In Utero Gender Dimorphism of Adiponectin Reflects Insulin Sensitivity and Adiposity of the Fetus

Circulating adiponectin reflects the degree of energy homeostasis and insulin sensitivity of adult individuals. Low abundance of the high molecular weight (HMW) multimers, the most active forms mediating the insulin‐sensitizing effects of adiponectin, is indicative of impaired metabolic status. The increase in fetal adiponectin HMW compared with adults is a distinctive features of human neonates. To further understand the functional properties of adiponectin during fetal life, we have evaluated the associations of adiponectin with insulin sensitivity, body composition, and gender. Umbilical cord adiponectin, adiponectin complexes, and metabolic parameters were measured at term by elective cesarean delivery. The associations between adiponectin, measures of body composition, and insulin sensitivity were evaluated in relation to fetal gender in 121 singleton neonates. Higher total adiponectin concentrations in female compared with male fetuses (34.3 ± 9.5 vs. 24.9 ± 8.6, P < 0.001) were associated with a 3.2‐fold greater abundance in circulating HMW complexes (0.20 ± 0.03 vs. 0.08 ± 0.03, P < 0.001, n = 9). Adiponectin was positively correlated with neonatal fat mass (r = 0.27, P < 0.04) and percent body fat in female fetuses (r = 0.28, P < 0.03) and with lean mass in males (r = 0.28, P < 0.03). There was no significant correlation between cord adiponectin and fasting insulin concentrations or fetal insulin sensitivity as estimated by homeostasis model assessment of insulin resistance (HOMA‐IR). The gender dimorphism for plasma adiponectin concentration and complex distribution first appears in utero. In sharp contrast to the inverse correlation found in adults, the positive relationship between adiponectin and body fat is a specific feature of the fetus.     

Assessment of immunization against recombinant human bone morphogenetic protein-2 (dibotermin alpha) on reproduction and development in rabbits

BACKGROUND: To determine if the fetus was affected by maternal antibodies to BMP‐2, the antibody response and developmental effects in fetuses from does immunized against recombinant human BMP‐2 were evaluated. METHODS: Female New Zealand White rabbits received four intramuscular injections (on premating days 1, 8, 22, and 43 [3 days before mating]) of saline and adjuvant (TiterMax^® Gold [control]) or recombinant human BMP‐2 (2 mg/dose) and adjuvant (treated). On GD 29, fetuses were examined, and maternal and fetal anti‐BMP‐2 titer levels and neutralizing activity were assessed. RESULTS: Anti‐BMP‐2 antibodies were detected in 17 of 18 treated does (127 of 151 fetuses), and low levels were detected in 2 of 16 control does (no fetal exposure observed). In general, levels of fetal anti‐BMP‐2 antibodies were similar to those in the does, and pregnancy did not boost the immune response to BMP‐2. There were no effects of immunization or anti‐BMP‐2 antibody titer levels on embryo–fetal viability, fetal weight, or fetal external, visceral, or skeletal development. Only a small number of fetuses (n = 4) displayed detectable neutralizing anti‐BMP‐2 antibodies, but there were no treatment‐related effects in those fetuses. CONCLUSIONS: The lack of embryo–fetal effects may be due to dosage effects of neutralizing anti‐BMP‐2 antibodies, timing of exposure (stage and duration) to neutralizing anti‐BMP‐2 antibodies, and/or redundancy of effects of the various BMPs. Birth Defects Res (Part B) 92:543–552, 2011. © 2011 Wiley Periodicals, Inc.     

Tissue-Specific and Minor Inter-Individual Variation in Imprinting of IGF2R Is a Common Feature of Bos taurus Concepti and Not Correlated with Fetal Weight

The insulin-like growth factor 2 receptor (IGF2R) is essential for prenatal growth regulation and shows gene dosage effects on fetal weight that can be affected by in-vitro embryo culture. Imprinted maternal expression of murine Igf2r is well documented for all fetal tissues excluding brain, but polymorphic imprinting and biallelic expression were reported for IGF2R in human. These differences have been attributed to evolutionary changes correlated with specific reproductive strategies. However, data from species suitable for testing this hypothesis are lacking. The domestic cow (Bos taurus) carries a single conceptus with a similar gestation length as human. We identified 12 heterozygous concepti informative for imprinting studies among 68 Bos taurus fetuses at Day 80 of gestation (28% term) and found predominantly maternal IGF2R expression in all fetal tissues but brain, which escapes imprinting. Inter-individual variation in allelic expression bias, i.e. expression of the repressed paternal allele relative to the maternal allele, ranged from 4.6−8.9% in heart, 4.3−10.2% in kidney, 6.1−11.2% in liver, 4.6−15.8% in lung and 3.2−12.2% in skeletal muscle. Allelic bias for mesodermal tissues (heart, skeletal muscle) differed significantly (P<0.05) from endodermal tissues (liver, lung). The placenta showed partial imprinting with allelic bias of 22.9−34.7% and differed significantly (P<0.001) from all other tissues. Four informative fetuses were generated by in-vitro fertilization (IVF) with embryo culture and two individuals displayed fetal overgrowth. However, there was no evidence for changes in imprinting or DNA methylation after IVF, or correlations between allelic bias and fetal weight. In conclusion, imprinting of Bos taurus IGF2R is similar to mouse except in placenta, which could indicate an effect of reproductive strategy. Common minor inter-individual variation in allelic bias and absence of imprinting abnormalities in IVF fetuses suggest changes in IGF2R expression in overgrown fetuses could be modulated through other mechanisms than changes in imprinting.     

Expression of miR-Let-7a,miR-15a,miR-16-1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment. The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin β-3 (Itg β3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.     

Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing

Vitrification is a novel cryopreservation method for mammalian blastocysts. This study was designed to compare different vitrification methods and slow freezing for their effects on survival rate and DNA integrity in mouse and human blastocysts. In Experiment 1, embryo survival and DNA integrity were compared between mouse blastocysts with collapsed and non‐collapsed blastoceles. In Experiment 2, embryo survival and DNA integrity were compared between vitrified and slow‐frozen mouse blastocysts. In Experiment 3, embryo survival and DNA integrity were compared between vitrified and slow‐frozen human blastocysts. Fresh blastocysts were used as controls in all experiments. Higher (P < 0.05) blastocyst survival rates were obtained in mouse blastocysts vitrified with collapsed versus intact blastoceles, although DNA‐integrity indices in the surviving blastocysts were the same among vitrified and fresh blastocysts. More mouse blastocysts (P < 0.05) survived after vitrification (100%) as compared to slow freezing (82.5%). DNA‐integrity indices examined in the surviving blastocysts were also higher (P < 0.001) in fresh (93.6%) and vitrified/warmed (93.7%) blastocysts than in slow‐frozen/thawed (75.8%) ones. More human blastocysts survived with a higher DNA‐integrity index after vitrification/warming than after slow freezing/thawing. These results indicate that higher survival rates can be obtained by vitrification of blastocele‐collapsed blastocysts, and that vitrification causes less cell apoptosis in both mouse and human blastocysts compared to slow freezing. Vitrification of blastocysts after blastocele collapse by single laser pulse supports a higher survival rate and less DNA apoptosis, suggesting that laser blastocele collapse is a safe procedure for blastocyst vitrification. Mol. Reprod. Dev. 79: 229–236, 2012. © 2011 Wiley Periodicals, Inc.     

Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction

The effects on subsequent fetal development of the presence or absence of serum at different times during IVC of ovine zygotes were studied. Zygotes, recovered from superovulated ewes 36h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media supplemented with either BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). Serum was present or absent during the first two and last 2 days of IVC giving four treatments (SOF-/SOF-; SOF-/SOF+;SOF+/SOF- and SOF+/SOF+). In total, 224 embryos, including 26 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Presence of serum during IVC had a biphasic effect on embryo development. The inclusion of serum during the first 2 days of IVC retarded early embryo development while the inclusion of serum during the last 2 days of IVC produced more blastocysts by day 6. These effects were independent of each other. The presence of serum during the first 2 days of IVC resulted in increased weights of gravid uterus, placenta, fetus, fetal heart and liver. The incidence of fetuses whose total or organ weights were greater than three standard deviations above the corresponding mean weights of control fetuses was also greater when serum was present during the first 2 days of IVC. However, even when serum was absent throughout IVC there was still an infrequent incidence of fetal weights greater than three standard deviations above the mean for control fetuses. These observations provide evidence that it is the early pre-compaction stages of embryo development that are particularly sensitive to perturbations leading to abnormal fetal development.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Evaluation of embryo-fetal development in rats housed in concrete or hwangto cages during pregnancy

BACKGROUND: Recently, it was reported that the 4‐week exposure of rats to a concrete building environment under cool temperatures (11.7–12.1°C) had adverse effects on the general health parameters. This study examined the potential effects of concrete and hwangto building environments on pregnant dams and embryo‐fetal development in Sprague‐Dawley rats. METHODS: Groups of 10 mated females were exposed to polycarbonate (control), concrete, or hwangto cages from gestational days (GD) 0 to 20 under cool temperatures (11.9–12.5°C). All the females underwent a caesarean section on GD 20, and their fetuses were examined for any morphological abnormalities. RESULTS: The temperatures in the cages were similar in all groups but the relative humidity in the concrete and hwangto groups were higher than in the control group. The concentration of volatile organic compounds in the hwangto group during the study period was lower than in the control group. In the concrete group, maternal effects manifested as an increase in the incidence of clinical signs, a lower body weight, and a decrease in the thymus and ovary weights. Developmental effects included increased post‐implantation loss and decreased litter size. In contrast, in the hwangto group, there were no exposure‐related adverse effects on the maternal and developmental parameters. CONCLUSIONS: Overall, the exposure of pregnant rats to a concrete building environment under cool temperatures has adverse effects on the clinical signs, body weight, organ weight, and embryo‐fetal development. On the other hand, exposure to a hwangto building environment does not have any adverse effects on pregnant dams or on embryo‐fetal development in rats. Birth Defects Res (Part B), 2008. © 2008 Wiley‐Liss, Inc.     

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.     

Combined effect of high-fat diet and copper deficiency during gestation on fetal copper status in the rat

We have previously shown that a low-copper (Cu) diet produced alterations in placental Cu transport and fetal Cu stores. Because Cu deficiency has been associated with lipid deposition in rat dam liver, we hypothesized that a high fat intake, a prevalent dietary habit in many populations, may worsen fetal Cu status and its closely linked iron (Fe) deposits. Pregnant rats were fed one of four diets during the second half of gestation: NFNCu: normal fat (7%), normal Cu (6 mg/kg); HFNCu: high fat (21%), normal Cu; NFLCu: normal fat, low Cu (0.6 mg/kg), and HFLCu: high fat, low Cu. One day before delivery, dams were anesthetized, and maternal as well as fetal plasma and tissues were obtained. Maternal, fetal, and placental weights were indistinguishable regardless of the group. Dam plasma Cu and placental Cu were lower in both LCu groups than in the NFNCu or the HFNCu groups. However, fetal plasma Cu was similar in all treatment groups. Dam and fetal liver Cu stores were reduced in the LCu groups compared to the NCu groups. This resulted in lower fetal/maternal liver Cu ratios in the NFLCu (1.79 ± 0.14,p < 0.05) and HFLCu (1.59 ± 0.21,p < 0.05) as compared to the NFNCu (4.12 ± 0.44) and the HFNCu (4.15 ± 0.27). Dam liver Fe was higher in the NFNCu than in HFNCu group (1.10 ± 0.8 vs. 0.89 ± 0.06 μmol/g,p < 0.05); fetal liver Fe from HFNCu and NFLCu dams was lower than that from NFNCu fetuses (NFNCu: 2.42 ± 0.14; HFNCu: 1.92 ± 0.15,p < 0.05; NFLCu: 1.81 ± 0.10,p < 0.01). Fetuses of the HFLCu group had a lower heart Fe than the NFNCu group (0.56 ± 0.03 vs. 44.0 ± 3.0 μg/g,p < 0.01). These data indicate that a maternal high-fat diet can potentially aggravate the effects of Cu deficiency by further altering fetal Cu and Fe tissue stores.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

Maternal exposure to multi-wall carbon nanotubes does not induce embryo-fetal developmental toxicity in rats

BACKGROUND: Although the potential risk of carbon nanotubes (CNTs) to humans has recently increased due to expanding production and widespread use, the potential adverse effects of CNTs on embryo–fetal development have not yet been determined. METHODS: This study investigated the potential effects of multi‐wall CNTs (MWCNTs) on pregnant dams and embryo–fetal development in rats. MWCNTs were administered to pregnant rats by gavage at 0, 40, 200, and 1,000 mg/kg/day. All dams were subjected to Cesarean section on day 20 of gestation, and the fetuses were examined for any morphological abnormalities. RESULTS: All animals survived to the end of the study. A decrease in thymus weight was observed in the high dose group in a dose‐dependent manner. However, maternal body weight, food consumption, and oxidant–antioxidant balance in the liver were not affected by treatment with MWCNTs. No treatment‐related differences in gestation index, fetal deaths, fetal and placental weights, or sex ratio were observed between the groups. Morphological examinations of the fetuses demonstrated no significant difference in incidences of abnormalities between the groups. CONCLUSIONS: The results show that repeated oral doses of MWCNTs during pregnancy induces minimal maternal toxicity and no embryo–fetal toxicity at 1,000 mg/kg/day in rats. The no‐observed‐adverse‐effect level of MWCNTs is considered to be 200 mg/kg/day for dams and 1,000 mg/kg/day for embryo–fetal development. In this study, the dosing formulation was not analyzed to determine the degree of reaggregation (or not), nor were blood levels of CNT's measured in the dosed animals to verify or characterize absorption. Birth Defects Res (Part B) 92:69–76, 2011. © 2011 Wiley‐Liss, Inc.     

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid‐gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re‐established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely‐flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non‐CpG cytosines of the retroelement promoter were highly methylated especially in the transgene‐active mid‐gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid‐gestational embryo.     

Alterations in methylation and expression levels of imprinted genes H19 and Igf2 in the fetuses of diabetic mice

The study aimed to reveal alterations in expression and methylation levels of the growth-related imprinted genes H19 and Igf2 in fetuses of diabetic mice. Diabetes was induced in female mice by intraperitoneal injection of streptozotocin. DNA and total RNA were extracted from fetuses obtained from diabetic and control dams on embryonic day (E) 14. Real-time RT-PCR analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.1% higher in diabetic fetuses than in those of control dams. In addition, the body weight of pups born to diabetic dams was 26.5% lower than that of the control group. The results indicate that maternal diabetes can affect fetal development by means of altered expression of imprinted genes. The modified genomic DNA methylation status of imprinting genes may account for the change in gene expression.     

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 μg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.     

Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage

The susceptibility of in vitro matured (IVM) porcine oocytes to be fertilized in vitro after vitrification was investigated. IVM oocytes were cryopreserved by solid surface vitrification (SSV) or treated with cryoprotectants (toxicity control, TC). Control oocytes were not treated or vitrified. Live oocytes in the three groups were in vitro fertilized (IVF) and then cultured (IVC) for 6 days. In vitro maturation and IVC were performed under 5% or 20% O(2) tension. The percentage of live oocytes in the SSV group was lower than those in the control and TC groups. Fertilization rates after SSV were significantly lower than in the control group. Significantly fewer penetrated oocytes formed male pronuclei in the SSV group than in the control and TC groups. Cleavage rates were significantly lower in the SSV group than in the control and TC groups. Blastocyst formation rates in the control and TC groups were similar, whereas only a single embryo developed to the blastocyst stage from 113 oocytes after vitrification. Blastocyst formation rates in the control group and in the TC group were significantly higher under 5% O(2) IVC than under 20% O(2) IVC. Oxygen tension during IVM had no effect on embryo development. The glutathione (GSH) content of vitrified oocytes was significantly lower than in the controls. In contrast, the H(2)O(2) level was higher in vitrified oocytes than in control oocytes. Vitrification caused parthenogenetic activation in 44.9% of unfertilized oocytes. This significant increase in parthenogenetic activation along with significantly dropped GSH level in vitrified oocytes may explain the decreased ability of the SSV group to form male pronuclei. These factors might have contributed to the poor developmental competence of vitrified oocytes.     

Global DNA hypomethylation is associated with NTD‐affected pregnancy: A case‐control study

BACKGROUND: Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD‐affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS: A hospital‐based case‐control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS: Sixty‐five NTD‐affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736‐fold increased risk for NTDs (95% confidence interval, 1.731–19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS: Global DNA hypomethylation in fetal brain tissue was associated with NTD‐affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. © 2010 Wiley‐Liss, Inc.