Selection of imprinted genes

We selected 23 imprinted genes from the Geneimprint, Genecards, and Genebank databases for BS-PCR amplification with < 15% CV. Table S1 lists the information on these 23 target genes. Among the 23 imprinted genes, 8 were maternally expressed, 14 were paternally expressed, and 1 was biparentally expressed.

Study participants

A total of 583 participants were enrolled in this study for the methylation analysis of placental imprinted genes. These participants were divided into screening and validation cohorts. The screening cohort consisted of 20 PAS cases, 20 PE cases, and 20 CON participants, while the validation cohort consisted of 136 PAS cases, 288 PE cases, and 99 CON participants. In addition, part of the samples were further used for the detection of placental oxidative DNA damage (28 PAS cases, 28 PE cases, and 24 CON participants), placental gene expression analysis (17 PAS cases, 31 PE cases, and 21 CON participants), and serological analysis of umbilical cord blood (30 PAS cases, 30 PE cases, and 24 CON participants). Fig. S1 demonstrates the experimental design.

A comparison of the clinical data showed that systolic blood pressure, diastolic blood pressure, and urine protein were significantly higher in the PE groups than in the PAS and CON groups in both the screening and validation cohorts (Tables 1 and 2; P < 0.05). Additionally, pregnancy outcome-related parameters, such as gestational week at birth, preterm birth rate, and birth weight, were significantly lower in both the PE and PAS groups than in the CON groups in both cohorts (Tables 1 and 2; P < 0.05).

Table 1 Clinical information of the patients in the screening cohortTable 2 Clinical information of the patients in the validation cohortAlternation in the methylation of the target imprinted genes in the screening cohort

Analysis of the methylation levels of the 23 imprinted genes revealed that only 8 genes showed methylation alterations in the PE and PAS groups. Figure 1 shows the average methylation levels of these eight imprinted genes. The data demonstrated that the average methylation level of MEST decreased in the PE group, while that of KCNQ1 increased in the PE and PAS groups compared with the CON group (P < 0.05 and P < 0.01; Fig. 1D and F).

Fig. 1

Average methylation levels of the imprinted genes in the screening cohort. A PEG3, B GNAS, C IGF2, D MEST, E DLK1, F KCNQ1, G RB1, and H PEG10. Vertical coordinates represent the average DNA methylation level in the analyzed DMR. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM; *P < 0.05 and **P < 0.01

Figure 2 demonstrates the methylation levels at each CpG site in the DMR of the imprinted genes in the screening cohort. The results showed that IGF2, GNAS complex locus (GNAS), MEST, DLK1, KCNQ1, and paternally expressed 3 (PEG3) had significantly different DNA methylation modifications at a single CpG site in the PE, PAS, and CON groups (P < 0.05; Fig. 2). Moreover, the methylation level at specific CpG sites of IGF2, MEST, and DLK1 decreased, while that of GNAS, KCNQ1, and PEG3 increased in the PAS and/or PE groups compared with the CON group (P < 0.05; Fig. 2).

Fig.2

Methylation levels at individual CpG sites in the DMR of the imprinted genes in the screening cohort. A IGF2, B GNAS, C MEST, D DLK1, E KCNQ1, F RB1, G PEG10, and H PEG3. Vertical coordinates represent the methylation level, and the horizontal axis represents specific CpG sites in the analyzed DMR. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001

Alternation in the methylation of the target imprinted genes in the validation cohort

Based on the previous results, methylation levels of KCNQ1, MEST, GNAS, and IGF2 were further analyzed in the validation cohort. Figure 3 presents the average DNA methylation levels of KCNQ1, MEST, GNAS, and IGF2 in the validation cohort. The data showed that the average methylation levels of KCNQ1 increased significantly, while that of IGF2 decreased significantly in the PE and PAS groups compared with the CON group (P < 0.001; Fig. 3A and D). In addition, the average methylation level of GNAS decreased significantly in PAS but was unaltered in the PE group compared with the CON group (P < 0.001; Fig. 3C).

Fig.3

Average methylation levels of the imprinted genes in the validation cohort. A KCNQ1, B MEST, C GNAS, and D IGF2. Vertical coordinates represent the average DNA methylation level in the analyzed DMR. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM; **P < 0.01 and ***P < 0.001

The analysis of the methylation levels at individual CpG sites in the DMR of KCNQ1, MEST, GNAS, and IGF2 showed that the majority of the CpG sites had significantly higher or lower methylation levels in the PAS group than in the CON group (Fig. 4). Meanwhile, in the PE group, the alterations were only found in KCNQ1, MEST, and IGF2 (Fig. 4).

Fig.4

Methylation levels at individual CpG sites in the DMR of the imprinted genes in the validation cohort. A KCNQ1, B MEST, C GNAS, and D IGF2.Vertical coordinates represent the methylation level, and the horizontal axis represents specific CpG sites in the analyzed DMR. M and P indicate maternal and paternal allele expression, respectively. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM; *P < 0.05 and #P < 0.01

Gene expression analysis of relevant imprinted genes in placental tissues

The expression levels of target imprinted genes in placental tissues were quantified using qPCR (Fig. 5). The results revealed that mRNA expression of DLK1, MEG3, IGF2, H19, and MEST was upregulated in the PE group (P < 0.05) and downregulated in the PAS group (P < 0.001; Fig. 5A–C, E, F, and H). Additionally, the mRNA expression of RB1 was also significantly different between the PE and PAS groups (P < 0.05; Fig. 5B).

Fig.5

mRNA expression levels of imprinted genes in the placental tissue of the PE, PAS, and CON groups. A DLK1, B RB1, C MEG3, D PEG3, E IGF2, F H19, G KCNQ1, H MEST, I PLAGL1, J IGF2/H19, and K DLK1/MEG3. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM;*P < 0.05 and ***P < 0.001

Additionally, we analyzed the expression of two pairs of contradicting maternal and paternal imprinting genes H19/IGF2 and MEG3/DLK1 in the PE, PAS, and CON groups (Fig. 5J, K). The expression ratios of H19/IGF2 were 4.73, 0.80, and 21.19 and that of MEG3/DLK1 were 2.26, 1.60, and 3.64 in the CON, PAS, and PE groups, respectively. The results indicated that the maternal imprinted genes were significantly over-expressed in PE patients, while the paternal imprinted genes were significantly over-expressed in PAS patients.

Analysis of oxidative DNA damage in placental tissue

The placental tissue samples were analyzed for oxidative DNA damage (Fig. 6). The results showed that the 8-OHdG levels were increased in both PE and PAS groups but were significantly high in the PE group (P < 0.01; Fig. 6A). Additionally, an association analysis indicated that DNA methylation of IGF2 was positively correlated with 8-OHdG levels in the PE (P = 0.1029) and PAS (P = 0.0023) groups, respectively (Fig. 6B and C).

Fig.6

Levels of placental imprinted gene methylation, oxidative damage, and fetal growth factors in the PE, PAS, and CON groups. A The 8-OHdG levels in the placental tissue of the PE, PAS, and CON groups. B, C Association between IGF2 DNA methylation and 8-OHdG levels in the PAS and PE groups. D–G The IGF2, DLK1, cortisol, and cotinine levels in the umbilical CBS of the PE, PAS, and CON groups. CON, normal term controls; PAS, placenta accreta spectrum; and PE, pre-eclampsia. Data are represented as the mean ± SEM;*P < 0.05, **P < 0.01, and ***P < 0.001

Detection of IGF2, DLK1, cortisol, and cotinine levels in umbilical CBS

The IGF2, DLK1, cortisol, and cotinine levels in the umbilical CBS samples of the PE, PAS, and CON groups were detected using ELISA (Fig. 6D–F). The results revealed that IGF2 and cortisol levels were significantly decreased in the PE and PAS groups compared with the CON group (P < 0.001; Fig. 6D and E), while DLK1 and cotinine levels were not significantly different between the three groups (Fig. 6F and G).