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.

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).

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.

Sequential (hFSH + recFSH) vs homogenous (hFSH or recFSH alone) stimulation: clinical and biochemical (cumulus cell gene expression) aspects.

FSH is a key hormone in the regulation of follicular development. Together with the EGF network, these molecules mediate oocyte maturation and competence in preparation for the action of LH. FSH isoforms regulate distinct biological pathways and have specific effects on granulosa cell function and maturation of the ovarian follicle. Their dynamic interactions occur during the follicular cycle; short-living forms are predominant in the pre-ovulatory phase, whereas long-acting molecules characterize the luteal-follicular transition. Recombinant FSH (rFSH) molecules have a reduced number of isoforms and are less acidic, with a shorter half-life. We have investigated sequential stimulation, comparing hFSH + rFSH, vs. rFSH alone and hFSH alone for the entire stimulation phase. Sequential stimulation leads to an E2 per MII oocyte ratio that is much lower than is seen during treatment with the two drugs individually. Although there is a positive tendency in favor of the sequential treatment, there was no significant difference in pregnancy rates, even taking frozen embryos into consideration. The cumulus cell transcriptome varies considerably between the treatments, although with no clear significance. When comparing pregnant vs. non-pregnant patients, in general a decrease in mRNA expression can be observed in the pregnant patients, especially in expression of folic acid receptor 1 and ovostatin 2. This indicates that material has been transferred from CC to the oocyte. However, a common observation in the literature is that variations in the transcriptome of the cumulus cells are highly dependent upon the patient genotype; the potential for applying this strategy as a basis for selecting embryos is, at the very least, questionable.

Culture systems: embryo co-culture.

During the 1970s, domestic animal biotechnology, i.e., embryo transfer in farm animals, was confronted with the problem of embryonic developmental arrest observed in vitro, especially during the cycle in which maternal to zygotic transition (MZT) cycle takes place. In farm animals, obtaining blastocysts is mandatory, as transfer at earlier stages results in expulsion of the embryo from the vagina. In humans, the first attempts to obtain blastocysts with classical culture media were disappointing, and the use of a coculture strategy was naturally tempting: the first significant results of successful blastocyst development were obtained in the early 1980s, using trophoblastic tissue as a feeder layer in order to mimic an autocrine embryotrophic system. The next supporting cell systems were based on oviduct epithelial cells and uterine cells in order to achieve a paracrine effect. Non-hormone dependence was then demonstrated with the use of prepubertal cells, and finally with the use of established cell lines of nongenital origin (African Green Monkey Kidney, Vero cells). The embryotrophic properties are linked to features of "transport epithelia." Vero cells have been extensively used in human ART, and most of our knowledge about the human blastocyst was gathered with the use of this technology. Coculture is still in current use, but with systems that employ autologous uterine cells. Results following the use of this technology in human ART are superior to those observed with the use of sequential media. The benefit is linked to the release of free radical scavengers and growth factors by the feeder cells. In animal biotechnology, an important part of the "precious embryos," i.e., those resulting from cloning technology, involves coculture with buffalo rat liver (BRL) cells or Vero cells.

Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients.

OBJECTIVE: To investigate DNA fragmentation by using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling in relation to World Health Organization parameters and computer-aided sperm analysis (CASA) in sperm to determine the possibility of obtaining a correlation among CASA parameters, sperm morphology, and DNA fragmentation. DESIGN: Sperm analysis according to World Health Organization parameters, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for sperm DNA fragmentation, and CASA for sperm movement. Prospective study. SETTING: All the patients were under clinical management, consulting for hypofertility at a fertility center in France. PATIENT(S): One thousand six hundred thirty-three men who were referred for infertility investigation, including a complete sperm analysis. INTERVENTION(S): Sperm analysis and DNA damage testing. MAIN OUTCOME MEASURE(S): Sperm morphology, DNA fragmentation, and movement characteristics. RESULT(S): One third of the patients had a TUNEL rate of >30%. Analysis of the 21 semen parameters tested revealed that 7 of them were significantly correlated with the TUNEL results. CONCLUSION(S): World Health Organization sperm parameters and DNA damage are complementary, rather than strongly linked. This should be considered to more fully understand the paternal contribution in assisted reproductive technologies failures.

Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (-19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment.

Paternal effect on embryonic development occurs as early as fertilization. Incorrect formation of the spermatozoon due to centrosome defects and abnormal concentrations of any components involved in the activation process lead to failure immediately or in the subsequent cell cycles. Sperm chromosomal abnormalities result in early embryo developmental arrests. Generally poor spermatozoa lead to poor blastocyst formation. Sperm DNA fragmentation may impair even late post-implantation development. The DNA repair capacity of the oocytes is of major importance. Early preimplantation development, i.e. until maternal to zygotic transition, is maternally driven. Maternal mRNAs and proteins are of major importance, as there is an unavoidable turnover of these reserves. Polyadenylation of these mRNAs is precisely controlled, in order to avoid too early or too late transcription and translation of the housekeeping genes. An important set of maternal regulations, such as DNA stability, transcriptional regulation and protection against oxidative stress, are impaired by age. The embryo biochemical endogenous pool is very important and may depend upon the environment, i.e. the culture medium. Paternal, maternal and environmental factors are unavoidable parameters; they become evident when age impairs oocyte quality.

Pregnancy and delivery after in vitro maturation of naked ICSI-GV oocytes with GH and transfer of a frozen thawed blastocyst: case report.

PURPOSE: To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. METHOD: All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37 degrees C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. RESULTS: The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. CONCLUSION: In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.

Blastocyst freezing.

Blastocyst are preimplantation embryos that have successfully passed the critical step of genomic activation and have so a high developmental potential, thus allowing to reduce the number of embryos transferred. Blastocyst culture and freezing have really started in IVF programs in the early nineties, when it was possible to obtain good blastocysts (essentially using coculture) on a large scale. Blastocysts freezing has been performed since the beginning using glycerol as cryoprotectant in slow protocols. The transition from coculture to sequential media has been a little bit delicate with a drop in the results. Few modifications including a change in the freezing curves and thawing at 37 degrees C have allowed to reach the same results with the two culture techniques. Vitrification with ethylene glycol (EG) has been proposed recently. However, the toxicity of the metabolites of EG for rat embryos has to be considered before moving to this technology on a large scale.

Pregnancy and delivery after stimulation with rFSH of a galatosemia patient suffering hypergonadotropic hypogonadism: case report.

PURPOSE: To determine if hypergonadotropic hypogonadism related to galactosemia could be linked to anomaly of the circulating FSH. A 26-year-old woman, suffering GALT (Galactoso-1-phosphate uridyltransferase) had a premature ovarian failure with amenorrhea since the age of 19. The circulating level for FSH was 83 and 34 mU/mL for LH. METHODS: After treatment with a hormonal substitution cycle including estradiol and progesterone, the patient underwent stimulations with recombinant FSH. The first cycle, one 16-mm diameter follicle and the second cycle one follicle of 17.5 mm of diameter were obtained at the time of ovulation induction. RESULTS: The patient conceived and delivered a female baby weighting 3.38 kg after the second stimulation protocol. CONCLUSIONS: The impact of galactosemia on the ovary seems rather related to the absence of recognition of circulating FSH by its receptor and not to a toxic alteration of the ovary by itself as it is currently reported. The rFSH treatment following hormonal substitution cycles allows to overcome infertility problems.

Preimplantation genetic diagnosis (PGD) in France.

In vitro fertilization is strongly regulated in France. An agreement is needed for the clinicians and the embryologists; and the activity is under the control of CNMBRDP, a governmental commission. PGD is even more controlled; an agreement is needed for embryo biopsy and for genetic/FISH testing. Only three centers are allowed to perform PGD. All the agreements are given for five years and submitted to renewal. Regulations are under the "Ethical" law which should have been re-examined in 1999, but still not redone. This leads to problems, especially in relation with the research on embryos. Evolution of the techniques and indications necessitates a follow-up by the representatives on a regular basis.

Mouse and bovine models for human IVF.

It is obvious that the first prerequisite is to define for what purpose a model is needed for humans. There are huge differences in reproductive physiology between the mouse, human and cow. As far as maturation is concerned, the plasticity of the mouse model is not the same in cows and humans. The final stages of oocyte maturation seem to be more finely regulated in cows and humans, where a minimum size of follicle is necessary to complete maturation in vitro. Bovine and human preimplantation embryos seem to be more similar in terms of biochemical and intrinsic paternal and maternal regulatory processes. Once again, interactions between the embryo and the corpus luteum are similar in cows and humans, but mouse and human embryo implantations are closer. Mouse oocytes and embryos should not be overlooked, but excessive generalization between mammalian species must be avoided.