Mitochondrial DNA methylation in placental tissue: a proof of concept study by means of prenatal environmental stressors

Figures & data

Table 1. Characteristics of the selected mother-newborn pairs, categorized by exposure group

Characteristics	Number (%) or mean (±SD or range)
	Control (n = 20)	Air Pollution (n = 20)	Smokers (n = 20)
Maternal
• Age (years)	31 (24–40)	32 (24–40)	31 (23–40)
• Pre-gestational BMI (kg/m^2)	23.9 (± 5.3)	22.9 (± 4.3)	24.0 (± 4.0)
• Maternal education
- Low	1 (5%)	4 (20%)	12 (60%)(*)
- Middle	8 (40%)	4 (20%)	7 (35%)
- High	11 (55%)	12 (60%)	1 (5%)
• Parity
- 1	9 (45%)	6 (30%)	7 (35%)
- 2	7 (35%)	12 (60%)	7 (35%)
- ≥ 3	4 (20%)	2 (10%)	6 (30%)
Newborn
• Gender
- Male	13 (65%)	13 (65%)	13 (65%)
- Female	7 (35%)	7 (35%)	7 (35%)
• Ethnicity
- European	15 (75%)	17 (85%)	19 (95%)
- Non-European	5 (25%)	3 (15%)	1 (5%)
• Gestational age (weeks)	39.2 (37–41)	39.0 (35–41)	39.3 (36–41)
• Apgar score (after 5 minutes)
- ≥ 8	2 (10%)	1 (5%)	2 (10%)
- 9	10 (50%)	5 (25%)	6 (30%)
- 10	8 (40%)	14 (70%)	12 (60%)
• Birth weight (g)	3394 (± 522)	3390 (± 401)	3149 (± 300) (†)
Exposure
• PM2.5 Exposure (µg/m^3)
- Third trimester	7.3 (± 1.0)	22.5 (± 3.0) (*)	11.7 (± 4.4) (*)
- Whole pregnancy	10.6 (± 1.7)	16.0 (± 1.4) (*)	13.3 (± 2.5) (*)
• BC Exposure (µg/m^3)
- Third trimester	0.7 (± 0.2)	1.9 (± 0.3) (*)	1.2 (± 0.4) (*)
- Whole pregnancy	0.9 (± 0.1)	1.8 (± 0.3) (*)	1.3 (± 0.4) (*)
• Smoking behaviour
- Number of pack years	/	/	15.1 (± 3.5)
- Number of cigarettes (/day)	/	/	13.2 (± 4.9)

Asterisks (*) indicate significantly differences in characteristics between the exposure groups (‘Air Pollution’ and ‘Smokers’) versus the ‘Control’ group at a 95% confidence level using a Chi-squared test for categorical variables and a Student’s t-test for numerical variables. Likewise, daggers (†) indicate borderline significant differences (p < 0.10). Abbreviations: PM_2.5: Particulate Matter with aerodynamic diameter smaller than 2.5 µm, BC: Black Carbon.

Figure 1. Mean mitochondrial DNA methylation levels as determined by bisulphite pyrosequencing (n = 20/group). The horizontal line is equivalent to the median. The lower and upper hinges represent the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR (= interquartile range) from the hinge, whereas the lower whisker extends from the hinge to the smallest value no further than 1.5 * IQR of the hinge. Data outside this range is represented as dots

Table 2. Differences in mtDNA methylation levels between exposure groups as determined by linear mixed models

	Control	Air Pollution group				Smoker group
DNA region	Mean	β (%)	Lower CI (%)	Upper CI (%)	p-value interaction	β	Lower CI (%)	Upper CI (%)	p-value interaction
D-loop	Ref	0.47	0.20	0.73	0.61	0.36	0.06	0.66	0.12
LDLR2	Ref	0.81	− 0.17	1.78	0.09	0.49	− 0.78	1.77	0.23

All models were adjusted for maternal education, parity, and birth weight. Models for the ‘Smokers’ group were additionally adjusted for exposure to PM_2.5 during the third trimester.

Table 3. Association between placental mtDNA methylation levels and newborn birth weight

	Birth weight (g)
DNA Region	β	Lower CI (%)	Upper CI (%)
D-loop
• Unadjusted	−125.63	−230.43	−20.83
• Adjusted	−106.98	−209.60	−4.36
LDLR2
• Unadjusted	0.02	−1.32	1.36
• Adjusted	−38.14	−165.78	89.50

Estimates (β) are presented for an IQR increase in mtDNA methylation (+0.42% for D-loop, +1.64% for LDLR2). Adjusted models included covariates for maternal age, gestational age, and parity.

Figure 2. Correlation between mitochondrial DNA content and absolute mitochondrial DNA methylation levels in placenta tissue for the D-loop (a) and LDLR2 (b) regions