Placental lipoprotein lipase DNA methylation alterations are associated with gestational diabetes and body composition at 5 years of age

Figures & data

Table 1. Clinical characteristics of mothers and offspring from first trimester of pregnancy to 5 y of age.

	NGT	GDM
Participant clinical data	n = 42	n = 24	P value
Maternal characteristics
1^st trimester of pregnancy
Age (years)	28.6 ± 3.7	29.0 ± 3.1	0.69
BMI (kg/m^2)	25.3 ± 5.3	26.1 ± 4.1	0.54
Fasting glucose (mmol/L)	4.37 ± 0.38	4.49 ± 0.42	0.27
2^n^d trimester of pregnancy
Fasting glucose (mmol/L)	4.29 ± 0.46	4.50 ± 0.52	0.18^b
Fasting insulin (mU/L)	10.42 ± 12.27	10.33 ± 5.30	0.97^c
HOMA-IR	2.11 ± 2.37	2.14 ± 1.33	0.96^b
2 h post-OGTT glucose concentration (mmol/L)	6.25 ± 0.76	8.51 ± 0.73	<0.001
Weight gain 2^n^d – 1^st of pregnancy (kg)	6.00 ± 2.52	5.55 ± 2.13	0.44
HDL (mmol/L)	1.79 ± 0.45	1.73 ± 0.30	0.53
LDL (mmol/L)	3.6 ± 0.91	3.28 ± 0.77	0.10
Cholesterol (mmol/L)	6.28 ± 1.03	5.98 ± 0.76	0.19
Triglycerides (mmol/L)	1.85 ± 0.65	2.13 ± 0.73	0.13
3^r^d trimester of pregnancy
Fasting glucose (mmol/L)	4.23 ± 0.80	4.24 ± 0.45	0.95^d
Fasting insulin (mU/L)	13.08 ± 7.80	9.02 ± 4.17	0.01^e
HOMA-IR	2.52 ± 1.89	1.71 ± 0.85	0.08^e
Weight gain 3^r^d – 2^n^d of pregnancy (kg)	6.00 ± 2.34	2.82 ± 2.26	<0.001
HDL (mmol/L)	1.74 ± 0.44	1.73 ± 0.29	0.89^d
LDL (mmol/L)	3.97 ± 1.10	3.62 ± 1.00	0.20^d
Cholesterol (mmol/L)	6.93 ± 1.24	6.57 ± 1.09	0.24^d
Triglycerides (mmol/L)	2.74 ± 0.95	2.67 ± 0.69	0.73^d
Newborns' characteristics
Gestational age (week)	39.5 ± 1.0	39.5 ± 1.5	0.63
Sex (Boys/Girls)	19/23	12/12	0.78
Birth weight (kg)^l	3.46 ± 0.35	3.33 ± 0.53	0.23
Chest circumference (cm)	33.52 ± 1.50	33.04 ± 2.05	0.43^f
Head circumference (cm)	34.41 ± 1.60	34.52 ± 1.44	0.77 g
Cord blood leptin (ng/mL)	10.75 ± 8.22	6.79 ± 4.60	0.08^h
Cord blood glucose (mmol/L)	4.58 ± 0.87	4.59 ± 0.89	0.81^i
Cord blood triglycerides (mmol/L)	0.44 ± 0.15	0.51 ± 0.27	0.83^j
C-Peptide (ng/mL)	129.2 ± 81.9	174.6 ± 83.0	0.05^k
Children' characteristics
Age (years)	5.9 ± 0.7	5.9 ± 0.7	0.73
Weight (kg)^l	20.39 ± 2.56	18.80 ± 2.23	0.03
Height (m)^l	0.86 ± 0.05	0.88 ± 0.04	0.56
BMI (kg/m^2)^l	15.05 ± 1.35	14.55 ± 0.84	0.21
Fat mass (%)^m	18.32 ± 2.50	17.62 ± 2.14	0.34
Lean mass (%)^m	81.28 ± 3.64	82.40 ± 2.15	0.27

NGT, normal glucose tolerance; GDM, gestational diabetes mellitus.

^a n = 63 (40 NGT and 23 GDM);

^b n = 47 (29 NGT and 18 GDM);

^c n = 61 (41 NGT and 20 GDM);

^d n = 64 (42 NGT and 22 GDM);

^e n = 60 (41 NGT and 19 GDM);

^f n = 61 (40 NGT and 21 GDM);

^g n = 61 (40 NGT and 21 GDM);

^h n = 58 (35 NGT and 23 GDM);

ⁱ n = 46 (29 NGT and 17 GDM);

^j n = 62 (38 NGT and 24 GDM);

^k n = 52 (29 NGT and 23 GDM);

^l Statistical analysis are performed with z-scores; n = 60 (38 NGT and 22 GDM); Continuous variables are mean ± s.d. Significant P values (≤ 0.05) are indicated in bold and trends (P < 0.1) are in italic.

Figure 1. Schematic representation of the LPL gene and localization of the three epigenotyped regions. Region 1, containing 3 CpG sites, is located within the proximal promoter of the LPL gene. Regions 2 and 3, containing respectively 9 and 8 CpG sites, are located in the first intron of LPL gene, downstream the first exon. The grey square represents a CpG island. *p < 0.05; **p > 0.01.

Table 2. Placental DNA methylation levels at the LPL loci in children exposed and not exposed to gestational diabetes mellitus during fetal development.

CpG	NGT	GDM^a
dinucleotides	n = 42	n = 24	P value^b	P value^c	P value^d
CpG1.1	38.65 ± 17.71	25.67 ± 10.57	0.008	0.003	0.004
CpG1.2	22.93 ± 13.41	21.51 ± 14.80	0.59	0.47	0.61
CpG1.3	36.50 ± 15.99	39.41 ± 19.08	0.59	0.63	0.57
Mean CpGs 2.1 to 2.3	36.87 ± 14.20	29.56 ± 10.65	0.05	0.04	0.04
Mean CpGs 2.4 to 2.6	39.52 ± 11.06	36.38 ± 11.81	0.52	0.47	0.38
Mean CpGs 2.7 to 2.9	23.85 ± 7.28	23.15 ± 9.11	0.98	0.95	0.97
CpG 3.1	54.88 ± 13.86	52.22 ± 16.33	0.71	0.63	0.66
CpG 3.2	32.01 ± 12.36	30.78 ± 10.60	0.82	0.69	0.59
CpG 3.3	33.59 ± 14.08	30.46 ± 9.75	0.71	0.48	0.37
CpG 3.4	44.09 ± 14.92	38.04 ± 12.52	0.11	0.06	0.06
CpG 3.5	32.45 ± 11.71	30.69 ± 10.13	0.48	0.45	0.49
Mean CpGs 3.6 to 3.8	31.99 ± 8.80	28.95 ± 9.91	0.32	0.31	0.14

NGT, normal glucose tolerance; GDM, gestational diabetes mellitus.

^a GDM diagnosed as 2 h post-75 g OGTT glucose ≥ 7.8 mmol/L.

^b Not adjusted P values;

^c P values adjusted for gestational age and sex;

^d P values adjusted for pre-pregnancy age and BMI, gestational age and sex of the children.

P values ≤ 0.05 are indicated in bold and P values <0.1 are in italic.

Table 3. Growth in children exposed and not exposed to gestational diabetes mellitus during fetal development.

	NGT	GDM^a
Children's body parameter	n = 42	n = 24	P value^b	P value^c
Birth weight z-scores	−0.01 ± 0.74	−0.31 ± 1.00	0.17	0.10
Weight z-scores^d	0.05 ± 0.62	−0.43 ± 0.87	0.03	0.03
Height z-scores	0.01 ± 0.03	−0.01 ± 0.04	0.56	0.63
BMI z-scores^e	−0.17 ± 0.74	−0.40 ± 0.76	0.21	0.15
Fat mass (%)^f	17.08 ± 4.56	16.40 ± 4.61	0.24	0.24
Lean mass (%)^f	82.53 ± 5.26	83.60 ± 4.55	0.22	0.25

NGT, normal glucose tolerance; GDM, gestational diabetes mellitus.

^a GDM diagnosed as 2 h post-75 g OGTT glucose ≥ 7.8 mmol/L.

^b P values adjusted for gestational age and sex (fat and lean masses).

^c P values adjusted for pre-gestational age and BMI and gestational age at birth and sex (fat and lean masses).

^d Weight and

^e BMI z-scores for GDM-exposed child were different from zero, according to a one-sample t-test.

^f n = 60 (38 NGT and 22 GDM).

P values ≤ 0.05 are indicated in bold and P values <0.1 are in italic.

Figure 2. Comparison of anthropometric profiles in GDM and NGT offspring with WHO recommendations. A. Children's weight and; B. BMI z-scores were compared to WHO references using a one-sample T-test (with 0 as comparing group). Dotted line represent the WHO growth chart reference for boys and girls. The mean z-score is indicated by a bold grey line.

Figure 3. Associations between birth weight and body composition at age 5. Spearman correlation was evaluated between birth weight and children's A. weight and; B. BMI. p values are adjusted for children's age and sex. p values ≤0.05 are indicated in bold and p values <0.1 are in italic.

Figure 4. Associations between placental DNA methylation levels at LPL CpG dinucleotide 3.4 and children's body composition. Spearman correlation between DNA methylation levels and A. birth weight and children's; B. weight; C. BMI; D. height; E. lean mass and; F. fat mass. ap values adjusted for children's age and sex; bp values adjusted for gestational age and sex. p values ≤0.05 are indicated in bold and p values <0.1 are in italic.