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.

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.

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.

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.

The negative impact of the environment on methylation/epigenetic marking in gametes and embryos: A plea for action to protect the fertility of future generations.

Life expectancy has increased since World War II, and this may be attributed to several aspects of modern lifestyles. However, now we are faced with a downturn, which seems to be the result of environmental issues. This paradigm is paralleled with reduced human fertility, decreased sperm quality, increased premature ovarian failure, and diminished ovarian reserve syndromes. Endocrine disruptor chemicals and other toxic chemicals, herbicides, pesticides, plasticizers, to mention a few, are a rising concern in today's environment. Some of these are commonly used in the domestic setting: cleaning material and cosmetics and they have a known impact on epigenesis and imprinting via perturbation of methylation processes. Pollution from polyaromatic hydrocarbons, particulate matter <10 and <2.5 µm, and ozone released into the air, all affect fertility. Poor food processing management is a source of DNA adduct formation, which impairs the quality of gametes. An important concern is the nanoparticles that are present in food and are thought to induce oxidative stress. Now is the time to take a step backward. Global management of the environment and food production is required urgently to protect the fertility of future generations.

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.

MTHFR isoform carriers. 5-MTHF (5-methyl tetrahydrofolate) vs folic acid: a key to pregnancy outcome: a case series.

PURPOSE: To evaluate the possibility of correcting metabolic defects in gametes and embryos due to methylene tetra hydrofolate reductase (MTHFR) isoforms C677T and A1298C, by supplementation with 5-methyl THF instead of synthetic folic acid. In these couples, high doses of folic acid lead to UMFA (un-metabolized folic acid) syndrome. METHODS: Thirty couples with fertility problems lasting for at least 4 years, such as recurrent fetal loss, premature ovarian insufficiency, or abnormal sperm parameters, with two thirds of them having failed assisted reproductive technology (ART) attempts were included in this program. For all couples, at least one of the partners was a carrier of one of the two main MTHFR isoforms. Most of the women had been previously treated unsuccessfully with high doses of folic acid (5 mg/day), according to what is currently proposed in the literature. The couples carrying one of the isoforms were treated for 4 months with 5-MTHF, at a dose of 600 micrograms per day, before attempting conception or starting another attempt at ART. The duration of treatment corresponding to an entire cycle of spermatogenesis is approximately 74 days. RESULTS: In this first series of 33 couples, one couple was not followed-up, and two are still currently under treatment. No adverse effects were observed. Thirteen of the couples conceived spontaneously, the rest needing ART treatment in order to achieve pregnancy. Only three couples have, so far, not succeeded. CONCLUSION: The conventional use of large doses of folic acid (5 mg/day) has become obsolete. Regular doses of folic acid (100-200 µg) can be tolerated in the general population but should be abandoned in the presence of MTHFR mutations, as the biochemical/genetic background of the patient precludes a correct supply of 5-MTHF, the active compound. A physiological dose of 5-MTHF (800 µg) bypasses the MTHFR block and is suggested to be an effective treatment for these couples. Moreover, it avoids potential adverse effects of the UMFA syndrome, which is suspected of causing immune dysfunction and other adverse pathological effects such as cancer (especially colorectal and prostate).

Association between the MTHFR-C677T isoform and structure of sperm DNA.

PURPOSE: The aim of this study is to evaluate whether the MTHFR contribution to male decreased fertility can be attributable to anomalies in sperm nucleus DNA structure in relation to defective methylation. METHODS: The presence of MTHFR C677T, contributing at most for male infertility, was determined from a venous blood sample, using real-time polymerase chain reaction (PCR). Sperm DNA fragmentation (SDF) and sperm nucleus decondensation index (SDI) measurements were performed using acridine orange and flow cytometry. SDF and SDI of men MTHFR C677T heterozygous or homozygous were compared to a general population of hypo-fertile patients RESULTS: SDF is not increased either in homozygous or heterozygous carriers of MTHFR C677T. In contrast, SDI is increased with a higher incidence in homozygous (p = 0.0006) than in heterozygous (p = 0.029) patients when compared with the control population. Using a critical threshold of 20% for either SDI or SDF assayed with our technique, the percentage of patients with results higher than this value is not significant with respect to fragmentation (0.128), but is significantly increased for decondensation (0.0003). CONCLUSIONS: Defective methylation linked to MTHFR may contribute to sperm pathogenesis via increased SDI. After DNA structure analysis, especially SDI, treatment with 5-methyl tetrahydrofolate (MTHF), the metabolite downstream from the action of MTHFR, should be recommended as a therapeutic approach. Patients with a high SDI should be tested for MTHFR isoforms as part of a healthcare policy.

Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction.

The negative effect of oxidative stress on the human reproductive process is no longer a matter for debate. Oxidative stress affects female and male gametes and the developmental capacity of embryos. Its effect can continue through late stages of pregnancy. Metabolic disorders and psychiatric problems can also be caued by DNA methylation and epigenetic errors. Age has a negative effect on oxidative stress and DNA methylation, and recent observations suggest that older men are at risk of transmitting epigenetic disorders to their offspring. Environmental endocrine disruptors can also increase oxidative stress and methylation errors. Oxidative stress and DNA methylation feature a common denominator: the one carbon cycle. This important metabolic pathway stimulates glutathione synthesis and recycles homocysteine, a molecule that interferes with the process of methylation. Glutathione plays a pivotal role during oocyte activation, protecting against reactive oxygen species. Assisted reproductive techniques may exacerbate defects in methylation and epigenesis. Antioxidant supplements are proposed to reduce the risk of potentially harmful effects, but their use has failed to prevent problems and may sometimes be detrimental. New concepts reveal a significant correlation between oxidative stress, methylation processes and epigenesis, and have led to changes in media composition with positive preliminary clinical consequences.

Improvement of gamete quality by stimulating and feeding the endogenous antioxidant system: mechanisms, clinical results, insights on gene-environment interactions and the role of diet.

Oxidative damage triggers extensive repair in gametes and thereafter in the zygote but it results in clinically relevant damage when affecting the maturation of the gametes chromatin, i.e. padlocking and epigenetic marking. It associates with defective DNA methylation and/or with oxidation of the methyl marks leading to derangement of gamete epigenetics, defects of chromatin condensation and aneuploidy. A proper feed to the one carbon cycle has the potential to stimulate the endogenous antioxidant defences, i.e. gluthatione synthesis, and to activate compensative homeostatic mechanisms restoring both the oxy-redox balance and DNA methylation, which are indeed strictly cross-regulated. This has been shown to produce measurable clinical improvements of male reproductive potential in pilot studies herein summarised. However, the effects of dietary habits and of supplementations are variable according to the individual genetic substrate, as genetic variants of several of the concerned enzymes occur with high frequency. Individual risk assessments and personalised interventions are still difficult to implement, in the meantime, a very varied diet may facilitate metabolic compensation in the majority of the cases. This review aims to report on the mechanisms of damage, on the opportunities to modulate the physiologic oxy-redox homeostasis by means of a varied diet or dietary supplements and on the open issues related to the genetic variability of the population.

The oviduct: a neglected organ due for re-assessment in IVF.

The oviduct has long been considered a 'pipeline', a tube allowing transit of spermatozoa and embryos; this perspective has been reinforced by the success of human IVF. Evidence accumulated over several decades, however, indicates that embryos can modulate the metabolism of tubal cells in their environment. Human IVF culture media is based on formulations that pass mouse embryo assays as quality control: the requirements of mouse embryos differ from those of human embryos, and therefore conditions for human IVF are far removed from the natural environment of the oviduct. The preimplantation environment, both in vitro and in vivo, is known to affect the health of offspring through mechanisms that influence imprinting. Recent studies also show that male accessory glands act in synergy with the oviduct in providing an optimal environment, and this represents a further perspective on the oviduct's contribution to harmonious embryo development and subsequent long-term health. The metabolism of the human embryo is far from being understood, and a 'return' to in-vivo conditions for preimplantation development is worthy of consideration. Although results obtained in rodents must be interpreted with caution, lessons learned from animal embryo culture must not be neglected.

A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index.

The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

Trends, fads and ART!

Morphological selection techniques of gametes and embryos are of current interest to clinical practice in ART. Although intracytoplasmic morphologically selected sperm injection (IMSI), time lapse imaging morphometry (TLIM) or quantification of chromosome numbers (PGS) are potentially useful in research, they have not been shown to be of statistically predictive value and, thus, have only limited clinical usefulness. We make the point that morphological markers alone cannot predict the success of the early embryo, which depends on the correct orchestration of a myriad of physiological and biochemical activation events that progress independently of the maternal or zygotic genome. Since previous attempts to identify metabolic markers for embryo quality have failed and there is no evidence that the intrinsic nature of gametes and embryos can be improved in the laboratory, embryologists can only minimize environmental or operator induced damage while these cells are manipulated ex vivo.

The importance of the one carbon cycle nutritional support in human male fertility: a preliminary clinical report.

BACKGROUND: Sperm chromatin structure is often impaired; mainly due to oxidative damage. Antioxidant treatments do not consistently produce fertility improvements and, when given at high doses, they might block essential oxidative processes such as chromatin compaction. This study was intended to assess the effect on male sub-fertility of a pure one carbon cycle nutritional support without strong antioxidants. METHODS: Male partners of couples resistant to at least 2 assisted reproductive technology (ART) attempts, with no evidence of organic causes of infertility and with either DNA fragmentation index (DFI) measured by Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) or nuclear decondensation index (SDI) measured by aniline blue staining exceeding 20%, were invited to take part in a trial of a nutritional support in preparation for a further ART attempt. The treatment consisted of a combination of B vitamins, zinc, a proprietary opuntia fig extract and small amounts of N-acetyl-cysteine and Vitamin E (Condensyl™), all effectors of the one carbon cycle. RESULTS: 84 patients were enrolled, they took 1 or 2 Condensyl™ tablets per day for 2 to 12 months. Positive response rates were 64.3% for SDI, 71.4% for DFI and 47.6% for both SDI and DFI. Eighteen couples (21%) experienced a spontaneous pregnancy before the planned ART cycle, all ended with a live birth. The remaining 66 couples underwent a new ART attempt (4 IUI; 18 IVF; 44 ICSI) resulting in 22 further clinical pregnancies and 15 live births. The clinical pregnancy rate (CPR) and the live birth rate (LBR) were 47.6% and 39.3% respectively. The full responders, i.e. the 40 patients achieving an improvement of both SDI and DFI, reported a CPR of 70% and a LBR of 57.5% (p<0.001). CONCLUSIONS: Nutritional support of the one carbon cycle without strong antioxidants improves both the SDI and the DFI in ART resistant male partners and results in high pregnancy rates suggesting a positive effect on their fertility potential.

Effect of antioxidants on sperm genetic damage.

According to worldwide statistics, between one in four and one in five couples have fertility problems. These problems are equally distributed between males and females. Modern lifestyle has obviously increased these problems: endocrine-disrupting chemicals, such as plastic polymer catalysts, alkylphenols, phthalates and so on, and cosmetic additives seem to be strongly involved in this fertility problem. Many of these compounds increase oxidative stress (OS) and thus impair spermatogenesis. The oocyte has only a finite capacity, decreasing with maternal age, to repair sperm-borne decays. To decrease this DNA repair burden, reducing the sperm DNA damages linked to OS is tempting. Antioxidant vitamins are often given haphazardly; they are not very efficient and potentially detrimental. A detailed analysis of the sperm nucleus is mandatory (DNA fragmentation or lack of nuclear condensation) prior to any treatment. Here we discuss new concepts in OS and the corresponding therapeutic approaches.