Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues

Figures & data

Figure 1. Circadian clock. Model of the circadian core oscillator, showing the main core clock genes and transcription/translation feedback loops, as well as clock (controlled) genes (CCGs) that link the molecular clock to oscillating output processes. The core clock proteins are depicted as circles (CLOCK, ARNTL, CRY, PER, NR1D1).

Figure 2. Flowchart of the study population. EOPE: early-onset preeclampsia; LOPE: late-onset preeclampsia; CTRL: uncomplicated controls; FGR: fetal growth restriction; PTB: preterm birth; UCL: umbilical cord leukocytes; HUVEC: human umbilical vein endothelial cells.

Figure 3. Selection of circadian clock and clock-controlled genes and CpGs. AKT1: gene; V-Akt: murine thymoma viral oncogene homolog1; ARNTL: aryl hydrocarbon receptor nuclear translocator-like; ARNTL2: aryl hydrocarbon receptor nuclear translocator-like 2; BHLHE40: basic helix-loop-helix family, member e40; BHLHE41: basic helix-loop-helix family, member e41; CLOCK: clock circadian regulator; CRTC1: CREB-regulated transcription coactivator 1; CRY1: cryptochrome circadian clock 1; CRY2: cryptochrome circadian clock 2; CSNK1E: casein kinase 1, epsilon; DBP: D site of albumin promoter (albumin D-box) binding protein; FGF21: fibroblast growth factor 21; FOXO1: forkhead box O1; FOXO3: forkhead box O3; GSK3B: glycogen synthase kinase 3 beta; MAPK1: mitogen-activated protein kinase 1; NPAS2: neuronal PAS domain protein 2; NR1D1: nuclear receptor subfamily 1, group D, member 1; NR1D2: nuclear receptor subfamily 1, group D, member 2; PER1: period circadian clock 1; PER2: period circadian clock 2; PER3: period circadian clock 3; PRDX1: peroxiredoxin 1; PRDX2: peroxiredoxin 2; PRDX3: peroxiredoxin 3; PRDX5: peroxiredoxin 5; PRDX6: peroxiredoxin 6; PRKAA1: protein kinase, amp-activated, alpha 1 catalytic subunit; PRKAB1: protein kinase, AMP-activated, beta 1 non-catalytic subunit; PRKACA: protein kinase, CAMP-dependent, catalytic, alpha; PRKCA: protein kinase C, alpha; RORA: RAR-controlled orphan receptor A; RORB: RAR-controlled orphan receptor B; RORC: RAR-controlled orphan receptor C; SIRT1: sirtuin 1; SIRT2: sirtuin 2; STRA13: stimulated by retinoic acid 13; TIMELESS: timeless circadian clock; WEE1, WEE1 G2 checkpoint kinase.

Table 1. Maternal and newborn characteristics.

Maternal characteristics (n = 112)	EOPE (n = 13)	LOPE (n = 16)	Uncomplicated controls (n = 36)	FGR (n = 27)	Spontaneous PTB (n = 20)
Age (years)	30.0 ± 4.7	33.3 ± 4.5	31.8 ± 5.1	29.7 ± 6.0	31.0 ± 4.8
Nulliparous, n (%)	11 (84.6)	13 (81.2)	11 (30.6)*#	17 (63.0)	10 (50.0)
Peak systolic BP (mmHg)	169.5 ± 24.2	151.4 ± 11.8	125.7 ± 12.2*#	129.9 ± 14.3*#	124.9 ± 14.6*#
Peak diastolic BP (mmHg)	98.2 ± 19.1	93.9 ± 11.2	74.8 ± 7.5*#	76.8 ± 9.9*#	76.6 ± 13.1*#
Cesarean section, n (%)	11 (84.6)	5 (31.3)*	10 (27.8)*	9 (33.3)*	4 (20.0)*
Geographical origin, n (%) Western Non-Western	12 (92.3) 1 (7.7)	9 (56.3) 7 (43.8)	30 (83.4) 6 (16.7)	17 (63.0) 10 (37.0)	18 (90.0) 2 (10.0)
Preconception BMI (kg/m^2)	24.7 (10.1)	24.1 (4.4)	23.7 (4.8)	23.0 (6.5)	23.8 (6.1)
Smoking (yes), n (%)	2 (18.2)	0 (0.0)	0 (0.0)	2 (8.0)	2 (10.5)1
Comorbidity (yes), n (%)	2 (15.4)	10 (62.5)	19 (52.8)	11 (40.7)	8 (40.0)
Newborn characteristics
Male gender, n (%)	3 (23.1)	7 (43.8)	21 (58.3)	15 (55.6)	10 (50.0)
Gestational age at birth^a (weeks)	30.7 (3.4)	37.4 (1.9)*	39.9 (1.9)*#	38.9 (2.6)*	35.4 (7.9)*#
Birth weight^a (grams)	1185 (481)	3183 (1244)*	3713 (551)*#	2630 (595)*#	2660 (1805)*#
Birth weight <10th percentile, n (%)	1 (7.7)	3 (18.8)	0 (0.0)#	27 (100.0)*#	0 (0.0)#

Data represented as mean (± standard deviation) with corresponding ANOVA testing to examine overall differences between EOPE, LOPE, uncomplicated controls, FGR and spontaneous PTB, followed by the post hoc Dunnett t-test for pairwise comparisons versus both EOPE and LOPE. Data represented as number (%) with corresponding chi-square/Fischer’s exact testing.

^aSkewed data represented as median (interquartile range) with corresponding Kruskal–Wallis testing and post hoc Mann–Whitney testing.

*p <0.05 versus EOPE pregnancies. ^# p <0.05 versus LOPE pregnancies.

ANOVA: analysis of variance; BP: blood pressure; BMI: body mass index; EOPE: early-onset preeclampsia; LOPE: late-onset preeclampsia; FGR: fetal growth restriction; PTB: preterm birth.

Figure 4. Differentially methylated CpGs for the three different tissues. An overall ANOVA was performed to test for differentially methylated CpGs (p < 0.05) between all study groups. UCL: umbilical cord leukocytes; HUVEC: human umbilical vein endothelial cells.

Figure 5. EOPE and LOPE compared to different control groups (ANCOVA). ANCOVA, adjusted for gestational age at delivery, batch effect, and leukocyte count for UCL only was performed. p Values are shown in the columns. EOPE: early-onset preeclampsia; LOPE: late-onset preeclampsia; CTRL: uncomplicated controls; FGR: fetal growth restriction; PTB: preterm birth; UCL: umbilical cord leukocytes; HUVEC: human umbilical vein endothelial cells; n.s.: not significant.

Figure 6. Absolute DNA methylation levels in the EOPE, LOPE and (un)complicated control groups depicted at the gestational age of delivery. EOPE: early-onset preeclampsia; LOPE: late-onset preeclampsia; CTRL: uncomplicated controls; FGR: fetal growth restriction; PTB: preterm birth. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 1^E-4, *****p < 1^E-5.