To our knowledge, this microarray analysis of surplus CVS specimens produced the earliest differential global gene expression of placentas in pregnancies destined for preeclampsia. Utilization of first trimester snap-frozen tissues with known pregnancy outcomes, oligonucleotide genechips, and subsequent prediction modeling distinguish our study from previous placental microarray investigations in preeclampsia. [14
] qRT-PCR of LAIR2 supported the microarray results. qRT-PCR expression levels in the additional unaffected samples (AS) replicated C group results, further verifying the microarray. qRT-PCR of CCK and CTAG2 trended in over- and under-expression consistent with the microarray results, and the lack of statistical differences are likely due to sample sizes. The variation in CTAG2 qRT-PCR expression patterns may indicate methylation in nonexpressors, denoted as “undetermined” among all 3 groups of samples. [28
] CCK was up-regulated 3.1 fold in placentas of preeclamptic pregnancies at 29–32 weeks in a previous study. [29
The IPA analyses suggested potential pathways in which some of the 36 genes probably function. Ten genes were not incorporated into any of the Function and Disease pathways produced by IPA, nor were they located in current literature searches in conjunction with preeclampsia: CTAG2, MUC15, OXGR1, SCARA5, MAGEB6, TNRC9, TMC4, DEPDC7, RUFY3, and LAIR2. We suggest functional groupings, other than immune/inflammation, integrating these with all other genes of interest (). The IPA findings were synthesized with hand searches of the literature to inform our interpretation of the dysregulated genes in relation to preeclampsia.
Functional groups other than immune incorporating IPA-omitted genes of interest in PE.
Overall, our results directly support the concept of the placental origins of the disorder [30
] and allow for targeted investigation of placental derived biomarkers in early
pregnancy. Assessment of cause rather than effect of preeclampsia is likely to have been more discernable in these first trimester placental tissues. The findings in this study suggest that impaired placentation in preeclampsia may be associated with an overall deficiency rather than an excess of gene expression, insofar as 31 of the 36 genes of interest were down-regulated. Preconceptional testing of susceptibility to preeclampsia could be developed from variants of the genes of interest. In addition, several produce secreted protein (), such that measurement of one or a combination of these biomarker proteins in maternal blood in the first trimester may prove to be a predictive screening test for preeclampsia.
Genes expressed in the CVS specimens can be interpreted as maternal and fetal. Innate immune responses at the maternal-fetal interface are likely to be represented. Remarkably, 12 of the 36 genes, 7 not previously associated with preeclampsia, are involved in immune dysregulation (). All of the immunoregulatory genes except S100A8 were down-regulated, implicating deficient, blocked, or impaired function. LAIR2, HPS3, and SART3 are immune-related genes () that were not incorporated by IPA into the immune pathway (; ).
The immune dysregulated cells may be trophoblasts, which are fetoplacental epithelial cells [31
] that act as a pregnancy-specific component of the innate immune system. [32
] By day 14 post conception, cytotrophoblasts (CTBs) have breached the chorionic basement membrane, switching from a proliferative to an invasive phenotype as extravillous trophoblasts (EVTs). [33
] Cellular Movement functions in IPA Networks 1 and 2, including inflammation, migration, and invasion, are known to be involved in CTB placentation processes (). The EVTs form cell columns contacting maternal immune cells in the decidua. [34
] From these columns, EVTs invade the uterine wall and remodel the maternal spiral arteries by displacing smooth muscle and endothelial cells. [35
] Normal trophoblast development differs from cancer in that proliferation ceases during invasion. [33
] Various genes associated with both of these processes were down-regulated in preeclampsia (). In the current analysis, no notable differential expression existed between PE and C in EVT epithelial integrins [36
] or human leukocyte antigens [38
] identified in other studies as dysregulated in CTBs of later gestation. Alternatively, some of the differentially expressed immuneregulatory genes may suggest abnormalities of fetoplacental Hofbauer cells, which are macrophages that populate the villous core. [34
The maternal innate immune system predominates at this stage of placental development with 70% of decidual leukocytes consisting of natural killer cells (NK), 20–25% macrophages and about 2% dendritic cells. [39
] Approximately 10% of decidual immune cells at this time are adaptive system T lymphocytes; no B cells are present. [40
] Thus, some of the immunoregulatory genes of interest could also be of maternal origin. Finally, one cannot exclude the potential contribution of circulating fetal or maternal immune cells in the placenta. [33
Surprisingly, a number of differentially expressed genes may be found in decidual stroma, including MUC15, [41
] IGFBP1, [42
] and PAEP. [43
] Although the goal of CVS is to obtain chorionic tissue for fetal genetic diagnosis, maternal decidual tissue is invariably present, as corroborated by our microarray analysis. Decidual tissue likely derives from placental septae projecting upwards from the basal plate towards the chorionic plate that contains an admixture of decidual cells, EVTs, and occasional trophoblast giant cells. [34
] On balance, the results suggest that preeclampsia may be associated with impaired decidualization. Whether this is etiological or secondary to suboptimal interaction with and stimulation by trophoblasts or maternal immune cells, or both is currently unknown. An alternative explanation, albeit less likely, is that there are fewer of these septae in early preeclampsia placentas, thus decreasing decidual tissue and consequently decidual gene expression in these CVS specimens.
In order to examine hypotheses concerning hypoxia inducible transcription factors and oxidative stress, a secondary analysis of expression fold changes was conducted with the caveat that a high rate of false positives could be expected (Table 1-Supplement
] A previous study, showing that gene expression of first trimester villous explant cultures incubated under 3% oxygen mimicked gene expression of various preeclamptic placentas at delivery, suggested that hypoxia may be involved in the pathogenesis [44
], but is not necessarily informative of etiology. Indeed, the concept that the placenta is hypoxic or over-expresses HIF-α protein during early gestation, thereby impairing trophoblast invasion in preeclampsia, [16
] is not corroborated by our microarray analysis of CVS tissue. We interrogated 26 genes proven to be HIF target genes [45
] and several have been shown to be over-represented in placentas delivered from preeclamptic women (Table 2-Supplement
] Only IGFBP1, WT1 and TH genes showed differential expression in one probe. Moreover, IGFBP1 and TH are typically up- and not down-regulated by hypoxia. [47
] Interestingly, EPAS1 or HIF2α () expression was markedly decreased, but did not consistently correlate with putative specific HIFα target genes (Table 3-Supplement
] suggesting adequate HIF2α protein levels, transcriptional activity or compensation despite markedly reduced HIF2α mRNA expression. We found no difference in HIF1α expression between preeclamptic and control samples by our microarray study.
Nor were we able to support the differential expression of oxidative stress regulated genes at this early stage of preeclamptic pregnancy. [19
] We interrogated fold changes in 11 genes previously shown to be regulated by oxidative stress (Table 4-Supplement
] and expression differences were nonsignificant. In fact, blood flow and oxygen delivery to the intervillous space begins around 10–12 weeks of gestation, 19 but expression profiles of the hypoxia (Table 2-Supplement
) and oxidative stress (Table 4-Supplement
) regulated genes do not support the concept of an undue delay or acceleration of this crucial physiological event, respectively. Thus, ischemia-hypoxia and oxidative stress due to reperfusion injury are likely to be later events in preeclampsia.
Noteworthy is that 17 of the 36 genes identified by the Naïve Bayes prediction model and J5 test were among the 152 identified by 2-fold FC analysis. Thus, there is considerable intersection of the two analytical approaches. The finding of aberrant decidualization in early placentas of preeclampsia revealed by the prediction modeling is bolstered by the FC analysis, insofar as FSTL3 (FC -2.56) [50
] and prolactin (FC -7.86) [52
] are down-regulated (Table 1-Supplement
). Additionally, marked downregulation of granulysin in the FC analysis (FC -23.51) further supports immune dysregulation in decidua. [53
Specimens obtained from surplus CVS revealed genomic differences in first trimester placentas of pregnancies eventuating in preeclampsia. The indication for CVS, advanced maternal age, may limit the present findings for younger women. Our samples were from a homogeneous racial group, consistent with the population undergoing CVS at the clinical site, which may limit the findings for groups other than Caucasian women. It must be mentioned, however, that CVS is the only method to directly access first trimester placental genome in the context of known pregnancy outcomes. The procedure is not offered to women without risk factors because CVS is associated with risk, e.g., 0.33% pregnancy loss. [54
] The specimens for this study provided a rare window into pregnancy disorders such as preeclampsia and to early normal placentation. Another precaution is that we presently lack microarray data on CVS tissues obtained from other obstetrical complications linked to abnormal placentation, e.g., normotensive IUGR and preterm labor. [46
] The results may not be specific only to preeclampsia in all women.
Many genes regulated at the post-transcriptional level could be important in the pathogenesis of preeclampsia. Because post-transcriptional changes are not directly interrogated by mRNA gene expression microarray, important pathogenetic mechanisms could be overlooked using this approach.
Finally, this microarray study could be considered a pilot investigation due to the relatively low sample number; yet, appropriate techniques for power analysis in microarray studies remain controversial. [55
] Previous microarray analyses used a range of 2 to 11 placentas from preeclamptic women. [14
] The laborious and time-consuming nature of specimen collection and stringent inclusion criteria affected our sample size. This limitation may be at least partially offset by the analytic methods within caGEDA. [24
This is the first known study of global gene expression in first trimester placental tissues of preeclamptic pregnancies, and it contributes to the systems biology of normal first trimester placentation. The 36 differentially expressed genes provide promising potential biomarkers of preeclampsia and clues to etiology, including dysregulation of maternal-fetal immune interaction and altered decidualization in first trimester placental tissue of women destined to develop preeclampsia. More specific hypotheses will require testing maternal versus fetal origins of the genes of interest. Clearly, we consider the maternal genome evident in our samples as a crucial component providing important insights, rather than as “contamination.” Individualized prevention strategies and treatments could follow from the genes identified. Validation studies using data from larger cohorts are warranted.