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.

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.

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.

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.

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.

Determinants of pregnancy rate in the donor oocyte model: a multivariate analysis of 450 frozen-thawed embryo transfers.

BACKGROUND: Conflicting results have been published about the determinants of pregnancy after oocyte donation (OD). We used the OD model to determine predictive factors of pregnancy in the recipient after frozen-thawed embryo transfer (FTET) in a specific series where all the embryos were cryopreserved without any prior selection for fresh transfer. METHODS: We report a retrospective study in a university tertiary care center. Multivariate analysis and logistic regression were used to identify predictive factors of pregnancy in a series of 450 OD FTET cycles in 198 infertile women between January 1992 and December 2006. RESULTS: The mean (+/-SD) recipient age was 35.7 (+/-4.5). Impaired ovarian function was the main indication for OD. The mean +/- SD (range) number of embryos transferred was 1.65 +/- 0.5 (1-3). Overall clinical pregnancy, implantation and delivery rates were 30, 18 and 23%, respectively. After univariate analysis, pregnancy rates were significantly higher in recipients under 35 years, in women with a body mass index (BMI) <30 kg/m(2), in women with an endometrial thickness of > or =8 mm, in amenorrheic women and in women not receiving pituitary down-regulation before endometrial preparation. Using multivariate analysis, the BMI, endometrial thickness and the use of pituitary down-regulation were independent predictors of pregnancy, regardless of age. CONCLUSIONS: This study supports that endometrial thickness of <8 mm, obesity and the use of GnRH analogue pituitary down-regulation before endometrial priming negatively impact pregnancy rates, independently of the recipient's age.

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.

Supporting the One-Carbon Cycle Restores Ovarian Reserve in Subfertile Women: Absence of Correlation with Urinary Bisphenol A Concentration.

Environmental endocrine disrupting chemicals (EDCs), including bisphenol A (BPA), induce DNA methylation errors and oxidative stress, and alter fertility. Animal studies have demonstrated that supporting the one-carbon cycle (1-CC) with appropriate dietary supplements can reduce the effects of EDCs. Anti-Mullerian hormone (AMH), a marker of ovarian functionality, has been tested in subfertile female patients, to control this hypothesis in humans. Fifty-five women with a history of 3-7 years of infertility, with at least two assisted reproductive technology (ART) treatment failures, and low serum levels of AMH were enrolled in the study. Before starting any further ART treatment, they were tested for AMH and for follicular count. A urinary control of BPA was proposed. Then a support of the 1-CC, already tested in other clinical studies, was initiated and continued for 4 months. At the end of this period, antral follicle count and serum AMH levels were re-evaluated. The AMH levels before and after treatment were compared using the Wilcoxon test (nonparametric test, non-Gaussian population). Out of the 55 patients, 35 accepted a BPA dosage in the urine. No correlation was found between BPA and serum AMH concentrations. Forty-nine patients followed the full treatment with 1-CC supplements, which resulted in increased AMH levels, independent of initial AMH levels and maternal age (in the range studied), p = 0.0001. Eight patients spontaneously conceived ongoing pregnancies within 3 months, at the end of the protocol. A support of the 1-CC can partly alleviate metabolic derangements induced by environment, as observed in animal models, and improve endocrine background in women.

Small supernumerary marker chromosomes derived from chromosomes 6 and 20 in a woman with recurrent spontaneous abortions.

In this report, we describe a case of multiple small supernumerary marker chromosomes (sSMC) presenting with recurrent abortions. Peripheral blood lymphocytes of a young, healthy and non-consanguineous couple who asked for genetic evaluation after two spontaneous miscarriages were obtained for karyotypes. Lymphocytes of the woman were analyzed by FISH techniques and DNA was extracted and used for array CGH investigation. Karyotyping revealed 48,XX,+2mar[24]/47,XX,+mar[5]/46,XX[3] for the woman and 46,XY for her husband. FISH analysis showed that the two sSMC consisted of chromosomes 6 and 20. Array CGH analysis showed gains of the 6p11.2q12 (9 Mb) and 20 p11.21 (3.3 Mb) chromosomal regions with a total of 42 genes present on both sSMC. Our findings support also the hypothesis that the modification of the expression of some genes involved in embryo implantation, like THBD gene, could be responsible in the recurrent abortions. This report underpins the necessity of array CGH for characterizing precisely sSMC and helping in genotype-phenotype correlations. Furthermore, a literature review on sSMC is included.

Materno-fetal cardiovascular complications in Turner syndrome after oocyte donation: insufficient prepregnancy screening and pregnancy follow-up are associated with poor outcome.

CONTEXT: Recombinant human GH treatment and oocyte donation (OD) have improved the quality of life in women with Turner syndrome (TS). However, life expectancy is reduced, mainly due to cardiovascular complications. Pregnancy may itself increase that risk and be associated with hazardous materno-fetal outcome. OBJECTIVE: The objective of this study was to evaluate the materno-fetal outcome of ongoing pregnancies beyond 20 wk of gestation obtained by OD in TS. DESIGN: This was a multicenter retrospective study including all assisted reproductive technology centers affiliated with the French Study Group for Oocyte Donation. RESULTS: Among 93 patients, only 37.6% were prescreened with echocardiography or thoracic magnetic resonance imaging. Maternal outcome was dominated by 37.8% of pregnancy-associated hypertensive disorders including preeclampsia in 54.8% and severe eclampsia in four patients. Prematurity occurred in 38.3% and was correlated with pregnancy-associated hypertensive disorder (P = 0.01). The frequency of in utero growth retardation was 27.5%. One fetal demise was linked to eclampsia. Two patients died from aortic rupture after cesarean section in a context of aortic root dilatation. Only 40% of pregnancies were associated with an absolutely normal materno-fetal outcome. CONCLUSIONS: OD pregnancies in TS who have not been managed following recent specific recommendations were at high risk for maternal death by aortic dissection and for preeclampsia and its complications (fetal distress and in utero growth retardation). These recommendations include previous echocardiography, thoracic magnetic resonance imaging, and overnight blood pressure monitoring associated with a tight follow-up during pregnancy. Until future assessment of these recent recommendations, pregnancies obtained in TS after OD must be still considered as very high-risk pregnancies.