Basal plate biopsy specimens were obtained from 36 placentas (14–40 weeks) from women who had normal pregnancies. RNA was isolated, processed and hybridized to HG-U133A and HG-U133B Affymetrix GeneChips. Surprisingly, the expression of very few genes was modulated during the 14- to 24-week interval. In contrast, hundreds of genes, including those already known to be regulated over gestation, were modulated between mid-pregnancy (14–24 weeks) and term. These data allowed us to identify molecules that play potentially important roles in the formation of the maternal-fetal interface during the second trimester or in preparation of this area for parturition.
Our analysis revealed a total of 418 genes/expressed sequence tags that were differentially regulated between term and mid-gestation. A heat map of the top 25 up and down regulated genes is shown [; complete heat map and data (13
)]. Based on GO annotations, the differentially expressed genes were involved in a variety of biological processes. At least one sixth were expressed sequence tags or hypothetical proteins and thus lacked annotations. Of the known differentially expressed genes, 17 were related to lipid metabolism, 10 were involved with formation or regulation of the extracellular matrix (ECM), 21 were immune effectors or modulators, 24 were transcription factors, and 6 had angiogenic/vasculogenic functions.
Heat map of the most highly up regulated (upper panel) and down regulated (lower panel) differentially expressed genes in the basal plate region at term in normal pregnancy
Ingenuity Pathway Analysis (IPA) software was used to further evaluate the participation of the differentially expressed genes in metabolic and signaling pathways. Analysis of genes with at least a twofold change highlighted two metabolic pathways—folate biosynthesis and N-glycan degradation involving mannose-containing structures. With regard to signaling, eleven of the differentially expressed genes mapped to the Wnt-β-catenin pathway. We also used the IPA software to map networks of the differentially expressed genes. The largest network contained genes that were involved in cell motility, cell-to-cell signaling/interaction and tissue development.
We were also interested to find that genes encoding molecules that are involved in immune defense are highly regulated. For example, defensin alpha 1 was upregulated about threefold at the RNA level at term as compared with the second trimester. Production of this antimicrobial peptide is constitutive in some cells (e.g., neutrophils) and induced in others (e.g., monocytes and CD8 T lymphocytes) in response to proinflammatory mediators (14
). The presence of defensins in human term placental tissue has been previously reported (15
). Increased expression at term could occur in preparation for labor and placental separation, which increase the risk of infection. In contrast, the expression of another antimicrobial molecule, granulysin, which localizes to the cytolytic granules of T cells, natural killer (NK) cells (16
) and certain dendritic cells (17
), is downregulated at term. We speculate that the decreased granulysin expression we observed parallels the decrease in T cell and NK cell numbers at the maternal-fetal interface at term (18
). The downregulation of Ly96 expression, another NK-cell-specific molecule, provides further support for this concept. Although the mechanisms that lead to the eventual disappearance of decidual leukocytes from the maternal-fetal interface are not known, the observed concurrent decrease in expression of chemotactic molecules, such as chemokine-like factor superfamily 6 (CKLFSF6) and secreted phosphoprotein 1 (SPP1), could be a related phenomenon.
A trophoblast-derived noncoding RNA (TncRNA) was one of the most interesting of the highly upregulated differentially expressed genes in the immune function category. This transcript, which directly suppresses MHC class II expression by interacting with the MHC IITA-PIII transactivator, likely accounts for the lack of trophoblast MHC class II expression (19
). As such, this molecule could play an important role in promoting maternal immunotolerance of the hemi-allogeneic fetus. Why TncRNA expression increases at term is unclear, but this phenomenon could be related to the continuing need to suppress MHC class II expression in trophoblasts, particularly as they are shed into maternal blood at the time of delivery. In this regard, it is interesting to note that expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which plays a role in regulating decidual immune responses, is also upregulated at term (20
Additionally, our group has also been interested in the functions of the myriad angiogenic factors that are produced at the maternal-fetal interface (3
). In broad terms, we know that these molecules have at least three targets—the intrinsic placental vasculature, the maternal vasculature, and the CTB subpopulation that executes an unusual epithelial-to-endothelial transition as the cells invade the uterine wall and remodel the maternal vasculature in this region. Thus, we anticipated that molecules involved in vasculogenesis/angiogenesis would be upregulated during the active phases of placentation, i.e., in the second trimester rather than at term, which is what we found. Consistent with our previously published work, the downregulated genes included angiopoietin (ANGPT)-2 (22
As with every microarray analysis, we made a number of interesting observations that warrant additional follow-up. For example, the cluster analysis showed a striking co-downregulation at term of ANGPT-2 and microcephalin (MCPH1). Interestingly, ANGPT-2 and MCPH1 genes are transcribed from opposite strands of the same region (chromosome 8p23.1). Their tight coexpression suggests that transcription from this area could be silenced at term, perhaps by local chromatin modifications or the recruitment of inhibitory protein complexes to the same promoter element. It will be interesting to determine if the pattern of co-expression of ANGPT-2 and MCPH1 occurs in other tissues or is specific to our data set. It is known that MCPH1 controls brain size in humans by regulating the proliferative and, hence, differentiative capacity of neuroblasts, ultimately exerting its effects through cell cycle regulators (23
). Furthermore, during human evolution there is evidence that strong genetic selection has been exerted on MCPH1 (25
). While the most obvious consequence is brain size, another interesting possibility is that placental form and function have been affected as well.
In summary, we found that gene expression patterns in the basal plate region change dramatically between the second trimester and term. Thus, it is important to control for this variable when studying the effect of pregnancy complications that occur during this timeframe. For our purposes, understanding the normal development and formation of the maternal-fetal interface is an important first step toward understanding PE-related changes.