Main

Intrauterine growth retardation (IUGR) is a common obstetrics complication, and is defined as a birthweight and/or length below the 10th percentile for the corresponding gestational age and an abdominal circumference that is less than the 2.5th percentile, with pathologic restriction of fetal growth (1). The prevalence of IUGR is about 8% worldwide (2). Increasing evidence supports that IUGR is related to increased prenatal morbidity and a higher susceptibility to some adult diseases in the offspring, such as the metabolic syndrome (3).

The etiology of IUGR is largely attributed to the adverse intrauterine environment during pregnancy. It is widely reported that maternal exposure to certain exogenous factors, such as maternal undernutrition and smoking, can inhibit fetal development by affecting the expression of developmental genes (4, 5). Epigenetics, which regulates the gene expression without changing the gene sequence, explains the relationship between gene expression and environmental signals (6). DNA methylation is a major epigenetic mechanism that can affect the stable maintenance of gene expression patterns (7). Reamon-Buettner et al. (8) observed that gestational protein deficiency could induce site-specific hypomethylation of the Wnt2 promoter region in the placenta and cause IUGR in rats. In our previous study, increased DNA methylation and decreased expression levels of several developmental genes (e.g., steroidogenic factor-1 and scavenger receptor class B type I) were observed in adrenals of IUGR fetal rats under prenatal caffeine ingestion (9, 10). Therefore, aberrant DNA methylation of developmental genes could be the underlying epigenetic mechanism for adverse intrauterine environment-induced IUGR.

Besides regulating gene expression in tissues and organs, DNA methylation status stably exists in body fluids (3, 7). As one of the readily available body fluids, maternal blood provides an avenue for signal exchange between the fetus and the mother (11). Thus, DNA methylation analysis in maternal peripheral blood may contribute to prenatal diagnosis or risk prediction, which has been proved in noninvasive prenatal diagnosis of trisomy 21 and congenital heart defects (12, 13). For IUGR, Kim et al. (14) first observed increased DNA methylation frequency of fetal-derived RASSF1A in maternal plasma of IUGR cases. However, no reports have focused on using high-throughput profiling of genomic DNA methylation in maternal blood to screen out IUGR.

In this study, we aimed to search for IUGR-related DNA methylation status in maternal peripheral blood. MeDIP array analysis was used to search for differentially methylated genes in the maternal blood of a nicotine-induced IUGR rat model. On the basis of the results of GO analysis, one placenta-associated gene was selected for bisulfite-sequencing PCR (BSP) confirmation in maternal blood. The role of the selected gene in IUGR was confirmed in the placentae. This study will provide evidence for the non-invasive prenatal screening of IUGR and for exploring the possible epigenetic mechanism of IUGR.

Methods

Chemicals and Reagents

Nicotine was obtained from Sigma-Aldrich (St Louis, MO). Isoflurane was purchased from Baxter Healthcare (Deerfield, IL). The DNeasy Blood & Tissue Kit was provided by Qiagen (Hilden, Germany). The pGEM-T Easy vector was from Promega (Madison, WI). The EZ DNA methylation kit (No. D5006) was provided by Zymo Research (Orange, CA). The Trizol reagent was purchased from Invitrogen (Carlsbad, CA). The primers were synthesized by Sangon Biotech (Shanghai, China). All chemicals and reagents were of analytical grade.

Animals and Treatment

Specific pathogen-free Wistar rats (20 females weighing 180-220 g vs. 10 males weighing 260–300 g) were obtained from the Experimental Center of Hubei Medical Scientific Academy (No. 2008-0005, Hubei, China). The animal studies were performed at the Center for Animal Experimentation of Wuhan University (Wuhan, Hubei, P.R. China), which has been accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The study protocol was designed in accordance with the Guidelines for Animal Research and was approved by the Ethical and Research Committee of the Medical College of Wuhan University.

The animals were housed under standard conditions and allowed free access to standard chow and water. They were allowed to acclimatize for at least one week before being subjected to experimental conditions. Each male rat was mated with two female rats, and the occurrence date of the vaginal plug or sperm in the vaginal smear was considered as gestational day (GD) 0. The pregnant rats were randomly assigned to two groups. The pregnant rats in the nicotine group were subcutaneously administered nicotine (1 mg/kg) twice per day from GD11 to GD20, and the control group was administered the same volume of vehicle (saline). On GD20, the pregnant rats were anesthetized with isoflurane and killed by decapitation. The maternal blood was collected in sodium citrate tubes. Each feto-placental unit was quickly removed from the uterus. The number of pregnant rats in each group was set to 8 (the litter size of each pregnant rat was 8–14). The fetuses were weighed after being dried on filter paper. Three placentae from each group were randomly selected for fixing in 10% neutral formalin solution for 24 h to prepare paraffin slices. The remaining placentae were stored at −80 °C. The IUGR rate was calculated according to the previously reported criteria for IUGR (4). All blood samples were stored at 4 °C and were subjected to genomic DNA extraction within 1 week.

Genomic DNA Extraction and MeDIP Array Analysis

Genomic DNA was extracted from maternal blood using the DNeasy Blood & Tissue kit according to the manufacturer’s protocol. Three samples from each group were selected randomly for DNA methylation array assays in KangChen Bio-tech (Shanghai, China). Rat DNA Methylation 385k CpG Plus Promoter Array from Roche NimbleGen (Madison, WI) was used to determine DNA methylation patterns in maternal blood. The t test was performed between the two groups to calculate the P values for each probe. Probes with P values <0.05 were taken as differentially methylated probes, which were further analyzed to identify the differentially methylated region (DMR)-associated genes. GO analysis was applied to determine the roles of these DMR-associated genes and to screen out developmental genes needed subsequent confirmation.

BSP Analysis of Syncytin b (Synb) in Maternal Blood

The methylation status of the Synb promoter was determined by the BSP method. Genomic DNA in maternal blood (3 control vs. 3 nicotine) was isolated with a DNeasy Blood & Tissue kit and then subjected to bisulfite modification using the EZ DNA methylation kit. The instruction manual of the EZ DNA methylation kit demonstrated that 500 pg–2 μg of DNA samples can be input in each reaction, and the bisulfite conversion efficiency could become higher than 99% under the optimal amount of DNA input, which is 200 ng–500 ng. Thus, we input 500 ng genomic DNA for every bisulfite conversion. The bisulfite-treated genomic DNA was amplified by PCR using the Thermal Cycler from Applied Biosystems (Foster City, CA). PCR products were electrophoretically separated and purified. The products were cloned into the pGEM-T easy vector for Sanger sequencing. Five clones were sequenced for each DNA sample. The percentage of methylation was calculated from the number of methylated CpG divided by the total CpG loci.

For PCR amplification, we used two pairs of primers (Synb-1 and Synb-2) covering almost the entire CpG-rich region of the proximal Synb promoter. The primers were designed by Methyl Primer Express 1.0 from Applied Biosystems. The primer sequences of Synb-1 (-325 bp~34 bp) were as follows: forward, 5′-TTTTGGGAAGGGATTTTTTTA-3′; and reverse, 5′-AAAAAACAAAACAAAAAACCCA-3′. Primers of Synb-2 (28 bp~422 bp) were as follows: forward, 5′-TATTTTTGGGTTTTTTGTTTTG-3′; and reverse, 5′-TCCTTCTCAAAATATATCACAAAA-3′. The reaction mixture contained 5 μl of 10 × PCR Buffer, 5 μl of genomic DNA template, 4 μl of 2.5 mM dNTPs, 1 μl of 10 μM of each primer, 0.5 μl of 5 U/μl Taq HS, and 34.5 μl of DEPC-H2O. The amplification conditions were as follows: initial denaturation at 98 °C for 2 min; 45 cycles of denaturation at 95 °C for 30 s, annealing at 61 °C (Synb-1) or 59 °C (Synb-2) for 30 s, and extension at 72 °C for 30 s; and a final elongation step at 72 °C for 10 min.

Hematoxylin and Eosin Staining in Placentae

For histopathological measurement, neutral formalin-fixed placentae were processed into 5-μm-thick paraffin sections using standard histological techniques. Sections were stained with hematoxylin and eosin (HE) and were observed under a light microscope.

RNA Extraction and Real-Time Reverse-Transcription PCR in Placentae

Total RNA was isolated from the placentae using Trizol reagent by following the manufacturer's protocol. Detailed protocols for reverse-transcription, primer design, and PCR condition have been published in our previous study (9). The mRNA content of Synb in each sample was normalized by the mRNA content of glyceraldehyde phosphate dehydrogenase (GAPDH). Primers for Synb (annealing at 60 °C) are as follows: forward primer, 5′-TCTGGGGACCACTCTTTACTG-3′; and reverse primer, 5′-GGGCTTTGAGAAGATTAGGG-3′. Primers for GAPDH (annealing at 60 °C) are as follows: forward primer, 5′-GCAAGTTCAACGGCACAG-3′; and reverse primer, 5′-GCCAGTAGACTCCACGACA-3′.

Statistical Analysis

SPSS 15.0 from SPSS Science Inc. (Chicago, IL) and Graphpad Prism 5.0 from GraphPad Software (La Jolla, CA) were used for data analysis. All measurement data were expressed as mean±SD and evaluated with Student’s t-test. Enumeration data, such as IUGR rates, were first calculated and then arcsine square-root transformed to make the data following normal distribution. The methylation frequencies of the two groups were compared statistically by Pearson’s χ2 test (15). Statistical significance was set at P<0.05.

Results

Developmental Parameters

As an important index for diagnosing IUGR, the fetal body weight significantly decreased in the nicotine group (P<0.01, Figure 1a) (16), and the nicotine group showed a higher IUGR rate compared with the control (P<0.01, Figure 1b). Thus, the nicotine-induced IUGR rat model was established successfully in this study.

Figure 1
figure 1

Effects of prenatal nicotine exposure on fetal weights and intrauterine growth retardation (IUGR) rates in rats on gestational day 20. (a) Fetal weights; (b) IUGR rates. Mean±SD, n=8 pregnant rats, **P<0.01 vs. control.

Full size image

MeDIP Array Analysis in Maternal Peripheral Blood

Genomic DNA was extracted from whole-blood samples and was analyzed by MeDIP array assays. Generally, there were 2,269 hypermethylated DMR-associated genes and 2,092 hypomethylated DMR-associated genes in the nicotine group. The hypermethylated and hypomethylated clusters related to fetal development, as per the GO analysis, are listed in Tables 1 and 2, respectively. Eight of the listed clusters contained both hypermethylated and hypomethylated genes, including epidermis development, limb development, muscle development, neurodevelopment, respiratory system development, sensory system development, skeletal development, and urogenital system development. Hypermethylated genes also demonstrated in clusters about digestive system development, endoderm development, heart development, immune system development, leukocyte development, liver development, mesoderm development, placental development, and vascular system development.

Table 1 Hypermethylated gene clusters related to fetal development in maternal peripheral blood on GO analysis
Full size table
Table 2 Hypomethylated gene clusters related to fetal development in maternal peripheral blood on GO analysis
Full size table

Aberrant Methylated Genes Related to Placental Development in Maternal Peripheral Blood

We searched for fetal development-related aberrant methylated genes among the clusters listed in Tables 1 and 2. The placenta serves as an essential barrier for fetal development, and placental dysfunction is taken as one of the predominant causes of IUGR. Thus, we analyzed genes related to placental development to screen out the gene for further analysis. There were four hypermethylated genes regulating placental development in the nicotine group (Table 3). The DNA methylation status of Synb demonstrated the most significantly change in this cluster.

Table 3 Differentially methylated region (DMR)-associated genes involved in placental development
Full size table

DNA Methylation Status of Synb Promoter in Maternal Peripheral Blood

BSP analysis was employed to validate the DNA methylation status of Synb in maternal peripheral blood. Figure 2a demonstrated the total methylation map of the CpG-rich regions (−325 bp~422 bp) of the rat Synb promoter in the two groups. As shown in Figure 2b, the total methylation rate of the Synb promoter in the nicotine group was significantly higher than that of the control (P<0.05). Among the 14 CpG sites, nt −52 and 160 showed increased frequency of single CpG methylation after nicotine treatment (P<0.05).

Figure 2
figure 2

DNA methylation status of syncytin b (Synb) proximal promoter in maternal peripheral blood. The methylation rate of CpG-rich regions (−325 to 422 bp) within the human Synb proximal promoter was detected by bisulfite-sequencing PCR (BSP). (a) The total methylation map of the human Synb proximal promoter. Black and white circles represent methylated and unmethylated CpGs, respectively. Each horizontal row of circles represents 14 CpG sites. (b) The total methylation rate of CpG-rich regions within the proximal promoter of the Synb gene. The results were evaluated with Pearson’χ2 test. n=3 pregnant rats, *P<0.05 vs. control.

Full size image

Histopathological Changes and Synb Expression in Placentae

Changes in placental structure were examined by HE staining. Syncytiotrophoblast hyperplasia and larger formation of syncytial knots were observed in the villous stroma of nicotine-treated placentae (Figure 3c), which were not evident in the control (Figure 3a). To confirm the role of Synb in placentae in our model, Synb expression was verified by real-time real-time reverse-transcription PCR in placentae. As shown in Figure 3e, prenatal nicotine exposure significantly reduced the Synb mRNA level to 64.9% of the control (P<0.01).

Figure 3
figure 3

Histopathological changes and syncytin b (Synb) expression in placentae. Paraffin sections were stained with hematoxylin and eosin (HE) and observed under a light microscope. (a) Control (× 100); (b) control (× 200); (c) nicotine (× 100); (d) nicotine (× 20). (e) Real-time reverse-transcription PCR was used to verify the mRNA expression of Synb. Each sample was normalized on the basis of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA content. Mean±SD, n=8 pregnant rats, **P<0.01 vs. control.

Full size image

Discussion

Establishment of Nicotine-Induced IUGR Rat Model

Maternal cigarette smoking is a common risk factor for IUGR, although ~20% of women continue smoking during pregnancy (17, 18). As one of the most aversive smoking products, nicotine can disturb fetal development and cause IUGR (18, 19). Both epidemiological investigations and animal experiments showed the association between prenatal nicotine exposure and increased IUGR rates (20, 21). Thus, prenatal nicotine exposure-induced IUGR is a recognized model and is widely used in IUGR-associated research. Usually, a period of 1–2 weeks is needed to establish a stable IUGR rat model through prenatal nicotine exposure (18). In this study, we referred to our previous studies and chose 1 mg/kg of nicotine exposure twice a day during GD11–GD20 to establish a typical IUGR rat model (4). Decreased fetal weights and increased IUGR rates were demonstrated in the nicotine group. It identified that the IUGR rat model was established successfully through prenatal nicotine exposure.

Analysis of Changed DNA Methylation in Maternal Blood

Early diagnosis and treatment is the goal of IUGR management (22). However, there is no definitively optimal method for screening and diagnosing IUGR during pregnancy. For example, ultrasound and Doppler flow, which require integration of multiple parameters and operator experience to identify IUGR, may fail to differentiate healthy but constitutionally small fetuses from IUGR, especially during the first trimester (11, 23). As a rapid, sensitive, and accurate method for IUGR screening, blood draw for nucleic acid analysis is characteristic of small cut, few complications, and tolerable pain, and can be regarded as a noninvasive method compared with amniocentesis (24). Meanwhile, placental and fetal nucleic acids can be detected in maternal blood from early pregnancy (22, 24). Therefore, measuring nucleic acids in maternal blood could also give us a better understanding of IUGR pathophysiology and underline the promise of DNA methylation status in maternal peripheral blood for IUGR screening. DNA methylation, a major epigenetic modification of nucleic acids, is more stable than RNA and more sensitive to environmental stimuli than DNA sequences (7, 25). Thus, DNA methylation patterns in maternal blood could be a promising method for prenatal IUGR screening.

Mammalian genomes contain a high frequency of CpG sites termed CpG islands. Most CpG islands are found in the proximal promoter regions of annotated genes in the mammalian genome. Methylation on the promoter CpG islands could result in chromatin remodeling and suppress gene expression (26). Thus, we used MeDIP array assays and the following GO analysis to evaluate the DNA methylation patterns of development-associated gene promoters in the maternal blood of IUGR rats. The results showed that the significantly changed DNA methylation frequencies were in genes involved in the development of multiple organs and major body systems, such as muscle and the nervous system. It suggested that the occurrence of IUGR may be the result of the combined impact of the aberrant DNA methylation patterns of the whole fetus.

The placenta, an essential barrier for fetal development, is vulnerable to many adverse intrauterine factors (such as smoking). It has been reported that placental dysfunction is one of the predominant causes of IUGR (22, 27). We have found that prenatal exposure to some xenobiotics (such as caffeine, nicotine, and ethanol) could lead to placental dysfunction and cause fetal exposure to excessive maternal glucocorticoids in rats (4, 28, 29). Excessive maternal glucocorticoid exposure has been reported to inhibit the development of multiple fetal organs or systems (such as skeletal growth, neuroendocrine system development) and induce IUGR in rats (29, 30). Thus, placental development may regulate fetal development by affecting the development of multiple fetal organs. In addition, placental dysfunction was reported to be related to the aberrant DNA methylation of genes regulating placental development (8). Thus, we intended to select a placenta-related gene for further confirmation.

Hypermethylated Genes Regulating Placental Development in Maternal Blood of IUGR

Among the genes regulating placental development, Synb, LRRC15, Met, and Tex19.1 showed significantly increased DNA methylation status. All of these four genes have an essential role in placental development, such as placental implantation and circulation. For example, LRRC15, which has a striking expression level within the cytotrophoblast cells of the placenta, has been reported to have an essential role during implantation (31). Ueno et al. demonstrated that loss of trophoblast c-Met could disrupt terminal differentiation and polarization of syncytiotrophoblasts, which could induce IUGR (32). According to the studies of Reichmann et al. (33), Tex19.1−/− mouse embryos exhibit intrauterine growth retardation and have small placentae due to a reduction in the number of spongiotrophoblast, glycogen trophoblast, and sinusoidal trophoblast giant cells. As the most significantly hypermethylated gene, Synb regulates the fusion and differentiation of trophoblast cells (34, 35, 36, 37). It has been reported that increased DNA methylation of human syncytin promoter in placentae could inhibit its expression and induce placental dysfunction and IUGR (38). Thus, we chose the DNA methylation status of Synb in maternal peripheral blood to perform further verification.

BSP analysis demonstrated hypermethylated Synb in the maternal blood of IUGR rats, which is in line with the results of MeDIP array assays. In addition, decreased Synb expression and histological damages were shown in nicotine-treated placentae. It suggested that DNA hypermethylation of the Synb promoter in maternal peripheral blood could reflect placental dysfunction and the occurrence of IUGR in rats under prenatal nicotine exposure. Although no animal research has evaluated DNA methylation or expression of syncytin in placentae or maternal blood in IUGR models, decreased syncytin expression has been widely observed in human placentae and in the maternal blood of IUGR cases (38, 39). Moreover, Ruebner et al. (40) have demonstrated that reduced syncytin expression correlated with the corresponding epigenetic hypermethylation in human placentae with IUGR or preeclampsia. Consequently, we speculated that Synb hypermethylation may be associated with various IUGR models rather than be specific to nicotine.

Conclusions

In summary, our study showed the IUGR-associated DNA methylation profile in maternal blood, which provides evidence for further studies on possible IUGR biomarkers. For example, hypermethylated Synb in maternal blood could reflect placental dysfunction and the occurrence of IUGR. Our present study underlines the promise of DNA methylation status in maternal peripheral blood for IUGR screening and gives us a better understanding of IUGR pathophysiology. Further studies need to be carried out on Synb methylation confirmation in early gestation and other IUGR models.