Vitamin B-12 and liver activity and expression of methionine synthase are decreased in fetuses with neural tube defects.

BACKGROUND: The risk of neural tube defects (NTDs) is influenced by nutritional factors and genetic determinants of one-carbon metabolism. A key pathway of this metabolism is the vitamin B-12- and folate-dependent remethylation of homocysteine, which depends on methionine synthase (MS, encoded by MTR), methionine synthase reductase, and methylenetetrahydrofolate reductase. Methionine, the product of this pathway, is the direct precursor of S-adenosylmethionine (SAM), the universal methyl donor needed for epigenetic mechanisms. OBJECTIVES: This study aimed to evaluate whether the availability of vitamin B-12 and folate and the expression or activity of the target enzymes of the remethylation pathway are involved in NTD risk. METHODS: We studied folate and vitamin B-12 concentrations and activity, expression, and gene variants of the 3 enzymes in liver from 14 NTD and 16 non-NTD fetuses. We replicated the main findings in cord blood from pregnancies of 41 NTD fetuses compared with 21 fetuses with polymalformations (metabolic and genetic findings) and 375 control pregnancies (genetic findings). RESULTS: The tissue concentration of vitamin B-12 (P = 0.003), but not folate, and the activity (P = 0.001), transcriptional level (P = 0.016), and protein expression (P = 0.003) of MS were decreased and the truncated inactive isoforms of MS were increased in NTD livers. SAM was significantly correlated with MS activity and vitamin B-12. A gene variant in exon 1 of GIF (Gastric Intrinsic Factor gene) was associated with a dramatic decrease of liver vitamin B-12 in 2 cases. We confirmed the decreased vitamin B-12 in cord blood from NTD pregnancies. A gene variant of GIF exon 3 was associated with NTD risk. CONCLUSIONS: The decreased vitamin B-12 in liver and cord blood and decreased expression and activity of MS in liver point out the impaired remethylation pathway as hallmarks associated with NTD risk. We suggest evaluating vitamin B-12 in the nutritional recommendations for prevention of NTD risk beside folate fortification or supplementation.

Fumonisin FB1 treatment acts synergistically with methyl donor deficiency during rat pregnancy to produce alterations of H3- and H4-histone methylation patterns in fetuses.

SCOPE: Prenatal folate and methyl donor malnutrition lead to epigenetic alterations that could enhance susceptibility to disease. Methyl-deficient diet (MDD) and fumonisin FB1 are risk factors for neural tube defects and cancers. Evidence indicates that FB1 impairs folate metabolism. METHODS AND RESULTS: Folate receptors and four heterochromatin markers were investigated in rat fetuses liver derived from dams exposed to MDD and/or FB1 administered at a dose twice higher than the provisional maximum tolerable daily intake (PMTDI = 2 µg/kg/day). Even though folate receptors transcription seemed up-regulated by methyl depletion regardless of FB1 treatment, combined MDD/FB1 exposure might reverse this up-regulation since folate receptors transcripts were lower in the MDD/FB1 versus MDD group. Methyl depletion decreased H4K20me3. Combined MDD/FB1 decreased H4K20me3 even more and increased H3K9me3. The elevated H3K9me3 can be viewed as a defense mechanism inciting the cell to resist heterochromatin disorganization. H3R2me2 and H4K16Ac varied according to this mechanism even though statistical significance was not consistent. CONCLUSION: Considering that humans are exposed to FB1 levels above the PMTDI, this study is relevant because it suggests that low doses of FB1 interact with MDD thus contributing to disrupt the epigenetic landscape.

A splicing variant leads to complete loss of function of betaine-homocysteine methyltransferase (BHMT) gene in hepatocellular carcinoma.

The remethylation of homocyteine into methionine is catalyzed either by methionine synthase (MTR) or by betaine-homocysteine methyltransferase (BHMT), in the liver. Choline/betaine deficiency and impaired BHMT pathway have been associated with hepatocellular carcinogenesis, in animal models. The molecular mechanisms that impair the BHMT pathway are unknown. We aimed to investigate BHMT, BHMT2, and MTR expression in HepG2 cells and human hepatocarcinoma tissues. Transcripts were quantified by RT-qPCR and splicing was assessed by analysis of exon junctions and sequencing of variants. Protein expression was studied by Western Blot, immunohistochemistry and enzyme activity. Tumor tissue was compared with surrounding healthy tissue. RT-qPCR of HepG2 cells and of tumor samples showed a strong decrease of transcripts of BHMT and BHMT2, compared to normal. MTR transcript levels were not different. The decreased BHMT expression resulted from the transcription of a splicing variant that produced a frameshift in exon 4, with a premature termination codon in exon 5 and a loss of function of the gene. This splicing variant did not fit with any mechanism resulting from known splicing consensus sequences and was not detected in normal adult and fetal liver. Consistently, BHMT activity was abolished in HepG2 and protein expression was not detectable in HepG2 and in 5 of the 6 tumor samples, compared to normal tissues. In conclusion, a transcription variant of exon 4 produces a loss of function of BHMT in human hepatocarcinoma. Whether this abnormal transcription of BHMT is part or consequence of liver carcinogenesis should deserve further investigations.

Ovarian tissue cryopreservation and subsequent spontaneous pregnancies in a patient with classic galactosemia.

OBJECTIVE: To report two consecutive spontaneous pregnancies in a compound heterozygous patient with classic galactosemia and a heterozygous partner, 6 years after ovarian tissue cryopreservation. DESIGN: Case report. SETTING: Tertiary health care center. PATIENT(S): A patient with classic galactosemia and strict adherance to a galactose-free diet. INTERVENTION(S): Right ovariectomy by laparoscopy and cryopreservation of cortical slices; metabolic follow-up. MAIN OUTCOME MEASURE(S): Genotyping, galactose-1-phosphate uridyltransferase (GALT) activity and erythrocyte galactose-1-phosphate determination, histology of ovarian cortex, pregnancy achievement. RESULT(S): Undetectable GALT activity; compound heterozygosity: association of c.563A>G (p.Gln188Arg) and a novel mutation c.982C>T (p.Arg328Cys); rare growing follicles and abnormally low primordial follicles; two uneventful spontaneous pregnancies without need for autografting of the cryopreserved tissue. CONCLUSION(S): The risk for ovarian failure is a frequent concern, but spontaneous pregnancies may occur, even repeatedly, in young patients with galactosemia. Thus, there is a need for more accurate predictive factors to guide the indication for ovarian tissue cryopreservation, the benefits and risks of which have to be balanced through a multidisciplinary approach.

Life-threatening methylenetetrahydrofolate reductase (MTHFR) deficiency with extremely early onset: characterization of two novel mutations in compound heterozygous patients.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzymatic component of the folate cycle, converting 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, the methyl donor for remethylation of homocysteine into methionine. Severe MTHFR deficiency is a rare recessive disease leading to major hyperhomocysteinemia, homocystinuria, and progressive neurological distress within the two first decades of life. More than 50 mutations have been reported so far in affected patients but only a few cases with very early onset of symptoms during the first weeks have been described, most of them showing a particular severe clinical course. We detected two novel mutations by direct sequencing of MTHFR in compound heterozygous patients with extremely low or undetectable enzyme activity; one of them had clinical onset during the first week of life and fatal issue at the age of six weeks. Prenatal diagnosis of his sibling allowed for early treatment with B vitamins and betaine and a favorable outcome. One of these mutations (c.523G>A) led to an Ala>Thr transition in the catalytic domain of the enzyme, the other (c.1166G>A) induced alternative splicing of exon 7 at the junction of the catalytic and regulatory domains. Both parents carried only one of these mutations and presented with moderate and intermediate hyperhomocysteinemia, respectively, without neurological symptoms. Severe MTHFR deficiency thus has to be taken into consideration when investigating neurological distress even in the newborn, regarding the need for an earliest possible treatment. Characterization of the relatives further allows for preventive measure to limit the risks of chronic hyperhomocysteinemia.

Local lymphocytic and epithelial activation in a case of autoimmune oophoritis.

OBJECTIVE: To further define the immunological tissular modifications in premature ovarian failure (POF). METHOD: The patient was followed up for premature ovarian failure and mild endometriosis associated with serum antiovarian antibodies. A laparoscopic ovarian biopsy was decided on to analyze the tissue and support the onset of immunosuppressive therapy. Immunohistochemistry was performed using monoclonal antibodies directed against T cell membrane markers, as well as activation molecules, to define the composition of the cellular infiltrate and the consequences on ovarian tissue. RESULT(S): A dense infiltration of activated T lymphocytes was observed in close contact with follicular epithelium expressing HLA-DR and CD40. CONCLUSION(S): This observation supports the role of cellular immunity in ovarian autoimmunity with features very similar to those reported in murine models and other human autoimmune endocrine pathologies.

Impact of folate and homocysteine metabolism on human reproductive health.

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.

Corticosteroids in patients with antiovarian antibodies undergoing in vitro fertilization: a prospective pilot study.

OBJECTIVE: Antiovarian autoantibodies (AOA) have been associated with reproductive failure, especially in in vitro fertilization (IVF) patients. Thus, the success rate of IVF might be improved by the use of corticosteroids. However, therapeutic trials with these drugs have yielded conflicting results, particularly because of heterogeneous inclusion criteria. Among women with previous IVF failure, we selected those who presented with a positive serum AOA assay, and analysed the efficacy of corticosteroids in improving the IVF outcome in these patients. METHODS: One hundred patients with serum AOA detected by ELISA and at least two previously failed IVF attempts were selected. These patients underwent a further IVF cycle with 0.5 mg/kg prednisolone, started on the first day of the treatment cycle. In patients who became pregnant, corticosteroids were administered until the end of the first trimester of pregnancy and then progressively discontinued. AOA were assessed before and after oocyte retrieval. Clinical data of the corticosteroid-treated cycle were compared with data from the preceding IVF cycle for each patient. RESULTS: No adverse effects resulting from corticosteroids were observed. Post oocyte retrieval antiovarian IgG were significantly lower in corticosteroid-treated attempts when compared with the preceding cycles. Twenty-six pregnancies resulted in the birth of 30 healthy children. The pregnancy rate, implantation rate, and live birth rate were 38.8%, 17.8%, and 26.5% respectively in prednisolone-treated cycles. CONCLUSION: This study confirms the usefulness of corticosteroids in improving the success rate in a subset of patients with previous IVF failure and significant serum AOA levels.

The influence of cigarette smoking on human sperm quality and DNA fragmentation.

The aim of the present study was to evaluate consequences of cigarette smoking on male gametes. In this prospective study, sperm parameters such as sperm density, motility, viability and normal morphology were measured according to the WHO criteria. In addition to these standard parameters, we analysed the degree of DNA fragmentation in spermatozoa using the TUNEL-assay with flow cytometry detection in 57 non-smokers and 51 smokers seeking for infertility counselling. The smoking intoxication was assessed by questionnaire and measured with the CO-Tester. We show that smokers' spermatozoa have a significantly higher DNA fragmentation than non-smokers (32% versus 25.9%, p<0.01). In contrast there is no significant difference in conventional parameters between smokers and non-smokers. The degree of sperm DNA fragmentation is not significantly correlated with any of the conventional parameters. These findings suggest that cigarette smoking may have deleterious effects on sperm nuclear quality and that sperm DNA fragmentation can therefore be considered as an independent parameter with diagnostic, prognostic, and strategic value in the treatment of infertility.

Gonadal antibodies interfering with female reproduction.

While the involvement of anti-ovarian antibodies (AOAs) is highly likely, yet still controversial, in patients with patent premature ovarian failure (POF), it is even more difficult--for several reasons--to ascertain the clinical significance of these antibodies in patients without obvious ovarian failure. First, AOAs form a heterogeneous group of antibodies recognizing several different antigenic targets such as granulosa and thecal cells, zona pellucida, oocyte cytoplasm, corpus luteum, as well as gonadotrophins and their receptors. Second, the detection of AOAs in various clinical situations does not readily imply a causal relationship between these antibodies and impaired ovarian function. Third, diagnostic tools for detecting AOAs and their molecular targets have to be improved to yield more reliable data and allow a better comprehension of the pathophysiology of AOAs. Preliminary results with immunosuppressive therapy in selected AOA patients have been encouraging, but randomized trials have to be performed.