The importance of preconception Hcy testing: identification of a folate trap syndrome in a woman attending an assisted reproduction program.

Methylation is a ubiquitous and permanent key biochemical process playing a major role in gametogenesis and embryogenesis in relation to epigenetics and imprinting. Methylation relies on a unique cofactor S-Adenosyl Methionine: SAM. Release of the methyl group onto target molecules is followed by liberation of S-Adenosyl Homocysteine (SAH), and then homocysteine (Hcy), both potent inhibitors of the methylation process. Defective recycling of homocysteine, leading to Hyperhomocysteinemia, is mainly due to reduced activity of MTHFR (Methylene TetraHydroFolate Reductase). However, we described here, in a woman attending an ART program, a rather rare syndrome: The Folate trap syndrome. Due to vitamin B12 deficiency (malabsorption), Hcy cannot be recycled to methionine by the methionine synthase. Transmethylation activity is weak and leads to Hhcy (Hyperhomocysteinhemia). Her Hhcy, over 16µM, was resistant to 5MTHF (5 Methyltetrahydrofolate) associated with a support of the one carbon cycle, a classical efficient treatment for elevated homocysteine. Treatment with Methylcobalamine (associated with adenosyl Cobalamine) allowed a Hcy drop down to 10 µM. Knowing the pleiotropic negative impact of Hcy on gametes, embryos and pregnancy in general, we strongly recommend a Hcy dosage in both members of couples seeking treatment for pregnancy.

Hyperhomocysteinemia in hypofertile male patients can be alleviated by supplementation with 5MTHF associated with one carbon cycle support.

Introduction: Homocysteine (Hcy) is a cellular poison, side product of the hydrolysis of S-Adenosyl Homocysteine, produced after the universal methylation effector S -Adenosylmethionine liberates a methyl group to recipient targets. It inhibits the methylation processes and its rising is associated with multiple disease states and ultimately is both a cause and a consequence of oxidative stress, affecting male gametogenesis. We have determined hyper homocysteinhemia (HHcy) levels can be reliably reduced in hypofertile patients in order to decrease/avoid associated epigenetic problems and protect the health of future children, in consideration of the fact that treatment with high doses of folic acid is inappropriate. Methods: Homocysteine levels were screened in male patients consulting for long-standing infertility associated with at least three failed Assisted Reproductive Technology (ART) attempts and/or repeat miscarriages. Seventy-seven patients with Hcy levels > 15 µM were treated for three months with a combination of micronutrients including 5- MethylTetraHydroFolate (5-MTHF), the compound downstream to the MTHFR enzyme, to support the one carbon cycle; re-testing was performed at the end of a 3 months treatment period. Genetic status for Methylenetetrahydrofolate Reductase (MTHFR) Single nucleotide polymorphisms (SNPs) 677CT (c.6777C > T) and 1298AC (c.1298A > C) was determined. Results: Micronutrients/5-MTHF were highly efficient in decreasing circulating Hcy, from averages 27.4 to 10.7 µM, with a mean observed decrease of 16.7 µM. The MTHFR SNP 677TT (homozygous form) and combined heterozygous 677CT/1298AC status represent 77.9% of the patients with elevated Hcy. Discussion: Estimation HHcy should not be overlooked in men suffering infertility of long duration. MTHFR SNPs, especially 677TT, are a major cause of high homocysteinhemia (HHcy). In these hypofertile patients, treatment with micronutrients including 5-MTHF reduces Hcy and even allows spontaneous pregnancies post treatment. This type of therapy should be considered in order to ensure these patients' quality of life and avoid future epigenetic problems in their descendants.

Metabolic enhancement of the one carbon metabolism (OCM) in bovine oocytes IVM increases the blastocyst rate: evidences for a OCM checkpoint.

The one carbon metabolism (OCM) has a primary role in the process of oocyte maturation. In this study bovine oocytes were cultured for 24 h, up to MII stage, with standard medium supplemented or not with 8 metabolic enhancers of the OCM and the MII and blastocyst rate were compared. Additional analyses were performed on matured oocytes, cumulus cells, zygotes and blastocysts. The OCM supplementation increased the blastocyst rate derived from in vitro fertilization. The mitochondrial mass and DNMT3a protein expression were increased whereas DNA fragmentation decreased in matured oocytes. DNA methylation in female pronucleus of zygotes was increased. The supplementation did not directly affect the redox balance as ROS and GSH in matured oocytes and homocysteine in the spent medium were unchanged. The supplementation of the oocytes with metabolic enhancers of the OCM may increase the yield from the culture, likely due to improved DNA methylation and epigenetic programming. The lack of effects on MII rate with huge differences appearing at the blastocyst stage suggest the existence of a OCM metabolic check point that hampers oocytes progression to blastocyst post-fertilization, if they were not properly primed at the time of maturation.

MTHFR SNPs (Methyl Tetrahydrofolate Reductase, Single Nucleotide Polymorphisms) C677T and A1298C Prevalence and Serum Homocysteine Levels in >2100 Hypofertile Caucasian Male Patients

Methylation is a crucially important ubiquitous biochemical process, which covalently adds methyl groups to a variety of molecular targets. It is the key regulatory process that determines the acquisition of imprinting and epigenetic marks during gametogenesis. Methylation processes are dependent upon two metabolic cycles, the folates and the one-carbon cycles. The activity of these two cycles is compromised by single nucleotide polymorphisms (SNPs) in the gene encoding the Methylenetetrahydrofolate reductase (MTHFR) enzyme. These SNPs affect spermatogenesis and oocyte maturation, creating cytologic/chromosomal anomalies. The two main MTHFR SNP variants C677T (c.6777C>T) and A1298C (c.1298A>C) together with serum homocysteine levels were tested in men with >3 years' duration of infertility who had failed several ART attempts with the same partner. These patients are often classified as having "idiopathic infertility". We observed that the genetic status with highest prevalence in this group is the heterozygous C677T, followed by the combined heterozygous C677T/A1298C, and then A1298C; these three variants represent 65% of our population. Only 13.1% of the patients tested are wild type (WT), C677C/A1298A). The homozygous 677TT and the combined heterozygote 677CT/1298AC groups have the highest percentage of patients with an elevated circulating homocysteine level of >15 µMolar (57.8% and 18.8%, respectively, which is highly significant for both). Elevated homocysteine is known to be detrimental to spermatogenesis, and the population with this parameter is not marginal. In conclusion, determination of these two SNPs and serum homocysteine should not be overlooked for patients with severe infertility of long duration, including those with repeated miscarriages. Patients must also be informed about pleiotropic medical implications relevant to their own health, as well as to the health of future children.

Biochemical Hazards during Three Phases of Assisted Reproductive Technology: Repercussions Associated with Epigenesis and Imprinting.

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques involved may have an impact on crucial stages of early embryo and fetal development highly vulnerable to epigenetic influence. In this paper, the key role of methylation processes in epigenesis, namely the essential biochemical/metabolic pathways involving folates and one-carbon cycles necessary for correct DNA/histone methylation, is discussed. Furthermore, potential contributors to epigenetics dysregulation during the three phases of assisted reproduction: preparation for and controlled ovarian hyperstimulation (COH); methylation processes during the preimplantation embryo culture stages; the effects of unmetabolized folic acid (UMFA) during embryogenesis on imprinting methyl "tags", are described. Advances in technology have opened a window into developmental processes that were previously inaccessible to research: it is now clear that ART procedures have the potential to influence DNA methylation in embryonic and fetal life, with an impact on health and disease risk in future generations. Critical re-evaluation of protocols and procedures is now an urgent priority, with a focus on interventions targeted toward improving ART procedures, with special attention to in vitro culture protocols and the effects of excessive folic acid intake.

T677T Methylenetetrahydrofolate Reductase Single Nucleotide Polymorphisms Increased Prevalence in a Subgroup of Infertile Patients with Endometriosis.

Background: Approximately 10% (190 million) of women worldwide are affected by endometriosis, ectopic deposits of endometrial tissue that create a major source of pain that affects lifestyle and reproductive function. The pathogenesis of endometriosis is an estrogen-dependent inflammatory process, influenced/catalyzed by oxidative stress and consequently defective methylation, with biochemical features centered around the folate and one-carbon cycles. We aimed to determine whether a link could be found between the two major methylenetetrahydrofolate reductase single nucleotide polymorphisms (MTHFR SNPs), c.677C>T and c.1298A>C, involved in methylation process/epigenetic marking failures, and endometriosis. Material and Methods: We studied a population of 158 patients in a group of >1500 referred for treatment of infertility. All the patients had experienced >2 failed assisted reproductive technology cycles and/or >2 miscarriages, a classical cohort for investigation in our group. Patients with endometriosis had at least stage 2+ disease confirmed by laparoscopy. Results: The prevalence of the homozygous c.677C>T isoform is doubled in the endometriosis group, 21.5% versus 10.2% in the non-endometriosis group (p > 0.01). Symmetrically, the percentage of patients in the endometriosis group with the wild type MTHFR significantly decreased by one-half (8.2%-17.2%) in the non-endometriosis group (p < 0.001). Conclusion: Determination of MTHFR c.677C>T should not be overlooked in patients with harmful endometriosis affecting their fertility. As folates metabolism is impaired in these MTHFR SNPs carrier patients, co-treatment with 5-methyl folate may constitute a successful (co)-treatment modality.

Folic Acid, Folinic Acid, 5 Methyl TetraHydroFolate Supplementation for Mutations That Affect Epigenesis through the Folate and One-Carbon Cycles.

Methylation is an essential biochemical mechanism that is central to the transmission of life, and crucially responsible for regulating gametogenesis and continued embryo development. The methylation of DNA and histones drives cell division and regulation of gene expression through epigenesis and imprinting. Brain development and its maturation also depend on correct lipid methylation, and continued neuronal function depends on biogenic amines that require methylation for their synthesis. All methylation processes are carried out via a methyltransferase enzyme and its unique co-factor S-adenosylmethionine (SAM); the transfer of a methyl group to a target molecule results in the release of SAH (SA homocysteine), and then homocysteine (Hcy). Both of these molecules are toxic, inhibiting methylation in a variety of ways, and Hcy recycling to methionine is imperative; this is achieved via the one carbon cycle, supported by the folates cycle. Folate deficiency causes hyperhomocysteinaemia, with several associated diseases; during early pregnancy, deficiency interferes with closure of the neural tube at the fourth week of gestation, and nutraceutical supplementation has been routinely prescribed to prevent neural tube defects, mainly involving B vitamins, Zn and folates. The two metabolic pathways are subject to single nucleotide polymorphisms that alter their activity/capacity, often severely, impairing specific physiological functions including fertility, brain and cardiac function. The impact of three types of nutraceutical supplements, folic acid (FA), folinic acid (FLA) and 5 Methyl THF (MTHF), will be discussed here, with their positive effects alongside potentially hazardous secondary effects. The issue surrounding FA and its association with UMFA (unmetabolized folic acid) syndrome is now a matter of concern, as UMFA is currently found in the umbilical cord of the fetus, and even in infants' blood. We will discuss its putative role in influencing the acquisition of epigenetic marks in the germline, acquired during embryogenesis, as well as the role of FA in the management of cancerous disease.

Modulating oxidative stress and epigenetic homeostasis in preimplantation IVF embryos.

Assisted reproductive technology is today considered a safe and reliable medical intervention, with healthy live births a reality for many IVF and ICSI treatment cycles. However, there are increasing numbers of published reports describing epigenetic/imprinting anomalies in children born as a result of these procedures. These anomalies have been attributed to methylation errors in embryo chromatin remodelling during in vitro culture. Here we re-visit three concepts: (1) the so-called 'in vitro toxicity' of 'essential amino acids' before the maternal to zygotic transition period; (2) the effect of hyperstimulation (controlled ovarian hyperstimulation) on homocysteine in the oocyte environment and the effect on methylation in the absence of essential amino acids; and (3) the fact/postulate that during the early stages of development the embryo undergoes a 'global' demethylation. Methylation processes require efficient protection against oxidative stress, which jeopardizes the correct acquisition of methylation marks as well as subsequent methylation maintenance. The universal precursor of methylation [by S-adenosyl methionine (SAM)], methionine, 'an essential amino acid', should be present in the culture. Polyamines, regulators of methylation, require SAM and arginine for their syntheses. Cystine, another 'semi-essential amino acid', is the precursor of the universal protective antioxidant molecule: glutathione. It protects methylation marks against some undue DNA demethylation processes through ten-eleven translocation (TET), after formation of hydroxymethyl cytosine. Early embryos are unable to convert homocysteine to cysteine as the cystathionine ß-synthase pathway is not active. In this way, cysteine is a 'real essential amino acid'. Most IVF culture medium do not maintain methylation/epigenetic processes, even in mouse assays. Essential amino acids should be present in human IVF medium to maintain adequate epigenetic marking in preimplantation embryos. Furthermore, morphological and morphometric data need to be re-evaluated, taking into account the basic biochemical processes involved in early life.

MTHFR (methylenetetrahydrofolate reductase: EC 1.5.1.20) SNPs (single-nucleotide polymorphisms) and homocysteine in patients referred for investigation of fertility.

PURPOSE: MTHFR, one of the major enzymes in the folate cycle, is known to acquire single-nucleotide polymorphisms that significantly reduce its activity, resulting in an increase in circulating homocysteine. Methylation processes are of crucial importance in gametogenesis, involved in the regulation of imprinting and epigenetic tags on DNA and histones. We have retrospectively assessed the prevalence of MTHFR SNPs in a population consulting for infertility according to gender and studied the impact of the mutations on circulating homocysteine levels. METHODS: More than 2900 patients having suffered at least two miscarriages (2 to 9) or two failed IVF/ICSI (2 to 10) attempts were included for analysis of MTHFR SNPs C677T and A1298C. Serum homocysteine levels were measured simultaneously. RESULTS: We observed no difference in the prevalence of different genetic backgrounds between men and women; only 15% of the patients were found to be wild type. More than 40% of the patients are either homozygous for one SNP or compound heterozygous carriers. As expected, the C677T SNP shows the greatest adverse effect on homocysteine accumulation. The impact of MTHFR SNPs on circulating homocysteine is different in men than in women. CONCLUSIONS: Determination of MTHFR SNPs in both men and women must be seriously advocated in the presence of long-standing infertility; male gametes, from MTHFR SNPs carriers, are not exempted from exerting a hazardous impact on fertility. Patients should be informed of the pleiotropic medical implications of these SNPs for their own health, as well as for the health of future children.

Methylation: An Ineluctable Biochemical and Physiological Process Essential to the Transmission of Life.

Methylation is a universal biochemical process which covalently adds methyl groups to a variety of molecular targets. It plays a critical role in two major global regulatory mechanisms, epigenetic modifications and imprinting, via methyl tagging on histones and DNA. During reproduction, the two genomes that unite to create a new individual are complementary but not equivalent. Methylation determines the complementary regulatory characteristics of male and female genomes. DNA methylation is executed by methyltransferases that transfer a methyl group from S-adenosylmethionine, the universal methyl donor, to cytosine residues of CG (also designated CpG). Histones are methylated mainly on lysine and arginine residues. The methylation processes regulate the main steps in reproductive physiology: gametogenesis, and early and late embryo development. A focus will be made on the impact of assisted reproductive technology and on the impact of endocrine disruptors (EDCs) via generation of oxidative stress.

5-Methyltetrahydrofolate reduces blood homocysteine level significantly in C677T methyltetrahydrofolate reductase single-nucleotide polymorphism carriers consulting for infertility.

PURPOSE: Methyltetrahydrofolate reductase (MTHFR) C677T (ala222Val) is a single-nucleotide polymorphism (SNP) that affects the formation of 5-methyltetrahydrofolate (5-MTHF), the active folate that allows the recycling of homocysteine (Hcy) to Methionine. Hcy is at the epicentre of oxidative stress and DNA methylation errors. This SNP often increases the circulating Hcy levels and consequently reduces the methylation process, which is involved in the epigenesis and imprinting of markings in gametes. This study aimed to investigate decreases in Hcy levels in MTHFR SNP carriers. PROCEDURE: Eighty-nine couples with fertility problems for at least 3 years were included in this program. The women were systematically tested for the MTHFR C 677T isoform. If the woman tested positive, testing of the male partner was proposed. All the carriers had well-controlled blood Hcy levels before and after treatment (600µg of 5-MTHF/day, with a backup of one carbon cycle during at least 3 months). FINDINGS: As expected, the circulating Hcy level was higher in the homozygous patients than in the heterozygous and wild-type patients. The treatments caused a significant decrease of the circulating Hcy in the SNP carriers group. CONCLUSIONS: Couples with a long history of infertility should be analysed for MTHFR SNP and homocysteine and should be treated with physiological doses of 5-MTHF instead of high doses of folic acid.

High doses of folic acid induce a pseudo-methylenetetrahydrofolate syndrome.

A 41-year-old Caucasian woman with a history of infertility dating from 2011 was identified as wild-type (no mutations) for methylenetetrahydrofolate reductase single nucleotide polymorphisms (MTHFR-SNPs). Previous treatment included three failed in vitro fertilization/intracytoplasmic sperm injection cycles as well as one failed cycle of in vitro fertilization/intracytoplasmic sperm injection with donated oocytes. Counseling for a further oocyte donation cycle included advice to take high doses of folic acid (5 mG per day). Prior to initiation of this cycle, in October 2017 she attended our unit for general gynecological assessment and was found to have a slightly increased level of homocysteine, 12.2 µmol/L. A further test in February 2018 showed an increase to 17.2 µmol/L. Folic acid was stopped, and she was treated with 5-MTHF (500 µG daily), which supports the one-carbon cycle. After 5 days of treatment, her homocysteine level dropped to a baseline level of 8.2 µmol/L. As previously described in mice, high doses of folic acid can induce a "pseudo MTHFR" syndrome in wild-type patients, leading to an elevated unmetabolized folic acid syndrome which results in increased serum levels of homocysteine.

DNA Methylation Patterns in the Early Human Embryo and the Epigenetic/Imprinting Problems: A Plea for a More Careful Approach to Human Assisted Reproductive Technology (ART).

An increasing number of publications indicate that babies born after IVF (in vitro fertilization) procedures have higher rates of anomalies related to imprinting/epigenetic changes, which may be attributed to suboptimal culture conditions. Appropriate maintenance of DNA methylation during the first few days of an in vitro culture requires a supply of methyl donors, which are lacking in current in vitro culture systems. The absence of protection against oxidative stress in the culture increases the risks for errors in methylation. A decrease in the methylation processes is sometimes observed immediately post fertilization, due to delays that occur during the maternal⁻zygotic transition period. Care should be exercised in ART (assisted reproductive technology) procedures in order to avoid the risk of generating errors in methylation during the in vitro culture period immediately post fertilization, which has an impact on imprinting/epigenetics. Formulation of IVF culture media needs to be re-assessed in the perspective of current knowledge regarding embryo physiology.