat Blount BC , Mack MM , Wehr CM , et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage , Proc Natl Acad Sci USA , 1997 , vol. 94 (pg. 3290 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Boers GH , Smals AG , Trijbels FJ , et al. Unique efficiency of methionine metabolism in premenopausal women may protect against vascular disease in the reproductive years , J Clin Invest , 1983 , vol. 72 (pg. 1971 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Botto LD , Yang Q . 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review , Am J Epidemiol , 2000 , vol. 151 (pg. 862 - 77 ) Google Scholar Crossref Search ADS PubMed WorldCat Brattstrom L , Israelsson B , Olsson A , et al. Plasma homocysteine in women on oral oestrogen-containing contraceptives and in men with oestrogen-treated prostatic carcinoma , Scand J Clin Lab Invest , 1992 , vol. 52 (pg. 283 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Calhaz-Jorge C , Costa AP , Santos MC , et al. Peritoneal fluid concentrations of interleukin-8 in patients with endometriosis depend on the severity of the disorder and are higher in the luteal phase , Hum Reprod , 2003 , vol. 18 (pg. 593 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Candito M , Magnaldo S , Bayle J , et al. Clinical B12 deficiency in one case of recurrent spontaneous pregnancy loss , Clin Chem Lab Med , 2003 , vol. 41 (pg. 1026 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Chadwick LH , McCandless SE , Silverman GL , et al. Betaine-homocysteine methyltransferase-2: cDNA cloning, gene sequence, physical mapping, and expression of the human and mouse genes , Genomics , 2000 , vol. 70 (pg. 66 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Chen Q , Ng V , Mei J , et al. Comparison of seminal vitamin B12, folate, reactive oxygen species and various sperm parameters between fertile and infertile males , Wei Sheng Yan Jiu , 2001a , vol. 30 (pg. 80 - 82 ) OpenURL Placeholder Text WorldCat Chen Z , Karaplis AC , Ackerman SL , et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition , Hum Mol Genet , 2001b , vol. 10 (pg. 433 - 43 ) Google Scholar Crossref Search ADS WorldCat Christensen B , Arbour L , Tran P , et al. Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects , Am J Med Genet , 1999 , vol. 84 (pg. 151 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Cosentino MJ , Pakyz RE , Fried J . Pyrimethamine: an approach to the development of a male contraceptive , Proc Natl Acad Sci USA , 1990 , vol. 87 (pg. 1431 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Coumans AB , Huijgens PC , Jakobs C , et al. Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion , Hum Reprod , 1999 , vol. 14 (pg. 211 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Czeizel AE , Vargha P . Periconceptional folic acid/multivitamin supplementation and twin pregnancy , Am J Obstet Gynecol , 2004 , vol. 191 (pg. 790 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Czeizel AE , Metneki J , Dudas I . The higher rate of multiple births after periconceptional multivitamin supplementation: an analysis of causes , Acta Genet Med Gemellol (Roma) , 1994 , vol. 43 (pg. 175 - 84 ) Google Scholar Crossref Search ADS PubMed WorldCat Del Bianco A , Maruotti G , Fulgieri AM , et al. Recurrent spontaneous miscarriages and hyperhomocysteinemia , Minerva Ginecol , 2004 , vol. 56 (pg. 379 - 83 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Delgado-Reyes CV , Wallig MA , Garrow TA . Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney , Arch Biochem Biophys , 2001 , vol. 393 (pg. 184 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Di Simone N , Maggiano N , Caliandro D , et al. Homocysteine induces trophoblast cell death with apoptotic features , Biol Reprod , 2003 , vol. 69 (pg. 1129 - 34 ) Google Scholar Crossref Search ADS PubMed WorldCat Dobson AT , Davis RM , Rosen MP , et al. Methylenetetrahydrofolate reductase C677T and A1298C variants do not affect ongoing pregnancy rates following IVF , Hum Reprod , 2007 , vol. 22 (pg. 450 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Doerksen T , Benoit G , Trasler JM . Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology , Endocrinology , 2000 , vol. 141 (pg. 3235 - 44 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Ebisch IM , van Heerde WL , Thomas CM , et al. C677T methylenetetrahydrofolate reductase polymorphism interferes with the effects of folic acid and zinc sulfate on sperm concentration , Fertil Steril , 2003 , vol. 80 (pg. 1190 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Ebisch IM , Peters WH , Thomas CM . Homocysteine, glutathione and related thiols affect fertility parameters in the (sub)fertile couple , Hum Reprod , 2006a , vol. 21 (pg. 1725 - 33 ) Google Scholar Crossref Search ADS WorldCat Ebisch IM , Pierik FH , De Jong FH , et al. Does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in men? , Int J Androl , 2006b , vol. 29 (pg. 339 - 45 ) Google Scholar Crossref Search ADS WorldCat Ericson A , Kallen B , Aberg A . Use of multivitamins and folic acid in early pregnancy and multiple births in Sweden , Twin Res , 2001 , vol. 4 (pg. 63 - 66 ) Google Scholar Crossref Search ADS PubMed WorldCat Eskes TK . Homocysteine and human reproduction , Clin Exp Obstet Gynecol , 2000 , vol. 27 (pg. 157 - 67 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Farag NH , Barshop BA , Mills PJ . Effects of estrogen and psychological stress on plasma homocysteine levels , Fertil Steril , 2003 , vol. 79 (pg. 256 - 60 ) Google Scholar Crossref Search ADS PubMed WorldCat Favier M , Faure P , Roussel AM , et al. Zinc deficiency and dietary folate metabolism in pregnant rats , J Trace Elem Electrolytes Health Dis , 1993 , vol. 7 (pg. 19 - 24 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Fenech M . The role of folic acid and Vitamin B12 in genomic stability of human cells , Mutat Res , 2001 , vol. 475 (pg. 57 - 67 ) Google Scholar Crossref Search ADS PubMed WorldCat Fowler B . The folate cycle and disease in humans , Kidney Int , 2001 , vol. 59 Suppl 78 (pg. S221 - 9 ) Google Scholar Crossref Search ADS WorldCat Fowler B . Homocysteine: overview of biochemistry, molecular biology, and role in disease processes , Semin Vasc Med , 2005 , vol. 5 (pg. 77 - 86 ) Google Scholar Crossref Search ADS PubMed WorldCat Friso S , Choi SW , Girelli D , et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status , Proc Natl Acad Sci USA , 2002 , vol. 99 (pg. 5606 - 11 ) Google Scholar Crossref Search ADS PubMed WorldCat Frosst P , Blom HJ , Milos R , et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase , Nat Genet , 1995 , vol. 10 (pg. 111 - 3 ) Google Scholar Crossref Search ADS PubMed WorldCat George L , Mills JL , Johansson AL , et al. Plasma folate levels and risk of spontaneous abortion , JAMA , 2002 , vol. 288 (pg. 1867 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Gmyrek GB , Sozanski R , Jerzak M , et al. Evaluation of monocyte chemotactic protein-1 levels in peripheral blood of infertile women with endometriosis , Eur J Obstet Gynecol Reprod Biol , 2005 , vol. 122 (pg. 199 - 205 ) Google Scholar Crossref Search ADS PubMed WorldCat Gris JC , Perneger TV , Quere I , et al. Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case-control study , Blood , 2003 , vol. 102 (pg. 3504 - 13 ) Google Scholar Crossref Search ADS PubMed WorldCat Gueant JL , Candito M , Andres E , et al. Familial pernicious anaemia with hyperhomocysteinaemia in recurrent early pregnancy loss , Thromb Haemost , 2004 , vol. 92 (pg. 1147 - 49 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Gueant JL , Gueant-Rodriguez RM , Anello G , et al. Genetic determinants of folate and vitamin B12 metabolism: a common pathway in neural tube defect and Down syndrome? , Clin Chem Lab Med , 2003 , vol. 41 (pg. 1473 - 7 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Gueant-Rodriguez RM , Gueant JL , Debard R , et al. Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations , Am J Clin Nutr , 2006 , vol. 83 (pg. 701 - 7 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Haggarty P , McCallum H , McBain H . Effect of B vitamins and genetics on success of in-vitro fertilisation: prospective cohort study , Lancet , 2006 , vol. 367 (pg. 1513 - 19 ) Google Scholar Crossref Search ADS PubMed WorldCat Hague WM . Homocysteine and pregnancy , Best Pract Res Clin Obstet Gynaecol , 2003 , vol. 17 (pg. 459 - 69 ) Google Scholar Crossref Search ADS PubMed WorldCat Hak AE , Polderman KH , Westendorp IC , et al. Increased plasma homocysteine after menopause , Atherosclerosis , 2000 , vol. 149 (pg. 163 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Hall JG. . Twinning , Lancet , 2003 , vol. 362 (pg. 735 - 43 ) Google Scholar Crossref Search ADS PubMed WorldCat Harmon DL , Woodside JV , Yarnell JW , et al. The common ‘thermolabile’ variant of methylene tetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia , QJM , 1996 , vol. 89 (pg. 571 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Hasbargen U , Lohse P , Thaler CJ . The number of dichorionic twin pregnancies is reduced by the common MTHFR 677C– > T mutation , Hum Reprod , 2000 , vol. 15 (pg. 2659 - 62 ) Google Scholar Crossref Search ADS PubMed WorldCat Henning BF , Tepel M , Riezler R , et al. Vitamin supplementation during weight reduction – favourable effect on homocysteine metabolism , Res Exp Med (Berl) , 1998 , vol. 198 (pg. 37 - 42 ) Google Scholar Crossref Search ADS PubMed WorldCat Herrero MB , de Lamirande E , Gagnon C . Nitric oxide is a signaling molecule in spermatozoa , Curr Pharm Des , 2003 , vol. 9 (pg. 419 - 25 ) Google Scholar Crossref Search ADS PubMed WorldCat Herrmann W . The importance of hyperhomocysteinemia as a risk factor for diseases: an overview , Clin Chem Lab Med , 2001 , vol. 39 (pg. 666 - 74 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Holm J , Hansen SI , Hoier-Madsen M . A high-affinity folate binding protein in human semen , Biosci Rep , 1991 , vol. 11 (pg. 237 - 42 ) Google Scholar Crossref Search ADS PubMed WorldCat Holm J , Hansen SI , Hoier-Madsen M , et al. Characterization of a high-affinity folate receptor in normal and malignant human testicular tissue , Biosci Rep , 1999 , vol. 19 (pg. 571 - 80 ) Google Scholar Crossref Search ADS PubMed WorldCat Hussein MR . Apoptosis in the ovary: molecular mechanisms , Hum Reprod Update , 2005 , vol. 11 (pg. 162 - 77 ) Google Scholar Crossref Search ADS PubMed WorldCat Isotalo PA , Wells GA , Donnelly JG . Neonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations , Am J Hum Genet , 2000 , vol. 67 (pg. 986 - 90 ) Google Scholar Crossref Search ADS PubMed WorldCat Jacques PF , Bostom AG , Williams RR , et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations , Circulation , 1996 , vol. 93 (pg. 7 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Jerzak M , Putowski L , Baranowski W . Homocysteine level in ovarian follicular fluid or serum as a predictor of successful fertilization , Ginekol Pol , 2003 , vol. 74 (pg. 949 - 52 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Kalla NR , Saggar SK , Puri R , et al. Regulation of male fertility by pyrimethamine in adult mice , Res Exp Med (Berl) , 1997 , vol. 197 (pg. 45 - 52 ) Google Scholar Crossref Search ADS PubMed WorldCat Kallen B . Use of folic acid supplementation and risk for dizygotic twinning , Early Hum Dev , 2004 , vol. 80 (pg. 143 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat Kapral A , Hyanek J , Zivny J , et al. Homocysteinemia and ovariectomy – initial experience with functional monitoring , Ceska Gyneko , 2002 , vol. 67 (pg. 328 - 32 ) OpenURL Placeholder Text WorldCat Kaufman RJ . Orchestrating the unfolded protein response in health and disease , J Clin Invest , 2002 , vol. 110 (pg. 1389 - 98 ) Google Scholar Crossref Search ADS PubMed WorldCat Kelly TL , Li E , Trasler JM . 5-aza-2′-deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome , J Androl , 2003 , vol. 24 (pg. 822 - 30 ) Google Scholar Crossref Search ADS PubMed WorldCat Kelly TL , Neaga OR , Schwahn BC , et al. Infertility in 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation , Biol Reprod , 2005 , vol. 72 (pg. 667 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Kilicdag EB , Bagis T , Tarim E , et al. Administration of B-group vitamins reduces circulating homocysteine in polycystic ovarian syndrome patients treated with metformin: a randomized trial , Hum Reprod , 2005a , vol. 20 (pg. 1521 - 8 ) Google Scholar Crossref Search ADS WorldCat Kilicdag EB , Bagis T , Zeyneloglu HB , et al. Homocysteine levels in women with polycystic ovary syndrome treated with metformin versus rosiglitazone: a randomized study , Hum Reprod , 2005b , vol. 20 (pg. 894 - 99 ) Google Scholar Crossref Search ADS WorldCat Kilic-Okman T , Guldiken S , Kucuk M . Relationship between homocysteine and insulin resistance in women with polycystic ovary syndrome , Endocr J , 2004 , vol. 51 (pg. 505 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Kucik J , Correa A . Trends in twinning rates in metropolitan Atlanta before and after folic acid fortification , J Reprod Med , 2004 , vol. 49 (pg. 707 - 12 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Landau B , Singer R , Klein T , et al. Folic acid levels in blood and seminal plasma of normo- and oligospermic patients prior and following folic acid treatment , Experientia , 1978 , vol. 34 (pg. 1301 - 2 ) Google Scholar Crossref Search ADS PubMed WorldCat Landy HJ , Keith LG . The vanishing twin: a review , Hum Reprod Update , 1998 , vol. 4 (pg. 177 - 83 ) Google Scholar Crossref Search ADS PubMed WorldCat Latacha KS , Rosenquist TH . Homocysteine inhibits extra-embryonic vascular development in the avian embryo , Dev Dyn , 2005 , vol. 234 (pg. 323 - 31 ) Google Scholar Crossref Search ADS PubMed WorldCat Lawrence de Koning AB , Werstuck GH , Zhou J . Hyperhomocysteinemia and its role in the development of atherosclerosis , Clin Biochem , 2003 , vol. 36 (pg. 431 - 41 ) Google Scholar Crossref Search ADS PubMed WorldCat Lawrence JM , Watkins ML , Chiu V , et al. Food fortification with folic acid and rate of multiple births, 1994–2000 , Birth Defects Res A Clin Mol Teratol , 2004 , vol. 70 (pg. 948 - 52 ) Google Scholar Crossref Search ADS PubMed WorldCat Lee NP , Cheng CY . Nitric oxide/nitric oxide synthase, spermatogenesis, and tight junction dynamics , Biol Reprod , 2004 , vol. 70 (pg. 267 - 76 ) Google Scholar Crossref Search ADS PubMed WorldCat Lee SJ , Kim KM , Namkoong S , et al. Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S -nitrosohomocysteine , J Biol Chem , 2005 , vol. 280 (pg. 5781 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Levy HL , Vargas JE , Waisbren SE , et al. Reproductive fitness in maternal homocystinuria due to cystathionine beta-synthase deficiency , J Inherit Metab Dis , 2002 , vol. 25 (pg. 299 - 314 ) Google Scholar Crossref Search ADS PubMed WorldCat Lewis SE , Aitken RJ . DNA damage to spermatozoa has impacts on fertilization and pregnancy , Cell Tissue Res , 2005 , vol. 322 (pg. 33 - 41 ) Google Scholar Crossref Search ADS PubMed WorldCat Li Z , Gindler J , Wang H . Folic acid supplements during early pregnancy and likelihood of multiple births: a population-based cohort study , Lancet , 2003 , vol. 361 (pg. 380 - 84 ) Google Scholar Crossref Search ADS PubMed WorldCat Lindenbaum J , Whitehead N , Reyner F . Oral contraceptive hormones, folate metabolism, and the cervical epithelium , Am J Clin Nutr , 1975 , vol. 28 (pg. 346 - 53 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Loverro G , Lorusso F , Mei L , et al. The plasma homocysteine levels are increased in polycystic ovary syndrome , Gynecol Obstet Invest , 2002 , vol. 53 (pg. 157 - 62 ) Google Scholar Crossref Search ADS PubMed WorldCat Lucock M . Folic acid: nutritional biochemistry, molecular biology, and role in disease processes , Mol Genet Metab , 2000 , vol. 71 (pg. 121 - 38 ) Google Scholar Crossref Search ADS PubMed WorldCat Lussana F , Zighetti ML , Bucciarelli P , et al. Blood levels of homocysteine, folate, vitamin B6 and B12 in women using oral contraceptives compared to non-users , Thromb Res , 2003 , vol. 112 (pg. 37 - 41 ) Google Scholar Crossref Search ADS PubMed WorldCat Malik NS , Matlin SA , Fried J , et al. The contraceptive effects of etoprine on male mice and rats , J Androl , 1995 , vol. 16 (pg. 169 - 74 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Malm J , Birn H , Frohm B , et al. A minor fraction of a high-affinity folate binding protein from the epididymis is associated with membranous vesicles and spermatozoa in human semen , Int J Androl , 2005 , vol. 28 (pg. 267 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Mann JR . Imprinting in the germ line , Stem Cells , 2001 , vol. 19 (pg. 287 - 94 ) Google Scholar Crossref Search ADS PubMed WorldCat Marques CJ , Carvalho F , Sousa M , et al. Genomic imprinting in disruptive spermatogenesis , Lancet , 2004 , vol. 363 (pg. 1700 - 2 ) Google Scholar Crossref Search ADS PubMed WorldCat Martinelli I , Taioli E , Ragni G , et al. Embryo implantation after assisted reproductive procedures and maternal thrombophilia , Haematologica , 2003 , vol. 88 (pg. 789 - 93 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Maul H , Longo M , Saade GR , et al. Nitric oxide and its role during pregnancy: from ovulation to delivery , Curr Pharm Des , 2003 , vol. 9 (pg. 359 - 80 ) Google Scholar Crossref Search ADS PubMed WorldCat Mayr CA , Ingersoll R , Wallock LM , et al. Folate levels and the effects of folate deficiency in the reproductive organs of male rats , FASEB J , 1999 , vol. 13 pg. A229 OpenURL Placeholder Text WorldCat Merki-Feld GS , Imthurn B , Keller PJ . Effects of two oral contraceptives on plasma levels of nitric oxide, homocysteine, and lipid metabolism , Metabolism , 2002 , vol. 51 (pg. 1216 - 21 ) Google Scholar Crossref Search ADS PubMed WorldCat Mijatovic V , van der Mooren MJ . Homocysteine in postmenopausal women and the importance of hormone replacement therapy , Clin Chem Lab Med , 2001 , vol. 39 (pg. 764 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Moger WH . Phospholipid methylation by intact rat Leydig cells , Biol Reprod , 1985 , vol. 33 (pg. 902 - 10 ) Google Scholar Crossref Search ADS PubMed WorldCat Mohanty D , Das KC . Effect of folate deficiency on the reproductive organs of female rhesus monkeys: a cytomorphological and cytokinetic study , J Nutr , 1982 , vol. 112 (pg. 1565 - 76 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Montgomery GW , Zhao ZZ , Morley KI , et al. Dizygotic twinning is not associated with methylenetetrahydrofolate reductase haplotypes , Hum Reprod , 2003 , vol. 18 (pg. 2460 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Mooij PN , Wouters MG , Thomas CM , et al. Disturbed reproductive performance in extreme folic acid deficient golden hamsters , Eur J Obstet Gynecol Reprod Biol , 1992 , vol. 43 (pg. 71 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Mudd SH . Vascular disease and homocysteine metabolism , N Engl J Med , 1985 , vol. 313 (pg. 751 - 3 ) Google Scholar Crossref Search ADS PubMed WorldCat Munoz-Moran E , Dieguez-Lucena JL , Fernandez-Arcas N , et al. Genetic selection and folate intake during pregnancy , Lancet , 1998 , vol. 352 (pg. 1120 - 1 ) Google Scholar Crossref Search ADS PubMed WorldCat Namour F , Olivier J , Abdelmouttaleb I , et al. Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood , Blood , 2001 , vol. 97 (pg. 1092 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Nelen WL . Hyperhomocysteinaemia and human reproduction , Clin Chem Lab Med , 2001 , vol. 39 (pg. 758 - 63 ) Google Scholar Crossref Search ADS PubMed WorldCat Nelen WL , Blom HJ , Steegers EA , et al. Hyperhomocysteinemia and recurrent early pregnancy loss: a meta-analysis , Fertil Steril , 2000 , vol. 74 (pg. 1196 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Orio F Jr , Palomba S , Di Biase S , et al. Homocysteine levels and C677T polymorphism of methylenetetrahydrofolate reductase in women with polycystic ovary syndrome , J Clin Endocrinol Metab , 2003 , vol. 88 (pg. 673 - 79 ) Google Scholar Crossref Search ADS PubMed WorldCat Ortega RM , Lopez-Sobaler AM , Andres P , et al. Changes in folate status in overweight/obese women following two different weight control programmes based on an increased consumption of vegetables or fortified breakfast cereals , Br J Nutr , 2006 , vol. 96 (pg. 712 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Outinen PA , Sood SK , Pfeifer SI , et al. Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells , Blood , 1999 , vol. 94 (pg. 959 - 67 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Papadopoulos V , Kamtchouing P , Drosdowsky MA , et al. Effects of the transmethylation inhibitor S -adenosyl-homocysteine and of the methyl donor S -adenosyl-methionine on rat Leydig cell function in vitro , J Steroid Biochem , 1987 , vol. 26 (pg. 93 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Paracchini V , Garte S , Taioli E . MTHFR C677T polymorphism, GSTM1 deletion and male infertility: a possible suggestion of a gene-gene interaction? , Biomarkers , 2006 , vol. 11 (pg. 53 - 60 ) Google Scholar Crossref Search ADS PubMed WorldCat Park JH , Lee HC , Jeong YM , et al. MTHFR C677T polymorphism associates with unexplained infertile male factors , J Assist Reprod Genet , 2005 , vol. 22 (pg. 361 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Peyrin-Biroulet L , Barraud H , Ancel D , et al. Folate metabolism and colorectal carcinogenesis , Gastroenterol Clin Biol , 2004 , vol. 28 (pg. 582 - 92 ) Google Scholar Crossref Search ADS PubMed WorldCat Poddar R , Sivasubramanian N , DiBello PM , et al. Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells: implications for vascular disease , Circulation , 2001 , vol. 103 (pg. 2717 - 23 ) Google Scholar Crossref Search ADS PubMed WorldCat van der Put NM , Gabreels F , Stevens EM , et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? , Am J Hum Genet , 1998 , vol. 62 (pg. 1044 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat Quere I , Bellet H , Hoffet M , et al. A woman with five consecutive fetal deaths: case report and retrospective analysis of hyperhomocysteinemia prevalence in 100 consecutive women with recurrent miscarriages , Fertil Steril , 1998 , vol. 69 (pg. 152 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Quere I , Mercier E , Bellet H , et al. Vitamin supplementation and pregnancy outcome in women with recurrent early pregnancy loss and hyperhomocysteinemia , Fertil Steril , 2001 , vol. 75 (pg. 823 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Raman R , Narayan G . 5-Aza deoxycytidine-induced inhibition of differentiation of spermatogonia into spermatocytes in the mouse , Mol Reprod Dev , 1995 , vol. 42 (pg. 284 - 90 ) Google Scholar Crossref Search ADS PubMed WorldCat Reik W , Walter J . Genomic imprinting: parental influence on the genome , Nat Rev Genet , 2001 , vol. 2 (pg. 21 - 2 ) Google Scholar Crossref Search ADS PubMed WorldCat Rey E , Kahn SR , David M . Thrombophilic disorders and fetal loss: a meta-analysis , Lancet , 2003 , vol. 361 (pg. 901 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Reyes-Engel A , Munoz E , Gaitan MJ . Implications on human fertility of the 677C– > T and 1298A– > C polymorphisms of the MTHFR gene: consequences of a possible genetic selection , Mol Hum Reprod , 2002 , vol. 8 (pg. 952 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Romerio SC , Linder L , Nyfeler J , et al. Acute hyperhomocysteinemia decreases NO bioavailability in healthy adults , Atherosclerosis , 2004 , vol. 176 (pg. 337 - 44 ) Google Scholar Crossref Search ADS PubMed WorldCat Roopnarinesingh R , Jackson B , Osman Z , et al. Homocysteine in assisted reproduction: does oestradiol influence homocysteine levels? , J Obstet Gynaecol , 2006 , vol. 26 (pg. 59 - 62 ) Google Scholar Crossref Search ADS PubMed WorldCat Rossato M . A genetic polymorphism and male fertility , Fertil Steril , 2004 , vol. 81 (pg. 1429 - 30 ) Google Scholar Crossref Search ADS PubMed WorldCat Rosselli M , Keller PJ , Dubey RK . Role of nitric oxide in the biology, physiology and pathophysiology of reproduction , Hum Reprod Update , 1998 , vol. 4 (pg. 3 - 24 ) Google Scholar Crossref Search ADS PubMed WorldCat Rousseaux S , Caron C , Govin J , et al. Establishment of male-specific epigenetic information , Gene , 2005 , vol. 345 (pg. 139 - 53 ) Google Scholar Crossref Search ADS PubMed WorldCat Said TM , Paasch U , Glander HJ , et al. Role of caspases in male infertility , Hum Reprod Update , 2004 , vol. 10 (pg. 39 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat Sanocka D , Jedrzejczak P , Szumala-Kaekol A , et al. Male genital tract inflammation: the role of selected interleukins in regulation of pro-oxidant and antioxidant enzymatic substances in seminal plasma , J Androl , 2003 , vol. 24 (pg. 448 - 55 ) Google Scholar Crossref Search ADS PubMed WorldCat Schachter M , Raziel A , Friedler S , Strassburger D , et al. Insulin resistance in patients with polycystic ovary syndrome is associated with elevated plasma homocysteine , Hum Reprod , 2003 , vol. 18 (pg. 721 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Scott JM , Weir DG . Folic acid, homocysteine and one-carbon metabolism: a review of the essential biochemistry , J Cardiovasc Risk , 1998 , vol. 5 (pg. 223 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Signore C , Mills JL , Cox C . Effects of folic acid fortification on twin gestation rates , Obstet Gynecol , 2005 , vol. 105 (pg. 757 - 62 ) Google Scholar Crossref Search ADS PubMed WorldCat Sills ES , Genton MG , Perloe M , et al. Plasma homocysteine, fasting insulin, and androgen patterns among women with polycystic ovaries and infertility , J Obstet Gynaecol Res , 2001 , vol. 27 (pg. 163 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Singh K , Singh SK , Sah R , et al. Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population , Int J Androl , 2005 , vol. 28 (pg. 115 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Stadtmauer L , Oehninger S . Management of infertility in women with polycystic ovary syndrome: a practical guide , Treat Endocrinol , 2005 , vol. 4 (pg. 279 - 92 ) Google Scholar Crossref Search ADS PubMed WorldCat Starkebaum G , Harlan JM . Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine , J Clin Invest , 1986 , vol. 77 (pg. 1370 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Steegers-Theunissen RP , Boers GH , Blom HJ , et al. Hyperhomocysteinaemia and recurrent spontaneous abortion or abruptio placentae , Lancet , 1992a , vol. 339 (pg. 1122 - 3 ) Google Scholar Crossref Search ADS WorldCat Steegers-Theunissen RP , Boers GH , Steegers EA . Effects of sub-50 oral contraceptives on homocysteine metabolism: a preliminary study , Contraception , 1992b , vol. 45 (pg. 129 - 39 ) Google Scholar Crossref Search ADS WorldCat Steegers-Theunissen RP , Steegers EA , Thomas CM , et al. Study on the presence of homocysteine in ovarian follicular fluid , Fertil Steril , 1993 , vol. 60 (pg. 1006 - 10 ) Google Scholar Crossref Search ADS PubMed WorldCat Steegers-Theunissen RP , Smith SC , Steegers EA , et al. Folate affects apoptosis in human trophoblastic cells , BJOG , 2000 , vol. 107 (pg. 1513 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Steegers-Theunissen RP , Van Iersel CA , Peer PG , et al. Hyperhomocysteinemia, pregnancy complications, and the timing of investigation , Obstet Gynecol , 2004 , vol. 104 (pg. 336 - 43 ) Google Scholar Crossref Search ADS PubMed WorldCat Stern LL , Mason JB , Selhub J , et al. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene , Cancer Epidemiol Biomarkers Prev , 2000 , vol. 9 (pg. 849 - 53 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Stuhlinger MC , Oka RK , Graf EE , et al. Endothelial dysfunction induced by hyperhomocyst(e)inemia: role of asymmetric dimethylarginine , Circulation , 2003 , vol. 108 (pg. 933 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Stuppia L , Gatta V , Scarciolla O , et al. The methylenetethrahydrofolate reductase (MTHFR) C677T polymorphism and male infertility in Italy , J Endocrinol Invest , 2003 , vol. 26 (pg. 620 - 2 ) Google Scholar Crossref Search ADS PubMed WorldCat Suhara T , Fukuo K , Yasuda O , et al. Homocysteine enhances endothelial apoptosis via upregulation of Fas-mediated pathways , Hypertension , 2004 , vol. 43 (pg. 1208 - 13 ) Google Scholar Crossref Search ADS PubMed WorldCat Szymanski W , Kazdepka-Zieminska A . Effect of homocysteine concentration in follicular fluid on a degree of oocyte maturity , Ginekol Pol , 2003 , vol. 74 (pg. 1392 - 6 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Tallova J , Tomandl J , Bicikova M , et al. Changes of plasma total homocysteine levels during the menstrual cycle , Eur J Clin Invest , 1999 , vol. 29 (pg. 1041 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Tamura T , Kaiser LL . The absorption of pteroylpolyglutamate and intestinal pteroylpolygammaglutamyl hydrolase activity in zinc-deficient rats , J Nutr , 1991 , vol. 121 (pg. 1042 - 6 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Tamura T , Picciano MF . Folate and human reproduction , Am J Clin Nutr , 2006 , vol. 83 (pg. 993 - 1016 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Thaler CD , Epel D . Nitric oxide in oocyte maturation, ovulation, fertilization, cleavage and implantation: a little dab'll do ya , Curr Pharm Des , 2003 , vol. 9 (pg. 399 - 409 ) Google Scholar Crossref Search ADS PubMed WorldCat Thaler CJ , Budiman H , Ruebsamen H , et al. Effects of the common 677 C > T mutation of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene on ovarian responsiveness to recombinant follicle-stimulating hormone , Am J Reprod Immunol , 2006 , vol. 55 (pg. 251 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Tsanadis G , Vartholomatos G , Korkontzelos I , et al. Polycystic ovarian syndrome and thrombophilia , Hum Reprod , 2002 , vol. 17 (pg. 314 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Ueland PM , Hustad S , Schneede J , et al. Biological and clinical implications of the MTHFR C677T polymorphism , Trends Pharmacol Sci , 2001 , vol. 22 (pg. 95 - 201 ) Google Scholar Crossref Search ADS WorldCat Vanselow J , Pohland R , Furbass R . Promoter-2-derived Cyp19 expression in bovine granulosa cells coincides with gene-specific DNA hypo-methylation , Mol Cell Endocrinol , 2005 , vol. 233 (pg. 57 - 64 ) Google Scholar Crossref Search ADS PubMed WorldCat Volek JS , Gomez AL , Love DM , et al. Effects of an 8-week weight-loss program on cardiovascular disease risk factors and regional body composition , Eur J Clin Nutr , 2002 , vol. 56 (pg. 585 - 92 ) Google Scholar Crossref Search ADS PubMed WorldCat Vollset SE , Gjessing HK , Tandberg A , et al. Folate supplementation and twin pregnancies , Epidemiology , 2005 , vol. 16 (pg. 201 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Vrbikova J , Bicikova M , Tallova J , et al. Homocysteine and steroids levels in metformin treated women with polycystic ovary syndrome , Exp Clin Endocrinol Diabetes , 2002 , vol. 110 (pg. 74 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Vrbikova J , Tallova J , Bicikova M , et al. Plasma thiols and androgen levels in polycystic ovary syndrome , Clin Chem Lab Med , 2003 , vol. 41 (pg. 216 - 21 ) Google Scholar Crossref Search ADS PubMed WorldCat Waller DK , Tita AT , Annegers JF . Rates of twinning before and after fortification of foods in the US with folic acid, Texas, 1996 to 1998 , Paediatr Perinat Epidemiol , 2003 , vol. 17 (pg. 378 - 83 ) Google Scholar Crossref Search ADS PubMed WorldCat Wallock LM , Tamura T , Mayr CA , et al. Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers , Fertil Steril , 2001 , vol. 75 (pg. 252 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Weiss N . Mechanisms of increased vascular oxidant stress in hyperhomocys-teinemia and its impact on endothelial function , Curr Drug Metab , 2005 , vol. 6 (pg. 27 - 36 ) Google Scholar Crossref Search ADS PubMed WorldCat Werler MM , Cragan JD , Wasserman CR , et al. Multivitamin supplementation and multiple births , Am J Med Genet , 1997 , vol. 71 (pg. 93 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Wijeyaratne CN , Nirantharakumar K , Balen AH , et al. Plasma homocysteine in polycystic ovary syndrome: does it correlate with insulin resistance and ethnicity? , Clin Endocrinol (Oxf) , 2004 , vol. 60 (pg. 560 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat Willmott M , Bartosik DB , Romanoff EB . The effect of folic acid on superovulation in the immature rat , J Endocrinol , 1968 , vol. 41 (pg. 439 - 45 ) Google Scholar Crossref Search ADS PubMed WorldCat Wong WY , Thomas CM , Merkus JM , et al. Male factor subfertility: possible causes and the impact of nutritional factors , Fertil Steril , 2000 , vol. 73 (pg. 435 - 42 ) Google Scholar Crossref Search ADS PubMed WorldCat Wong WY , Merkus HM , Thomas CM , et al. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial , Fertil Steril , 2002 , vol. 77 (pg. 491 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Wouters MG , Boers GH , Blom HJ , et al. Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss , Fertil Steril , 1993 , vol. 60 (pg. 820 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Wouters MG , Moorrees MT , van der Mooren MJ , et al. Plasma homocysteine and menopausal status , Eur J Clin Invest , 1995 , vol. 25 (pg. 801 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat Wulffele MG , Kooy A , Lehert P , et al. Effects of short-term treatment with metformin on serum concentrations of homocysteine, folate and vitamin B12 in type 2 diabetes mellitus: a randomized, placebo-controlled trial , J Intern Med , 2003 , vol. 254 (pg. 455 - 63 ) Google Scholar Crossref Search ADS PubMed WorldCat Xu C , Bailly-Maitre B , Reed JC . Endoplasmic reticulum stress: cell life and death decisions , J Clin Invest , 2005 , vol. 115 (pg. 2656 - 64 ) Google Scholar Crossref Search ADS PubMed WorldCat Yarali H , Yildirir A , Aybar F , et al. Diastolic dysfunction and increased serum homocysteine concentrations may contribute to increased cardiovascular risk in patients with polycystic ovary syndrome , Fertil Steril , 2001 , vol. 76 (pg. 511 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat Yilmaz M , Biri A , Bukan N , et al. Levels of lipoprotein and homocysteine in non-obese and obese patients with polycystic ovary syndrome , Gynecol Endocrinol , 2005 , vol. 20 (pg. 258 - 63 ) Google Scholar Crossref Search ADS PubMed WorldCat Zetterberg H . Methylenetetrahydrofolate reductase and transcobalamin genetic polymorphisms in human spontaneous abortion: biological and clinical implications , Reprod Biol Endocrinol , 2004 , vol. 2 (pg. 7 - 14 ) Google Scholar Crossref Search ADS PubMed WorldCat Zetterberg H , Regland B , Palmer M , et al. Increased frequency of combined methylenetetrahydrofolate reductase C677T and A1298C mutated alleles in spontaneously aborted embryos , Eur J Hum Genet , 2002 , vol. 10 (pg. 113 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Zetterberg H , Zafiropoulos A , Spandidos DA , et al. Gene–gene interaction between fetal MTHFR 677 C > T and transcobalamin 776 C > G polymorphisms in human spontaneous abortion , Hum Reprod , 2003 , vol. 18 (pg. 1948 - 50 ) Google Scholar Crossref Search ADS PubMed WorldCat Zhang X , Li H , Jin H , et al. Effects of homocysteine on endothelial nitric oxide production , Am J Physiol Renal Physiol , 2000 , vol. 279 (pg. F671 - 8 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Zhang C , Cai Y , Adachi MT , et al. Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response , J Biol Chem , 2001 , vol. 276 (pg. 35867 - 74 ) Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
TI - Impact of folate and homocysteine metabolism on human reproductive health
JF - Human Reproduction Update
DO - 10.1093/humupd/dml063
DA - 2007-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/impact-of-folate-and-homocysteine-metabolism-on-human-reproductive-aZfe93Q4D9
SP - 225
EP - 238
VL - 13
IS - 3
DP - DeepDyve
ER -