Placental DNA methylation changes in gestational diabetes mellitus

Figures & data

Table 1. Participants’ characteristics in the study (N = 48).

Variables	GDM(n = 24)	Control(n = 24)	p-value
	Mean ± SD or n(%)
Maternal age (years) ^a	28.50 ± 2.15	27.29 ± 4.42	0.237
Pre-pregnancy BMI (kg/m^2) ^a	22.21 ± 3.98	20.87 ± 2.18	0.154
Education ^b			0.219
<high school	0	2(8.33)
high school or equivalent	10(41.67)	5(20.83)
> high school	14(58.33)	17(70.84)
Prenatal systolic pressure (mmHg) ^a	113.83 ± 12.25	114.17 ± 10.26	0.919
Prenatal diastolic pressure (mmHg) ^a	68.54 ± 7.81	70.83 ± 6.93	0.288
Fasting glucose (mmol/L) ^a	4.79 ± 0.62	4.33 ± 0.29	0.002
1 hr OGTT (mmol/L) ^a	9.69 ± 1.58	7.40 ± 1.21	<0.001
2 hr OGTT (mmol/L) ^a	8.26 ± 2.07	5.81 ± 1.11	<0.001
TC (mmol/L) ^a	6.04 ± 1.14	6.08 ± 0.99	0.895
TG (mmol/L) ^a	3.34 ± 1.33	3.75 ± 1.74	0.365
HDL (mmol/L) ^a	1.88 ± 0.29	1.77 ± 0.34	0.214
LDL (mmol/L) ^a	3.60 ± 1.31	3.59 ± 0.97	0.994
ApoA1 (g/L) ^a	1.70 ± 0.21	1.66 ± 0.20	0.501
ApoB (g/L) ^a	1.17 ± 0.32	1.16 ± 0.22	0.860
Lp(a) (mg/L) ^a	179.09 ± 175.07	198.13 ± 219.12	0.744
Gestational age at delivery (weeks) ^a	39.40 ± 0.90	39.77 ± 0.90	0.156
Infant sex ^b			0.146
Boy	13(54.17)	8(33.33)
Girl	11(45.83)	16(66.67)
Birth weight (g) ^a	3462.50 ± 421.51	3287.92 ± 270.77	0.095

Pearson Chi-Square tests for categorical variables, and independent-samples t-test for continuous variables. Data are expressed as ^a mean ± standard error of the mean, or as ^b count (percentage).

Abbreviation: GDM, gestational diabetes mellitus; BMI, body mass index; FG, fasting glucose; OGTT, oral glucose tolerance test; TC, total cholesterol; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein; ApoA1, Apolipoprotein A1; ApoB, Apolipoprotein B; Lp(a), Lipoprotein (a).

Figure 1. Comparison of distribution and methylation levels of all differentially methylated genes across the human genome between five GDM samples and five healthy controls. Up-regulated and down-regulated genes after screening are indicated in green and yellow, respectively. The outer ring with numbers 1 to 22 respectively represents 22 autosomes. The inner ring with red and blue pattern shows the methylation levels of DMGs. Circle graph of DMGs throughout autosomal chromosome showing -log10 (p-value).

Figure 2. (a) Proportion of differentially methylated CpG sites identified by RRBS analysis from 5 GDM samples and 5 healthy controls in relation to genomic location across the human genome. UTR: untranslated region; CDS: coding domain sequence. (b) The volcano plot of all differentially expressed genes comparing 4 GDM samples with 4 control groups (p-value <0.05) including up-regulated genes (yellow dots) and down-regulated genes (green dots) by RNA-Seq analysis.

Figure 3. The Venn graph integrated RRBS and RNA-Seq results showing the whole number of aberrantly methylated-differentially expressed genes overlapped including five up-regulated genes (a) and nineteen down-regulated genes (b). (c) A total of 24 screened genes were shown in heatmap according to the expression levels between four GDM samples and four healthy controls, which were also signed in Figure 1 identifying their location in chromosomes.

Figure 4. Graph of differentially methylated regions in TRIM67 gene assessed by pyrosequencing (a) and diagram plots of nucleotide sequence of three representative transcription factors binding sites (b, c, d). (a) CpG sites 3, 5, 7 and 9 exhibited significant hypermethylation levels (blue solid circular) in TRIM67 promoter between GDM samples and healthy controls. Most representative TF (b)GATA2, (c)TEAD4, and (d)NR2F2 in TRIM67 promoter were selected and identified by JASPAR database. The size of the capital letters at each position is proportional to the frequency of nucleotide bases.

Figure 5. Comparison with the methylation level of TRIM67 CpG sites between 24 GDM samples and 24 healthy controls (NC) by pyrosequencing analysis. Pearson Chi-square test, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Table 2. Basic information for the top five candidate differentially expressed genes between 24 GDM samples and 24 healthy controls.

ID	Genes	log2FC	t	B	Location	FDR p-value	Expression
ENSG00000128709	HOXD9	3.3599	2.5548	−4.2906	Chr2:176,122,719–176,124,937	0.0331	Down
ENSG00000119283	TRIM67	2.5555	2.7175	−4.2606	Chr1:231,162,058–231,221,565	0.0257	Down
ENSG00000128918	ALDH1A2	2.2201	0.8034	−4.6246	Chr15:57,953,429–58,065,711	0.041	Down
ENSG00000183117	CSMD1	−2.425	−1.3792	−4.5472	Chr8:2,935,353–4,995,035	0.027	Up
ENSG00000125618	PAX8	1.3525	2.457	−4.3092	Chr2:113,215,997–113,278,921	0.0387	Down

Table 3. Detail information for representative predicted transcription factors in the promoter region of TRIM67 predicted using JASPAR database.

Matrix ID	TF	Class	Score	Relative score	Location of TFBS in TRIM67 promoter
MA0809.1	TEAD4	TEA domain factors	5.388	0.835	Ch1: 231,161,366–231,161,375
MA0036.1	GATA2	C3 zinc finger-type factors	6.232	0.976	Ch1: 231,161,508–231,161,512
MA1111.1	NR2F2	Nuclear receptors with C4 zinc fingers	7.041	0.826	Ch1: 231,161,403–231,161,413

Abbreviation: TF, transcription factors; TFBS, transcription factor binding sites; Ch, Chromosome.

Figure 6. The Gene Ontology (GO) enrichment bar graph of up-regulated genes (red bars) and down-regulated genes (green bars) among all methylated-differentially expressed genes.

Figure 7. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways bubble chart of all methylated-differentially expressed genes. Data are presented as enriched scores expressed as -log₁₀(p-value).

Table 4. Spearman’s correlation analysis showing the association between differentially methylated CpG sites of TRIM67 and fasting glucose, 1 h OGTT, 2 h OGTT, and Lp(a) between 24 GDM samples and 24 healthy controls.

TRIM67 CpG sites	FG(mmol/L)		1 h OGTT(mmol/L)		2 h OGTT(mmol/L)		Lp(a)(mg/L)
	Rho	p-value	Rho	p-value	Rho	p-value	Rho	p-value
Site3	0.154	0.295	0.448	0.001	0.325	0.024	−0.284	0.053
Site5	0.241	0.098	0.565	<0.001	0.427	0.002	−0.372	0.01
Site7	0.24	0.101	0.465	0.001	0.396	0.005	−0.219	0.139
Site9	0.209	0.153	0.266	0.067	0.216	0.141	−0.443	0.002

Spearman’s correlation coefficient (Rho) is shown with significance at p < 0.05.

Abbreviation: FG, fasting glucose; OGTT, oral glucose tolerance test; Lp(a), Lipoprotein (a).

Table 5. Linear regression analysis of differentially methylated TRIM67 CpG sites and glucose/lipid metabolism, adjusting for age and BMI between 24 GDM samples and 24 healthy controls.

Variables	FG (mmol/L)		1 h OGTT (mmol/L)		2 h OGTT (mmol/L)		Lp(a) (mg/L)
	β(95%CI)	p-value	β(95%CI)	p-value	β(95%CI)	p-value	β(95%CI)	p-value
Site3	−0.017(−0.063;0.029)	0.460	0.020(−0.118;0.158)	0.768	0.001(−0.169;0.171)	0.994	−12.31(−28.75;4.13)	0.138
Site5	0.022(0.001;0.043)	0.042	0.193(0.051;0.336)	0.009	0.099(−0.076;0.274)	0.261	1.33(−15.03;17.69)	0.870
Site7	−0.001(−0.054;0.051)	0.962	−0.009(−0.168;0.150)	0.913	0.125(0.034;0.215)	0.008	15.29(−2.96;33.54)	0.098
Site9	0.023(−0.014;0.059)	0.211	−0.034(−0.144;0.076)	0.537	−0.052(−0.187;0.084)	0.447	−16.27(−29.05;-3.49)	0.014
Age	0.027(−0.017;0.071)	0.223	0.038(−0.094;0.171)	0.562	0.129(−0.035;0.292)	0.119	7.41(−8.29;23.11)	0.346
BMI	0.037(−0.012;0.087)	0.136	0.179(0.029;0.328)	0.020	0.107(−0.077;0.292)	0.245	−5.733(−23.02;11.55)	0.506

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.