In spite of the many suggestions that epigenetic changes might be involved in adverse pregnancy outcomes,6, 8, 27
no genome-wide study has searched for epigenetic abnormalities in preeclampsia and IUGR. In this study, we profiled the DNA methylation of placentas from preeclampsia and IUGR pregnancies and their control counterparts using Illumina GoldenGate Methylation Cancer panel I array. Although the array mainly targets cancer-related genes, the pseudomalignant nature of the placenta makes it suitable for this study.28
Among the 1505 CpG loci targeted by the array, 34 loci were identified as hypomethylated in EOPET but none was differentially methylated in LOPET. The different epigenetic profiles in EOPET and LOPET placentas support the hypothesis that the two forms of preeclampsia are caused by different mechanisms.29, 30
EOPET, which is often associated with IUGR, is a severe form of preeclampsia (76% of our cases were associated with IUGR). It is suggested to be initiated by abnormal placentation, caused by reduced perfusion with increased apoptosis of trophoblasts.2, 30, 31
In contrast, LOPET, which is considered to be a maternal syndrome, is a mild form of preeclampsia. It is usually associated with normal placental development and a predisposed maternal constitution, such as hypertension or diabetes.2, 30
Epigenetic change may have a role in EOPET by altering gene expression and, as a consequence, normal placental development. Epigenetic changes may also result from hypoxic conditions associated with preeclampsia or an altered trophoblast composition in these placentas. Hypomethylation was found in many gene promoter regions in EOPET, but there was no difference in the global DNA methylation level as indirectly assessed by methylation at the LINE1 repeat sequence compared with other groups of placentas (data not shown). As LINE1 methylation is a measure of global methylation, these results imply that CpG hypomethylation observed is a gene-specific effect. Interestingly, many of the associated genes, such as the imprinted gene CDKN1C
, are known to be important for normal placentation.32
To control for maternal and gestational age factors, the sample size used for array profiling in this study is small (8 to 10 samples per group). The small sample size likely explains why we do not find an association between polymorphic DNA methylation of TUSC3
and LOPET as we did in a previous investigation of over 100 placentas (P
This latter study was focused on the identification of epipolymorphisms and did not involve the statistical comparison of all methylation sites between groups, as was conducted for this study.
Recently, we reported a reduction of methylation at the H19
imprinting control region in IUGR-associated placentas, but we did not find altered methylation at CDKN1C
or other imprinted genes in IUGR and/or preeclampsia.18
The discrepancy can be attributed to the different ways of grouping samples, because we divided preeclampsia cases into PET and PET+IUGR previously without considering the effect of gestational changes on DNA methylation. Global changes of gene expression have been previously reported in association with gestational age.33
Our current findings suggest this is important also in regard to DNA methylation. Thus, the gestational age-dependent profile is important to evaluate and control for when considering any methylation change identified as a potential biomarker. This is particularly important in the study of preeclampsia as such pregnancies tend to be delivered early and comparisons with term births may be inappropriate.
The DNA methylation differences of CpGs in CAPG
were confirmed in an independent set of 26 placentas with EOPET and gestational age-matched control pregnancies. Among these four genes, TIMP3
had the largest difference in DNA methylation level with an over 15% reduction in EOPET compared with control placentas. A previous study showed that TIMP3
gene expression can be regulated by promoter DNA methylation in the placental tissues.34
Although our assays target CpG sites upstream of the CpG island where previous groups analyzed (), we also found a significant inverse correlation between its DNA methylation and gene expression in placentas. Therefore, hypomethylation of the TIMP3
promoter may alter its gene expression in EOPET.
is a family member of the matrix metalloproteinase inhibitors, which have an important function in regulating a wide range of physiological processes such as cell growth, invasion, migration transformation and apoptosis. This gene is highly expressed in placenta and suggested to be important for implantation and decidualization by regulating trophoblast invasion.35, 36
Elevated expression of many TIMPs, including TIMP3
, has been reported in preeclamptic placentas.37, 38
The hypomethylation of the TIMP3
promoter found in this study may increase TIMP3
expression and, in turn, reduce the invasiveness of trophoblast during placental development, which leads to placental hypoperfusion in EOPET. Intriguingly, hypermethylation of the TIMP3
promoter has been reported in choriocarcinoma and hydatidiform mole, conditions that have increased trophoblast invasiveness,34, 39
which further supports the inverse relationship between TIMP3
promoter methylation and trophoblast invasiveness. It has also been shown that TIMP3 could inhibit angiogenesis by blocking the vascular endothelial growth factor from binding its receptor,25
a well-known defect that is found in the trophoblast of preeclamptic pregnancies.40
Although the cause of the epigenetic modification is unknown, it may be related to the hypoxic environment of the cells.41, 42
expression was increased in the first-trimester trophoblasts on hypoxic treatment.43
This implies that the increased expression of TIMP3
under hypoxic condition, a hallmark in preeclamptic trophoblast, may be mediated by the epigenetic alteration on its promoter.
Early detection of preeclampsia is necessary for effective treatment. We identified several genes with hypomethylation in their promoter regions. In particular, the significant reduction of DNA methylation in TIMP3 promoter of EOPET placentas could be useful as a biomarker for the disorder. Importantly, this site showed no significant change of DNA methylation by gestational age and there was a good intra-placental correlation in DNA methylation values. If further study shows that this methylation change is also conserved earlier in pregnancy, then measuring the DNA methylation level of TIMP3 in chorionic villus sampling (CVS) from pregnant women could reflect subsequent risk for EOPET.
Recent advances in measuring circulating fetal DNA from maternal plasma opens up an additional approach for non-invasive prenatal diagnosis.44
This strategy takes advantage of the fact that during pregnancy, 3 to 6% of cell-free DNA in maternal blood plasma is derived from the placenta.44
Therefore, one can detect abnormalities in the fetal DNA directly from the maternal blood without going through invasive methods such as amniocentesis and CVS. It has been shown that there is an over fivefold increase in circulating fetal DNA in the maternal plasma of preeclamptic pregnancies compared with their control counterparts as estimated by measuring the placental-specific unmethylated SERPINB5
is not differentially methylated between normal and preeclamptic placentas. The same extent of increase in circulating fetal DNA can also be found in preeclamptic maternal plasma by measuring SRY
suggesting that SERPINB5
is not a specific marker for preeclampsia. As TIMP3
is significantly hypomethylated in EOPET placentas the detection of an increased level of unmethylated TIMP3
cell-free DNA in the maternal plasma could provide increased sensitivity for the non-invasive screening of pregnancies for EOPET. Importantly, it possesses the same characteristics as SERPINB5
for being a potential universal non-invasive prenatal diagnostic marker: its methylation is specifically reduced in placenta but it is completely methylated in other tissues, including blood samples. SERPINA3
, another gene in the SERPIN family, has been reported to be hypomethylated in severe preeclampsia, but the extent of methylation and its potential for being a clinical marker have not been examined thoroughly.8
We suggest, therefore, that the level of unmethylated TIMP3
DNA in maternal plasma could be a useful biomarker for early detection of severe preeclampsia.
In summary, we report the application of DNA methylation analysis to the elucidation of abnormal placental development associated with preeclampsia. Although DNA methylation at critical sites can reflect the availability of a gene for transcription, which may lead to altered expression depending on other regulatory factors present, it has a number of advantages over expression studies. First, it may be more resistant to the transient changes in gene expression associated with labor and delivery,12, 13, 14
as well as the effects of placental storage before sample processing.15
Although we did in this case observe an inverse association between TIMP3
methylation and expression, expression studies at term may not always reflect that which occurred during relevant periods of development. Second, the trend to hypomethylation of a variety of genes in EOPET suggests that loss of methylation may generally be involved in the response to hypoxia. Finally, DNA methylation differences provide an alternative approach for pre-symptomatic diagnosis of at risk pregnancies.