Idiopathic Male Infertility Is Strongly Associated with Aberrant Promoter Methylation of Methylenetetrahydrofolate Reductase (MTHFR)

Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected.Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene (NG_013351: 1538-1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility (P-value for trend  = 0.0007). Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.Hypermethylation of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation

Thyroid Disruption by Di-n-Butyl Phthalate (DBP) and Mono-n-Butyl Phthalate (MBP) in Xenopus laevis

BACKGROUND: Di-n-butyl phthalate (DBP), a chemical widely used in many consumer products, is estrogenic and capable of producing seriously reproductive and developmental effects in laboratory animals. However, recent in vitro studies have shown that DBP and mono-n-butyl phthalate (MBP), the major metabolite of DBP, possessed thyroid hormone receptor (TR) antagonist activity. It is therefore important to consider DBP and MBP that may interfere with thyroid hormone system. METHODOLOGY/PRINCIPAL FINDINGS: Nieuwkoop and Faber stage 51 Xenopus laevis were exposed to DBP and MBP (2, 10 or 15 mg/L) separately for 21 days. The two test chemicals decelerated spontaneous metamorphosis in X. laevis at concentrations of 10 and 15 mg/L. Moreover, MBP seemed to possess stronger activity. The effects of DBP and MBP on inducing changes of expression of selected thyroid hormone response genes: thyroid hormone receptor-beta (TRβ), retinoid X receptor gamma (RXRγ), alpha and beta subunits of thyroid-stimulating hormone (TSHα and TSHβ) were detected by qPCR at all concentrations of the compounds. Using mammalian two-hybrid assay in vitro, we found that DBP and MBP enhanced the interactions between co-repressor SMRT (silencing mediator for retinoid and thyroid hormone receptors) and TR in a dose-dependent manner, and MBP displayed more markedly. In addition, MBP at low concentrations (2 and 10 mg/L) caused aberrant methylation of TRβ in head tissue. CONCLUSIONS: The current findings highlight potential disruption of thyroid signalling by DBP and MBP and provide data for human risk assessment

Endocrine effects of methoxylated brominated diphenyl ethers in three in vitro models

a b s t r a c t Methoxylated brominated diphenyl ethers (MeO-BDEs) in aquatic environments have been found to be primarily of natural origin in the marine environment and not from biotransformation of synthetic PBDEs. Two of the eight MeO-PBDEs (2 0 -MeO-BDE-68 and 6-MeO-BDE-47) that were detected in anchovy from the Yangtze River Delta, were natural products from marine organisms. So 2 0 -MeO-BDE-68 and 6-MeO-BDE-47 were chosen to study the potential to modulate androgen, estrogen, or thyroid hormone receptor-(AR, ER, ThR) mediated responses by use of reporter gene assays. 2 0 -MeO-BDE-68 was antiandrogenic at 50 lM, estrogenic at 10 lM and antiestrogenic at 10 and 50 lM (IC 50 = 4.88 lM). 2 0 -MeO-BDE-68 enhanced luciferase expression by 5 nM T3 at 50 lM. 6-MeO-BDE-47 exhibited potent antiandrogenicity at 1 lM and greater (IC 50 = 41.8 lM) and possessed estrogenic activity at 10 lM and antiestrogenic activity at 10 and 50 lM (IC 50 = 6.02 lM)

Adjusted ORs (95% CIs) for idiopathic male infertility by methylation patterns of <i>MTHFR</i>.

a<p>Control: fertile men.</p>b<p>Case 1: idiopathic infertile men with normozoospermia.</p>c<p>Case 2: idiopathic infertile men with oligozoospermia.</p>d<p>Case all: the sum of Case 1 and Case 2.</p>e<p>U: unmethylation (only the unmethylated allele).</p>f<p>U/M: hemimethylation (both methylated and unmethylated alleles).</p>g<p>M: full methylation (only the methylated allele).</p><p>*<i>P</i><0.05 compared with the unmethylation of <i>MTHFR</i>.</p

Characteristics of idiopathic infertile males and fertile controls.

a<p>Control: fertile men.</p>b<p>Case 1: idiopathic infertile men with normozoospermia.</p>c<p>Case 2: idiopathic infertile men with oligozoospermia.</p>d<p>Case all: the sum of Case 1 and Case 2.</p>e<p>BMI: body mass index.</p>f<p>Values are given as median and interquartile range (IQR).</p><p>*<i>P</i><0.05 when compared between case and control groups.</p

The CpG island promoter region of the <i>MTHFR</i> gene.

<p>The CpG Island Searcher Program identified a CpG island within the <i>MTHFR</i> gene promoter region, downstream of the transcriptional start site. Arrow represents the transcriptional start site, blue line depicts the CpG island and the vertical bars represent a CpG site. Black lines and yellow lines represent the location of the MSP primers and BSP primers, respectively.</p

Frequencies of <i>MTHFR</i> hypermethylation (both hemimethylation and full methylation) in case and control groups.

<p>Significant differences were marked with *<i>P</i><0.05 compared with the control.</p

Methylation status of the promoter of <i>MTHFR</i> in genomic DNA prepared from ejaculated human sperm.

<p>(A) Representative results of the MSP analysis of <i>MTHFR</i>. DNA obtained from the sperms was amplified with primers specific to the unmethylated (U) or the methylated (M) of <i>MTHFR</i> after treatment with sodium bisulfite. (a) Fertile controls a1–a13 showed only the unmethylated allele. Others showed both methylated and unmethylated alleles. (b) Idiopathic infertile males with normozoospermia (Case 1) b1–b9 showed only the unmethylated allele. Idiopathic infertile males with normozoospermia b10–b14 showed both methylated and unmethylated alleles. Other showed only the methylated allele. (c) Idiopathic infertile males with oligozoospermia (Case 2) c1–c8 showed only the unmethylated allele. Idiopathic infertile males with oligozoospermia c9–c13 showed both methylated and unmethylated alleles. Others showed only the methylated allele. (B) Representative results of bisulfite-PCR sequencing of <i>MTHFR</i>. Filled and open circles represent methylated and unmethylated CpGs, respectively. L: molecular weight markers; P: positive control (universal methylated DNA).</p