Search

We have characterized the uterine expression of DNA methyltransferases (DNMTs) during early pregnancy in mice and determined whether a folate-deficient diet (FDD) can affect DNMTs in this context. Within endometrial cells, expressions of DNMT (cytosine-5) 1 (Dnmt1),Dnmt3a, andDnmt3bwere significantly elevated during the prereceptive phase of pregnancy but generally returned to baseline levels during receptive and postimplantation periods. As such, the transcription of DNMT genes is temporally regulated during early pregnancy. When comparisons were made between implantation sites (IS) and inter-IS on day 5 of pregnancy, lower levels ofDnmt3awere detected at IS. Comparisons between IS and inter-IS did not reveal significant expression differences for other DNMT genes. When tissue sections were examined, DNMT3A was specifically lower in the stroma of IS. Reduced DNMT1 and DNMT3B levels were also observed in the luminal and glandular epithelia of IS, whereas no obvious differences in the stroma were detected. In pseudo-pregnant mice subjected to a FDD, levels ofDnmt1andDnmt3a(but notDnmt3b) were significantly upregulated in endometrial tissues, as compared with controls. When tissues from these folate-deficient mice were examined, DNMT1 levels were elevated in both the luminal and glandular epithelia, whereas DNMT3A was upregulated in the luminal epithelium and the stroma. A slight increase in DNMT3B levels was detected in the glandular epithelium. These results indicate that DNMTs may regulate the transcription of endometrial genes associated with embryo implantation and that levels of DNMTs are affected by dietary folate in mice.

Activated microglia producing reactive nitrogen species, inflammatory factors, reactive oxygen species (ROS) and other neurovirulent factors, can lead to the development of neurodegenerative diseases. Certain compounds can inhibit the activation of microglia. However, the mechanisms remain unclear. In the present study, we investigated the inhibitory effect of geniposide on the production of ROS and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated N9 murine microglial cells through the p38, ERK1/2 and nuclear factor-κB (NF-κB) signaling pathways. After the N9 cells were pre-treated with the vehicle or geniposide and exposed to LPS for the time indicated, the MTT conversion test was used to assess cell viability. Suitable concentrations were chosen and adjusted according to the experiments. Extracellular nitric oxide (NO) release was measured by Griess reaction. The formation of ROS and intracellular NO was evaluated by fluorescence imaging. NOS activities were determined using commercially available kits. The morphology of the N9 cells was examined by hematoxylin and eosin staining. The expression of iNOS mRNA was examined by RT-PCR. The protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2 and NF-κB, inhibitory factor-κB-α (IκB-α) were determined by western blot analysis. The results showed that geniposide attenuated the activation of N9 cells and inhibited the overproduction of NO, intracellular ROS and the expression of iNOS induced by LPS in the cells. In addition, geniposide blocked the phosphorylation of p38, ERK1/2 and inhibited the drop-off of IκB induced by LPS in the cells. These data indicate that geniposide has therapeutic potential for the treatment of neurodegenerative diseases, and that it exerts its effects by inhibiting inflammation.

The single nucleotide polymorphism (SNP) of the vangl1 gene is highly correlated with Neural Tube Defects (NTDs), a group of severe congenital malformations. It is hindered by the lack of a quantitative detection method. We first propose the use of a DNA biosensor to detect the missense single nucleotide polymorphism (rs4839469 c.346G>A p.Ala116Thr) of the vangl1 gene in this work. Polypyrrole (PPy) and streptavidin were integrated to modify a gold electrode. We took advantage of the PPy's good biocompatibility and excellent conductivity. To further accelerate the electron transfer process at the electrode surface, polyamidoamine dendrimer-encapsulated gold nanoparticles (Au-PAMAM) were used, because Au-PAMAM possess a large number of amino groups to load capture probes (CP). Using the biotin-streptavidin system, the Au-PAMAM-CP bionanocomposite probe, which can detect the target DNA, was conjugated to the electrode surface. Under optimal conditions, the DNA biosensor exhibited a wide linear range of 0.1–100 nM with a low detection limit of 0.033 nM (S/N=3). The results suggest that this approach has the potential to be used in clinical research.

The harmful effects of the environmental carcinogen, benzo[a]pyrene (B[a]P), on mammalian neurodevelopment and behavior as yet remain unclear. Several studies have suggested that B[a]P impairs learning and memory. In the present investigation, we investigated the effects of subchronic exposure to B[a]P on rats. Male rats received daily injection of B[a]P (0, 1.0, 2.5, and 6.25 mg/kg, i.p.) or vehicle for 13 weeks. Employing the Morris water maze (MWM) test, we observed that rats exposed to either 2.5 mg/kg or 6.25 mg/kg B[a]P had modified behavior compared to controls as indicated by the increased mean latencies, the decreased number of crossing platform and the decreased swimming time in the target area. B[a]P treatment decreased the levels of malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), acetylcholine (ACh), choline acetyltransferase (ChAT), and increased the activity of acetylcholinesterase (AChE). Endogenous monoamine levels, norepinephrine (NE), adrenaline (A), dopamine (DA) and 5-hydroxytryptamine (5-HT) and their selected metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in hippocampus were measured using high performance liquid chromatography (HPLC). B[a]P at both doses, 2.5 and 6.25 mg/kg, increased NE, DA, DOPAC and 5-HT content in the hippocampus. Our results suggested a close link between the modified levels of neurotransmitters in the hippocampus and the impaired behavioral performance, indicating that B[a]P is a potential neurotoxic pollutant.

Tert-butyldiphenylsilyl (TBDPS) was testified to be an appropriate orthogonal protecting group for novel 7-hydroxyl-functionalized 8-aza-7-deaza-2'-deoxyadenosine analogues. It was stable in partial and complete hydrogenation reactions used for the different linker preparation. The corresponding phosphoramidites and hydroxyl-functionalized oligodeoxynucleotides were synthesized and identified. The thermal effect of the hydroxyl group with different linkers on DNA duplexes was evaluated. It provided a feasible strategy for the preparation of hydroxyl-functionalized DNAs for the nucleic acid research.

In order to depict crowd dynamics in the case of smoke effect, a novel evacuation model is presented based on the extended cellular automaton. In the model, the moving probability of the target position in the next time step is given by combining distance gain and smoke gain at first. Through considering individual kinship and group effect, group disbanding and regrouping rules are defined, and the optimal evacuation route planning is determined. At last, the simulation was conducted to study the key factors influencing evacuation efficiency. The results showed that when the group size was small and the smoke spreading velocity was slow, priority selection of the shortest route strategy was good for reducing evacuation time, and large-scale group should be avoided as much as possible in evacuation process.

Tert-butyldiphenylsilyl (TBDPS) was found to be an effective orthogonal protecting strategy for the 5-substituted hydroxyl groups of de novo synthesized deoxyuridine analogues 1-3 and 7-(3-hydroxypropynyl)- of 8-aza-7-deazadeoxyadenosine 4 for their incorporation into oligodeoxynucleotides by phosphoramidite chemistry. It could be completely cleaved under normal and ultra-mild deprotection conditions applied to DNA synthesis, without extra cleaving operation. The new phosphoramidites were coupled as usual with high yields. The new modified oligodeoxynucleotides were characterized by MALDI-TOF and enzymatic cleavage analysis. The thermal stability and conformation of these hydroxyl-functionalized DNA duplexes were evaluated.

Mammalian target of rapamycin (MTOR) is a protein kinase that plays a central role in cell growth and proliferation. It is a part of the signaling network transmitting growth factor signaling to translational control. Previous studies have shown that MTOR is involved in embryo implantation, but its expression in the uterus and its role in implantation are unclear. Here, we have investigated the expression and role of MTOR in mouse uterus during early pregnancy. RT-FQ PCR showed that the mRNA levels of Mtor in endometria of pregnant mice were higher than those of nonpregnant mice. The mRNA levels in the pregnant mice gradually increased from D3 of pregnancy, reached maximum on D5, and then declined afterward. In situ hybridization and immunohistochemical analysis showed that the mRNA and protein of MTOR were mainly located in stromal cells. The levels of the expressed MTOR protein correlate with those of mRNA. The number of implantation sites was greatly decreased by the intrauterine injection with rapamycin in the early morning of D4 of the pregnancy. These findings suggest that MTOR may play an important role in embryo implantation in mice.

Hemiasterlin is a tripeptide with highly alkylated unnatural amino acids. It acts as a potent tumor cell growth inhibitor. From the comparison of the N-terminal between hemiasterlin and its analogues, a further modification was conducted on this position for a SAR study. Some unnatural amino acids with aryl or ureido groups were introduced into the N-terminal of hemiasterlin analogues to improve their hydrophobicity/hydrophilicity. Here 14 hemiasterlin analogues were synthesized. And their activities against tumor cell lines were evaluated. Discussions on SAR preliminarily indicated that no matter whether the N-terminals come from the aryl or alkyl units, sufficient steric bulk, lipophilicity and methylation of the N-terminal should be crucial factors to the cytotoxic activity.

The aberrant expression of Vangl1 is highly correlated with dysontogenesis, especially for neural tube defects. Therefore, the ultrasensitive detection of Vangl1 would provide a new approach for the specific early diagnostics in dysembryoplasia. However, no quantitative detection method is currently available. Herein, we describe the development of a new approach to fill this assay gap. We utilized C60-templated AuPt bimetallic nanoclusters for signal amplification because the promising C60 nanomaterial provides a large surface area for the in site reduction of bimetallic nanocomposites as well as excellent conductivity. To further amplify the electrochemical signal, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) and a derivative of 3,4,9,10-perylenetetracarboxylicdianhydride (PTC-NH2) were selected for modification of the electrode to provide more amino groups for the immobilization of antibodies and to enhance the conductivity. The electrochemical signal was primarily derived from the catalysis of H2O2 by C60-AuPt. Chronoamperometry was applied to record the electrochemical signals. Under optimal conditions, the prepared immunosensor exhibited a wide linear range from 0.1 pg mL(-1) to 450 pg mL(-1) and a low detection limit of 0.03 pg mL(-1). Moreover, the proposed method exhibited good stability and recovery, suggesting its potential for use in clinical research.

The mechanism of birth defects induced by folate deficiency was focused on mainly in fetal development. Little is known about the effect of folate deficiency on the maternal uterus, especially on decidual angiogenesis after implantation which establishes vessel networks to support embryo development. The aim of this study was to investigate the effects of folate deficiency on decidual angiogenesis. Serum folate levels were measured by electrochemiluminescence. The status of decidual angiogenesis was examined by cluster designation 34 (CD34) immunohistochemistry and the expression of angiogenic factors, including vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), and VEGF receptor 2 (VEGFR2) were also tested. Serum levels of homocysteine (Hcy), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), progesterone (P4), and estradiol (E2) were detected by Enzyme-linked immunosorbent assay. The folate-deficient mice had a lower folate level and a higher Hcy level. Folate deficiency restrained decidual angiogenesis with significant abnormalities in vascular density and the enlargement and elongation of the vascular sinus. It also showed a reduction in the expressions of VEGFA, VEGFR2, and PLGF. In addition, the serum levels of P4, E2, LH, and PRL were reduced in folate-deficient mice, and the expression of progesterone receptor (PR) and estrogen receptor α (ERα) were abnormal. These results indicated that folate deficiency could impaire decidual angiogenesis and it may be related to the vasculotoxic properties of Hcy and the imbalance of the reproductive hormone.

In this paper, a simple, ultrasensitive and label-free electrochemical α2,6-sialylated glycans biosensor based on reduced graphene oxide-tetraethylene pentamine-1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) hybrids was developed. Due to the abundance of amino groups from reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) and the electrostatic interaction of BMIMPF6, bimetallic gold platinum alloy nanoparticles (AuPtNPs) were densely adsorbed onto the surface of the nanocomposite, providing a large surface area available for the immobilization of Sambucus nigra agglutinin (SNA). AuPtNPs have excellent conductivity and catalytic activity, which can promote electron transfer between the electrolyte solution and the surface of electrode and can enhance the sensitivity of biosensor. SNA, which specifically binds α2,6-sialylated glycans, was covalently immobilized on AuPtNPs for specific detection of α2,6-sialylated glycans in human serum. Under optimal experimental conditions, amperometric response changes were used to detect α2,6-sialylated glycans with a broad linear range of 10 fg mL(-1) -1 μg mL(-1) and a low detection limit of 3 fg mL(-1) (S/N=3). When applied to spiked serum samples, the recovery of the developed biosensor ranged from 100.8% to 101.4%, suggesting that the electrochemical biosensor would be suitable for the practical detection of α2,6-sialylated glycans.

Previous studies have shown that microRNAs are involved in the process of implantation. They play an important role in cell growth and proliferation. DROSHA is the microRNA-processing enzyme and is required for the maturation of microRNAs. However, its expression and function during early pregnancy in mice still remain unclear. In the present study, we analyzed the expression pattern of DROSHA in the mouse uterus during early pregnancy, pseudopregnancy, artificially induced decidualization, and in the ovariectomized mouse uterus using real-time quantitative polymerase chain reaction, Western blotting analyses, and immunohistochemistry. We found that DROSHA was spatiotemporally expressed in decidualizing stromal cells during early pregnancy and in pseudopregnant mice in which decidualization was artificially induced. In the ovariectomized mouse uterus, the expression of DROSHA was upregulated after progesterone treatment. In a stromal cell culture model, the expression of DROSHA gradually increased with the progression of stromal decidualization. Taken together, our findings suggest that DROSHA is involved in stromal decidualization and may play an important role in embryo implantation in mice.

Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice.

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.

A simplified electrochemical multianalyte immunosensor for the simultaneous detection of high sensitivity C-reactive protein (hsCRP) and soluble CD40 ligand (sCD40L) that uses reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) that directly adsorbs metal ions as labels is reported. rGO-TEPA contains a large number of amino groups and has excellent conductivity, making it an ideal template for the loading of Pb(2+) and Cu(2+), which greatly amplifies the detection signals. The signals could be directly detected in a single run through differential pulse voltammetry (DPV), and each biorecognition event produces a distinct voltammetric peak. The position and size of each peak reflects the identity and the level of the corresponding antigen. Primarily designed for an application in a sandwich-type immunoassay based on Pb(2+) and Cu(2+) labels, two main challenges are accomplished with the herein presented nanosheets: fabrication of the template and the amination process for Pb(2+) and Cu(2+) adsorption. To further improve the analytical performance of the immunosensor, Au@bovine serum albumin (BSA) nanospheres synthesized through a "green" synthesis route were used as a sensor platform, which not only provides a biocompatible microenvironment for the immobilization of antibodies but also amplifies the electrochemical signals. Under optimal conditions, hsCRP and sCD40L could be assayed in the range of 0.05 to 100 ng mL(-1) with detection limits of 16.7 and 13.1 pg mL(-1) (S/N=3), respectively. The assay results on clinical serum samples with the proposed immunosensor were in acceptable agreement with those using the standard single-analyte test of the enzyme-linked immunosorbent assay (ELISA). This novel immunosensing system provides a simple, sensitive and low-cost approach for a multianalyte immunoassay.

For the first time, a simple, ultrasensitive and label-free electrochemical monocyte chemotactic protein-1 (MCP-1) immunosensor based on orderly oriented conductive wires has been developed. A conductive wire, which is similar to an electron-conducting tunnel, was designed with Au nanoparticles (AuNPs) joined to Au@Pt core-shell microspheres via a cysteamine (CA) crosslinker. To enhance the sensitivity of the immunosensor, Au nanoparticles were electrodeposited onto the gold electrode, and CA was self-assembled via strong Au-S covalent bonds, providing an appropriate surface and promoting electron transfer. Next, Au@Pt core-shell microspheres with large surface area were grafted onto the modified electrode to immobilize more MCP-1 antibodies. MCP-1 is an initiating factor and biomarker of atherosclerotic diseases. Under optimal experimental conditions, differential pulse voltammetry (DPV) current changes were used to detect MCP-1 with a broad linear range of 0.09-360 pg mL(-1) and a low detection limit of 0.03 pg mL(-1) (S/N=3). The proposed immunosensor exhibited good selectivity, reproducibility and reusability. When applied to spiked serum samples, the data for the developed immunosensor were in agreement with an enzyme linked immunosorbent assay, suggesting that the electrochemical immunosensor would be suitable for practical detection.

STUDY QUESTION Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans. SUMMARY ANSWER The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1. WHAT IS KNOWN ALREADY GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported. STUDY DESIGN, SIZE, DURATION Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM. PARTICIPANTS, MATERIALS, SETTING, METHODS Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM. LIMITATIONS, REASONS FOR CAUTION The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.

This study investigated the expression pattern of Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in the endometrium of early-stage pregnant mice and its function during embryo implantation. The expression of KRAS was measured at the mRNA level using real-time polymerase chain reaction (PCR) and at the protein level using immunohistochemistry and western blotting. The expressions of KRAS mRNA and protein were not significantly different in the endometrium of pseudopregnant and early-stage pregnant mice. However, the immunohistochemistry results showed that KRAS was highly expressed in the decidualizing stromal cells on days 5-7 of mouse pregnancy and was enhanced in the epithelial cells as pregnancy progressed. The expression of KRAS protein was higher after the stromal cell was artificially decidualized in vivo and in vitro. Stromal cell proliferation was attenuated after down-regulating KRAS expression. After silencing KRAS in the mouse uterus, the embryo implantation rate was significantly reduced (P < 0.005). We speculate that KRAS may regulate the stromal cell proliferation and differentiation progress and then affect the embryo implantation process. This study reveals that KRAS plays an important role in regulating the embryo implantation process.

The synthesis of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl nucleosides (1b, 2b, and 3b) were described and their conformation in solution as well as in the solid state was determined In addition to this, building blocks 10a,b and 13a,b were prepared and employed in solid-phase oligonucleotide synthesis. For compounds 1a and 1b the lactime proton is protected to avoid unresolved degradation of its phosphoramidites 10a,b. UV-melting studies have been carried out to assess the thermal stability of oligonucleotides containing compounds 1a,b, and 3a,b.

The 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl ortho-aza-purine and -pyrimidine nucleosides manifest an unusually rigid sugar N conformation in solution.

Oligonucleotides incorporating the 7-propynylderivatives of 8-aza-7-deaza-2¢-deoxyguanosine (3b)and 8-aza-7-deaza-2¢-deoxyadenosine (4b) weresynthesized and their duplex stability was com-pared with those containing the 5-propynyl deriva-tives of 2¢-deoxycytidine (1) and 2¢-deoxyuridine (2).For this purpose phosphoramidites of the 8-aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine) nucleo-sides were prepared and employed in solid-phasesynthesis. All propynyl nucleosides exert a positiveeffect on the DNA duplex stability because of theincreased polarizability of the nucleobase and thehydrophobic character of the propynyl group. Thepropynyl residues introduced into the 7-position ofthe 8-aza-7-deazapurines are generally more stabil-izing than those at the 5-position of the pyrimidinebases. The duplex stabilization of the propynylderivative 4b was higher than for the bromo nucleo-side 4c. The extraordinary stability of duplexescontaining the 7-propynyl derivative of 8-aza-7-deazapurin-2,6-diamine (5b) is attributed to theformation of a third hydrogen bond, which is appar-ently not present in the base pair of the purin-2,6-diamine 2¢-deoxyribonucleoside with dT.INTRODUCTIONThe thermal stability of oligonucleotide duplexes depends ontheir length and their base pair composition (1). Efforts wereundertaken to increase the duplex stability by chemicalmodification of the DNA constituents. The sugar moiety (2)or the nucleobase (1,3–7) of the DNA constituents as well asthe oligonucleotide backbone (8) were structurally modified.These changes have broadened the application of oligonucle-otides in the field of antisense technology (9–11) or inhybridization techniques used for diagnostic purposes (12).All approaches focused either on strengthening the hydrogenbonds between the bases and/or the increase of the stackinginteractions among them. Our search for more stable base pairsled us to the pyrazolo[3,4-d]pyrimidine (8-aza-7-deazapurine)2¢-deoxyribonucleosides (13–16), which can be considered asideal shape mimics of the parent purine nucleosides. Their 7-halogenated and alkynylated derivatives induce a positiveeffect on the base pair stability of duplex DNA (1,17,18).Among the various groups introduced into oligonucleotidesfor duplex stabilization, the propynyl group found particularattention (19–21). This group was introduced in the 5-positionof the pyrimidine nucleosides, e.g. in 2¢-deoxycytidine (1)(19–23) and 2¢-deoxyuridine (2) (24–26) or in the 7-positionof pyrrolo[2,3-d]pyrimidine nucleosides (27). Also, 8-propy-nylated 2¢-deoxyadenosine and 2¢-deoxyguanosine derivativeswere studied (26,28). However, 8-substituted purine deriva-tives destabilize DNA due to steric hindrance. As the 7-substituents of 8-aza-7-deazapurines have steric freedomwithin a DNA-duplex, propynyl residues were now introducedinto the 7-position of oligonucleotides incorporating pyrazo-lo[3,4-d]pyrimidines (8-aza-7-deazapurines) (1,13,18). Thisstudy reports on the synthesis of 7-propynyl derivatives of the8-aza-7-deaza-2¢-deoxyguanosine (3b) and the corresponding2¢-deoxyadenosine derivative (4b). The work also paysattention to the extraordinary stability of duplexes incorpor-ating the propynyl derivative of 8-aza-7-deazapurin-2,6-diamine 2¢-deoxyribonucleoside (5b) (18) and compares thestabilities of ‘dA–dT’ versus ‘dG–dC’ base pairs replacingeither one or two of the propynylated base analogs shown inScheme 1.MATERIALS AND METHODSGeneralFor general remarks see Seela and Becher (1). Thin-layerchromatography (TLC) was performed on TLC aluminiumsheets coated with silica gel 60 F

The 8-aza-7-deazapurin-2,6-diamine 2′-deoxyribonucleoside (2a)–dT base pair is more stable than the 2-amino-2′-deoxyadenosine (1)–dT pair. Halogeno or propynyl substituents introduced in the 7-position of the 8-aza-7-deazapurine system (2b–d) lead to an additional duplex stabilization. Pyrazolo[3,4-d]pyrimidine nucleosides related to 2′-deoxyadenosine or 2′-deoxyguanosine show a similar behavior. The base pair stabilization is comparably small when identical substituents are present in the 5-position of 2′-deoxyuridine. The extraordinary stability of the 2b–d–dT base pairs is caused by better proton donor properties of the two amino groups of the 8-aza-7-deazapurin-2,6-diamine moiety and the better base stacking induced by the 7-substituents compared to that of 2-aminoadenine.

The Hotoda’s sequence substituted with TBDPS via 5′-end nucleobase existed as parallel quadruplex structure and exhibited inhibitory activities in an HIV-1 envelop proteins mediated cell–cell fusion assay. This result demonstrated that the 5′-aromatic groups of the Hotoda’s sequence are allowed to have a large spatial freedom and remain to be optimized for its role in the binding to HIV-1 envelop proteins.

We characterised DNA methylation and gene expression of four tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4, DR5, DcR1 and DcR2 in three choriocarcinoma (JAR, JEG-3, BeWo) and two transformed (HTR-8/SVneo and HPT-8) cell lines. DR4 mRNA was detected in JAR, JEG-3, BeWo and HTR-8/SVneo cells, whereas DR5 was present in all detected cells. DcR1 transcripts were expressed only in JAR, JEG-3 and BeWo cells, whereas DcR2 transcripts were detected only in HTR-8/SVneo and HPT-8 cells. Hypermethylated DR4 promoter was observed in JAR, JEG-3, BeWo and HTR-8/SVneo cells, hypermethylated DcR1 promoter in HTR-8/SVneo and HPT-8 cells and hypermethylated DcR2 promoter in JAR, JEG-3 and BeWo cells. Restoration of DR4, DcR1 and DcR2 expression with decreased DNA methylation of these genes was induced by the DNA demethylation agent 5-aza-2′-deoxycytidine (5-aza-CdR) in trophoblast cells, whereas DR5 expression did not exhibit any change. Significant negative correlation between the expression and DNA methylation of these genes was also observed. In all tested cell lines, only HPT-8 demonstrated sensitivity to TRAIL-induced apoptosis. Combined treatment with 5-aza-CdR and TRAIL resulted in apoptosis in JAR, JEG-3, BeWo and HTR-8/SVneo cells but not in HPT-8 cells. The results indicate that DNA methylation is associated with TRAIL receptor expression and might be involved in trophoblast apoptosis.

T-Hg-T base pair formation has been demonstrated to be compatible with duplex DNA context, with considerable thermal stability contribution. Here, the T-Hg-T stem in two small DNAzymes 8-17 and 10-23 was studied for its structural and functional roles. The recognition arm 5' to the cleavage site of 10-23 DNAzyme complex and the stem in the catalytic loop of 8-17 DNAzyme could be replaced by consecutive T-Hg-T stem of different length. The linear relationship between the activity of the complex 10-23DZ-6T+D19-6T and the concentration of Hg(2+) demonstrated that the T-Hg-T stem contributes thermal stability of the recognition arm binding. The effect of T-Hg-T stem in the catalytic core of 8-17 DNAzyme and the position-dependent effect in 10-23 DNAzyme demonstrated that T-Hg-T base pair is not compatible with canonical base pairs in playing the functions of nucleic acids.

Nucleoside analogues with imidazolyl and histidinyl groups were synthesized for site-specific modification on the catalytic core of 10-23 DNAzyme. The distinct position-dependent effect of imidazolyl group was observed. Positive effect at A9 position was always observed. The pH- and Mg(2+)-dependence of the imidazolyl-modified DNAzymes suggested that imidazolyl group in 10-23 DNAzyme probably plays a dual role, its hydrogen bonding ability and spacial occupation play the favorable influence on the catalytic conformation of the modified DNAzymes. This research demonstrated that the catalytic performance of DNAzymes could be enhanced by incorporation of additional functional groups. Chemical modification is a feasible approach toward more efficient DNAzymes for therapeutic and biotechnological applications.

The racial/ethnic disparities in DNA methylation patterns indicate that molecular markers may play a role in determining the individual susceptibility to diseases in different ethnic groups. Racial disparities in DNA methylation patterns have been identified in prostate cancer, breast cancer and colorectal cancer and are related to racial differences in cancer prognosis and survival.

Oligonucleotides containing halogenated `purine' and pyrimidine bases were synthesized. Bromo and iodo substituents were introduced at the 7-position of 8-aza-7-deazapurine-2,6-diamine (see 2b,c) or at the 5-position of uracil residues (see 3b,c). Phosphoramidites were synthesized after protection of 2b with the isobutyryl residue and of 2c with the benzoyl group. Duplexes containing the residues 2b or 2c gave always higher Tm values than those of the nonmodified counterparts containing 2′-deoxyadenosine, the purine-2,6-diamine 2′-deoxyribonucleoside (1), or 2a at the same positions. Six 2b residues replacing dA in the duplex 5′-d(TAGGTCAATACT)-3′ (11)⋅5′-d(AGTATTGACCTA)-3′ (12) raised the Tm value from 48 to 75° (4.5° per modification (Table 3)). Contrary to this, incorporation of the 5-halogenated 2′-deoxyuridines 3b or 3c into oligonucleotide duplexes showed very little influence on the thermal stability, regardless of which `purine' nucleoside was located opposite to them (Tables 4 and 5). The positive effects on the thermal stability of duplexes observed in DNA were also found in DNA⋅RNA hybrids or in DNA with parallel chain orientation (Tables 8 and 9, resp.).

The toxicity of sodium fluoride (NaF) to female fertility is currently recognized; however, the mechanisms are unclear. Previously, we reported a reduction in successful pregnancy rates, ovarian atrophy and dysfunction following exposure to NaF. The purpose of this study was to elucidate the underlying molecular mechanisms. Female Sprague-Dawley rats (10 rats/group) received 100 or 200mg/L NaF in their drinking water for 6 months or were assigned to an untreated control group. Apoptotic indices and oxidative stress indicators in blood and ovarian tissue were analyzed following sacrifice. The results confirmed the NaF-induced ovarian apoptosis, with concomitant activation of oxidative stress. Further investigations in ovarian granular cells showed that exposure to NaF activated extracellular regulated protein kinase (ERK) and c-Jun NH2 kinase (JNK), disrupting the ERK and JNK signaling pathways, while p38 and PI3K remained unchanged. These data demonstrated that oxidative stress may play a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways.

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating literatures have indicated that T cells are involved in this process. The first signal mediated by T-cell receptor/CD3 complex and the second signal delivered by costimulatory molecules are essential for the differentiation of T cell into an effector cell. Expression and function of CD28, an important costimulatory molecule, during early pregnancy in mice is still unclear. In the present study, we investigated the expression pattern of CD28 in mouse uterus during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, Western blotting, in situ hybridization, and immunohistochemistry (IHC). We found that injection of the uterine horn with CD28 antisense oligodeoxynucleotides leads to a decreased number of implantation sites. The expression pattern of CD3 protein examined by IHC is similar to that of CD28. These findings suggest that CD28 participates in the process of embryo implantation in mice, which might play its role through delivering the second costimulatory signal.

Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation.

Benzo(a)pyrene (BaP) is a ubiquitous environmental pollutant. Studies have demonstrated it to be an endocrine-disrupting chemical that can cause adverse effects on the female reproductive system. However, the effect of BaP on early pregnancy has not been reported. We investigated the effect of BaP on endometrial receptivity and embryo implantation. Pregnant mice were dosed with BaP at 0.2, 2 and 20 mg/kg/day from day 1 (D1) to day 5 (D5) of gestation. Exposure to BaP impaired the morphology of the endometrium and decreased the number of implantation sites (p0.2=0.006, p2=0.167, p20=0.003). Levels of estrodiol (p0.001, for three treatment group compare with control group) and progesterone-4 in plasma were elevated in BaP-treatment groups (p0.20.001, p20.001, p20=0.032). Expression of estrogen receptor-α was up-regulated (p0.2=0.002, p2=0.131, p20=0.024) whereas expression of the progesterone receptor was down-regulated (p0.20.001, p2=0.064, p20=0.021). Levels of receptivity-related genes HoxA10 (p0.20.001, p2=0.135, p200.001) and E-cadherin (p0.2=0.002, p2=0.624, p20=0.137) were changed by BaP. These results revealed that BaP can disrupt the balance of estrogen and progesterone, influence expression of their receptors and downstream related genes, lead to changes in endometrium receptivity, and reduce of the number of implantation sites.

To study the expression of Toll-like receptors (TLRs) mRNA in human trophoblast HTR-8/SVneo cells and the changes in indoleamine 2,3-dioxygenase (IDO) mRNA expression in response to TLR ligand stimulation.The expressions of TLRs and IDO mRNA in human HTR-8/SVneo cells were tested by RT-PCR, and the changes in IDO mRNA levels after exposure to TLR3, TLR4, TLR7/8, and TLR9 ligands were quantitatively analyzed with real-time PCR.IDO and TLR1-10 mRNAs were expressed in HTR-8/SVneo cells. As the cell culture time extended, IDO mRNA expression level tended to increase within 48 h. After stimulation with the TLR ligands, the expression of TLR-3 mRNA was down-regulated while the expression of TLR-4, 7, 8, and 9 mRNA up-regulated. Stimulation of the cells with poly(I:C) lowered the expression of IDO mRNA while IFN-γ increased its expression.The expression of IDO mRNA is associated with the nutrition of the maternal-fetal interface. Stimulation with the TLR ligands affects the expression of IDO and TLR mRNA expressions in the cells, which verifies the functional activity of TLRs and suggests a role of IDO in TLR pathway-dependent antiviral immunity.

Successful mouse embryo implantation requires a receptive uterus and an activated blastocyst. A large number of genes, cytokines, and other factors are involved in the process. MicroRNAs (miRNAs) regulate the expression of many genes, and previous studies have investigated the relationship between miRNA expression and embryo implantation. In this study, we show that mmu-microRNA-200a (mmu-miR-200a) is expressed in a spatiatemporal manner during implantation in mouse uterus and found that phosphatase and tensin homolog (PTEN), SON, and programmed cell death 4 (Pdcd4) are the target genes of mmu-miR-200a by bioinformatics analysis. In vitro gain and loss of function experiments confirm that PTEN, a critical gene for cell proliferation and apoptosis, is the target gene of mmu-miR-200a. Our experiments also show that injection of the uterine horn with mmu-miR-200a lentivirus leads to a decreased implantation rate. Collectively, our results suggest that mmu-miR-200a affects embryo implantation by regulating PTEN protein expression. Thus, clarifying the physiological functions of uterine miRNAs will help to elucidate the embryo implantation process and may even contribute to curing infertility and inventing new contraceptives.

Background This study attempts to determine the antimicrobial resistance profiles of obligate anaerobic bacteria that were isolated from a periodontal abscess and to evaluate the prevalence of resistance genes in these bacteria. Methods Forty-one periodontal abscess samples were cultivated on selective and non-selective culture media to isolate the oral anaerobes. Their antibiotic susceptibilities to clindamycin, doxycycline, amoxicillin, imipenem, cefradine, cefixime, roxithromycin, and metronidazole were determined using the agar dilution method, and polymerase chain reaction assays were performed to detect the presence of the ermF, tetQ, nim, and cfxA drug resistance genes. Results A total of 60 different bacterial colonies was isolated and identified. All of the isolates were sensitive to imipenem. Of the strains, 6.7%, 13.3%, 16.7%, and 25% were resistant to doxycycline, metronidazole, cefixime, and amoxicillin, respectively. The resistance rate for both clindamycin and roxithromycin was 31.7%. Approximately 60.7% of the strains had the ermF gene, and 53.3% of the amoxicillin-resistant strains were found to have the cfxA gene. Two nim genes that were found in eight metronidazole-resistant strains were identified as nimB. Conclusions In the present study, the Prevotella species are the most frequently isolated obligate anaerobes from periodontal abscesses. The current results show their alarmingly high resistance rate against clindamycin and roxithromycin; thus, the use of these antibiotics is unacceptable for the empirical therapy of periodontal abscesses. A brief prevalence of four resistance genes in the anaerobic bacteria that were isolated was also demonstrated.

10–23 DNAzyme has been used as a very good model for exploring the potential of functional nucleic acids. Its 15-mer catalytic core has been demonstrated to be the optimized in vitro selection result. However, with the introduction of protein-like functional groups through 2′-deoxyguanosine analogues, its two highly conserved guanine residues (G2 and G14) can be modified for more efficient 10–23 DNAzyme analogues. This result implies that chemical modifications of functional nucleic acids with well-designed nucleoside analogues of each canonical residue could be used as the first step in the efforts toward more powerful functions of nucleic acids.

The molecular structures of enzyme mimics may be modified to optimize their catalytic properties. In this study, to generate artificial enzyme mimics, Watson-Crick base paired DNA duplexes were designed as scaffolds which were assembled by nucleotides modified with specific functional groups. This process allowed various functional groups to be precisely assembled at different sites on the duplexes. By using this strategy, the 5-[2-(1H-imidazolyl-4)-(E)-ethylene]-2'-deoxythymidine (1) analog with the 5-substituted imidazolyl group was incorporated into single strands of DNA. Upon DNA duplex formation, several combinations of the imidazolyl group were formed. Using p-nitrophenyl acetate as the substrate of the catalytic reaction, we evaluated the hydrolysis capabilities of the imidazolyl assemblies. The catalytic ability was closely related to the distribution of imidazolyl groups in the DNA duplex. The most effective catalytic center was that of the duplex O5-O6 construct with three imidazolyl groups. This construct displayed bell-shaped pH-dependent and Mg(2+)-independent kinetic curves, which are typical characteristics of imidazolyl-mediated catalytic reactions.

Abnormal cell proliferation is a main driver of tumor formation and development, which involves the deletion, mutation, and downregulation of tumor suppressor genes. One study recently demonstrated that miR-200a plays an oncogenic role by inhibiting phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression. In the human endometrial adenocarcinoma cell line HEC-1B, suppression of miR-200a expression inhibited cell proliferation and promoted apoptosis, whereas its over-expression had no effect on proliferation and apoptosis. Furthermore, inhibition or over-expression of miR-200a increased or reduced the expression of PTEN, respectively, with no change in PTEN mRNA levels. These effects were achieved by directly targeting miR-200a to the 3′ untranslated region of the PTEN mRNA to inhibit its translation. Taken together, we propose that in HEC-1B cells, miR-200a functions as an oncogene, affecting proliferation and apoptosis by regulating the expression of the tumor suppressor PTEN at the translational level.

For the evacuation dynamics in indoor space, a novel crowd flow model is put forward based on Linear Fractional Stable Motion. Based on position attraction and queuing time, the calculation formula of movement probability is defined and the queuing time is depicted according to linear fractal stable movement. At last, an experiment and simulation platform can be used for performance analysis, studying deeply the relation among system evacuation time, crowd density and exit flow rate. It is concluded that the evacuation time and the exit flow rate have positive correlations with the crowd density, and when the exit width reaches to the threshold value, it will not effectively decrease the evacuation time by further increasing the exit width.

In the title compound, C(10)H(11)BrFN(5)O(4).C(3)H(6)O.H(2)O, the N-glycosylic bond torsion angle, chi, is anti [-108.0 (4) degrees ]. The sugar pucker is N-type [C2'-exo, (2)E with P = 346.5 (4) degrees and tau(m) = 34.5 (2) degrees ], and the conformation around the C-C bond linking the CH(2) group and the furan ring is -sc [torsion angle gamma = -70.0 (4) degrees ].

Modification on the catalytic core of 10-23 DNAzyme with protein-like functional groups is a potential approach to obtain its more efficient analogs. In our efforts for this purpose, a lead structure (DZ-2-9) with 8-aza-7-deaza-2'-deoxyadenosine at the A9 position in its catalytic core was obtained. Here we report our structure-activity relationship studies on this lead structure. Various functional groups of different chemical properties were introduced through the 7-substituents of 8-aza-7-deaza-2'-deoxyadenosine to DZ-2-9. The functional groups capable of forming hydrogen bonds, like amino and hydroxyl groups, are more favorable for catalytic rate enhancement than the large groups with spacial occupation, like phenyl and tert-butylphenyl groups, and the flexible alkyl linkage was the more preferred choice for optimizing their positive effect. Furthermore, they exerted positive effect cooperatively with the N8 atom. These results give us a clear hint in the design of compounds for A9 substitution of 10-23 DNAzyme for more efficient DNAzymes.

During the reproductive cycle, a number of genes controlling endometrial changes are regulated by DNA methylation, a common epigenetic modification. Because dietary folate affects DNA methylation, we determined whether a folate-deficient diet (FDD) alters DNA methylation in endometria of pseudopregnant mice, focusing on the homeobox A10 (Hoxa10) promoter. Mice were given an FDD or control diet for 40 to 45 days and examined on day 5 of pseudopregnancy. Compared to control mice, FDD mice had lower folate levels in liver and serum (P = .004). However, the FDD did not significantly affect DNA methylation within the cytosine-guanine dinucleotide (CpG)-rich Hoxa10 promoter, even when specific CpG sites were examined (P > .05). In endometrial tissue sections, the localization of anti-Hoxa10 staining was unchanged in FDD mice. Therefore, folate deficiency did not significantly affect promoter methylation or expression of Hoxa10.

Background Folate, one of the B vitamins, provides the one-carbon units required for methylation. Folate deficiency has been associated with many pathologies. However, much less is known about the effect of it on human reproduction, especially on implantation. The establishment of uterine receptivity is crucial for successful embryo implantation. Gene expression can be influenced by both heredity and epigenetics such as DNA methylation. However, it is not known whether the methylation and expression of genes related to uterine receptivity can be affected by folate levels. To explore whether folate deficiency affected the epigenetic regulation of genes related to uterine receptivity, and their influence on implantation, we investigated the methylation and expression of cadherin 1 (Cdh1), progesterone receptor (Pgr) and estrogen receptor 1 (Esr1) genes during implantation and the implantation efficiency using a folate-deficient pregnant mouse model. Methods Serum folate levels of pregnant mice were measured using the electro-chemiluminescence immunoassay. The methylation status of Cdh1, Pgr and Esr1 promoter regions was determined by methylation-specific PCR and bisulfite sequencing. The expression of Cdh1, Pgr and Esr1 in the implantation-site endometrium was examined by real-time PCR, western blot and immunohistochemistry. The number and the morphology of pinopodes, important morphological markers of endometrial receptivity, were examined using scanning electron microscopy. The number of implantation sites demarcated by distinct blue bands was recorded. Results Serum folate levels of the folate-deficient group were lower (5.42 ± 1.35 ng/ml, n= 42) than those of the normal group (24.13 ± 4 .26 ng/ml, n= 37; P = 0.003). Here we show that the methylation status and mRNA levels of Esr1 were decreased (P= 0.021, P= 0.045, respectively), while the Cdh1 and Pgr expression levels were slightly but not significantly elevated and the methylation status did not vary in the folate-deficient mice compared with the wild type. Neither the number nor morphology of pinopodes was affected by folate deficiency. Furthermore, folate deficiency did not affect the number of implantation sites in mice. Conclusions This study demonstrates for the first time that, unlike the effects on Esr1, folate deficiency in mice does not influence the methylation and expression of Pgr and Cdh1, two genes shown to be essential for uterine receptivity and embryo implantation. Embryo implantation in mice appears to be unaffected by a deficiency in folate, suggesting that abnormalities in a pregnancy caused by folate deficiency start to develop after implantation.