The methylation profile of the HERVWE1 promoter region, as determined by the pyrosequencing assay.

<p>There were 5 CG sites in HERVWE1 promoter region. The pyrosequencing assay was applied to investigate the five CG sites methylation profile. The bar chart showed the five CG sites methylation level. All of the CG sites methylation ratios were lower than 40%. The most hypermethylated site was the 3rd CG site. The most hypomethylated site was the 5th CG site. The black, darkgrey, and lightgrey bars were represented the smaller discordant twin group, larger discordant twin group, and singleton group respectively. The 1st, 2nd, and mean methlation level in smaller discordant twin group were lower than that in larger discordant twin group (P<0.01) and singleton group (P<0.01).</p

5-MC staining in placentas from discordant twins and singletons.

<p>In eukaryotic genomes, DNA methylation always occurs on a cytosine base, the fifth carbon of which is linked to a methyl group (-CH3). IHC staining with an anti-5-MC antibody can quantitatively detect the intra-nuclear 5-MC density and intensity. A–H show placental tissues with anti-5-MC IHC staining. The slices were incubated in a primary sheep polyclonal anti-5-methyl cytosine IgG antibody (diluted 1∶200) overnight. A goat anti-sheep secondary antibody (diluted 1∶400) was applied sequentially. The slices were developed with an immunoperoxidase system. 5-MC was mainly detected in the nucleus. Positive cells were stained brown. Negative cells were stained blue after counterstaining with hematoxylin. (A) The larger infant in discordant group (magnification 10×20). (B) The larger infant in discordant group (magnification 10×40). (C) The smaller infant in discordant group (magnification 10×20). (D) The smaller infant in discordant group (magnification 10×40). (E) A singleton infant (magnification 10×20). (F) A singleton infant (magnification 10×40). (G) The negative control (magnification 10×20). This singleton tissue slice was stained with 1% gelatin instead of the anti-5-MC antibody. (H) The negative control (magnification 10×40). Syncytiotrophoblast and cytotrophoblast cells are marked as S and C respectively. Stromal cells are marked as ST.</p

<i>HERVWE1</i> and <i>HCS</i> mRNA levels among the groups (<i>GAPDH</i>, <i>β-actin</i>, and <i>PCNA</i> as internal reference genes respectively).

<p>The values shown indicate the relative quantitation of <i>HERVWE1</i> and <i>HCS</i> mRNAs, which were standardized to <i>GAPDH</i>,<i>β-actin</i>,<i>and PCNA</i> respectively, and normalized to the singleton control group.</p><p>The data are shown as the means ± standard deviations.</p>†<p>P<0.01 vs. other four groups.</p

The correlation between DNMT3b3 transcript levels and HERVWE1 promoter region methylation level.

<p>The scatterplots showed the correlation between the HERVWE1 promoter region methylation level and the DNMTs transcript profile in discordant twin groups and singleton group. Using GAPDH as internal reference gene, DNMT3b3 transcript level was positively correlated with the HERVWE1 promoter region methylation level (A–C). Using β-actin as internal reference gene, DNMT3b3 transcript level was positively correlated with the HERVWE1 promoter region the 1st CG site methylation level (D).</p

The correlation between HERVWE1 promoter region methylation level and HERVWE1 transcript levels.

<p>The scatterplots showed the correlation between the HERVWE1 transcript profile and the HERVWE1 promoter region methylation level in discordant twin groups and singleton group. Using GAPDH as internal reference gene, the HERVWE1 transcript level was significant negatively correlated with the HERVWE1 promoter region methylation level (A–C). Using β-actin as internal reference gene, the HERVWE1 transcript level was significant negatively correlated with the HERVWE1 promoter region methylation level (D–F).</p

5-MC staining scores for the discordant twin and singleton group.

<p>Data are shown as the mean scores ± standard deviations.</p>*<p>P<0.01 vs. larger twin in discordant group;</p>†<p>P<0.01 vs. singleton group.</p

The correlation between the HERVWE1 staining score and the HERVWE1 mRNA level.

<p>The scatterplots showed the correlation between the HERVWE1 transcript profile and the HERVWE1 staining score in all five groups. Using GAPDH as internal reference gene, the HERVWE1 transcript level was significant positively correlated with the HERVWE1 staining score (spearman correlation coefficient 0.229, P<0.05).</p

DNMTs transcript levels among discordant twins and singleton group.

<p>The values shown indicated the relative quantitation of the DNMT mRNAs, which were standardized to GAPDH (A) and β-actin (B), normalized to the singleton control group. The DNMT 3b3 mRNA decreased and DNMT 3b7 mRNA increased in smaller discordant twin group compared with the larger discordant twin group and singleton group.</p

Univariate analysis of characteristics of new PTB cases among the migrant population and their association with delayed initial health-seeking (IHS).

<p>Univariate analysis of characteristics of new PTB cases among the migrant population and their association with delayed initial health-seeking (IHS).</p

Characteristics of new PTB cases among the migrant population recruited in Shanghai, Guangdong and Jiangsu, China, in 2008.

*<p>Median (1st quartile, 3rd quartile).</p