The placenta-specific proteome

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Figure 1. The distribution of all genes across the five categories based on transcript abundance in placenta as well as in all other tissues.

354 genes show some level of elevated expression in the placenta compared to other tissues. The three categories of genes with elevated expression in placenta compared to other organs are shown in Table 1. The list of genes defined as tissue enriched in placenta (n=78), suggests a relation to pregnancy and functions well in-line with the function and properties of the placenta.

Table 2. The 12 genes with the highest level of enriched expression in placenta. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. "mRNA (tissue)" shows the transcript level as�TPM values,�TS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Some of the proteins predicted to be membrane-spanning are intracellular, e.g. in the Golgi or mitochondrial membranes, and some of the proteins predicted to be secreted can potentially be retained in a compartment belonging to the secretory pathway, such as the ER, or remain attached to the outer face of the cell membrane by a GPI anchor.

The placenta transcriptome

An analysis of the expression levels of each gene made it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 76% of the mRNA molecules derived from placenta correspond to housekeeping genes and only 12% of the mRNA pool corresponds to genes categorized as placenta enriched, group enriched, or placenta enhanced. Thus, most of the transcriptional activity in the placenta relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.

Gene Ontology-based analysis of all the elevated genes (n=354) in placenta indicated a clear overrepresentation of proteins associated with female pregnancy and estrogen biosynthetic/metabolic pathways.

Protein expression of genes elevated in placenta

In-depth analysis of the elevated genes in placenta using antibody-based protein profiling allowed us to create a map of where these proteins are expressed with regards to the different cellular compartments that constitute the placenta, i.e. cytotrophoblast cells, syncytiotrophoblast cells, extravillous trophoblast cells, decidual cells and blood vessels.

Proteins specifically expressed in cytotrophoblasts

The placenta begins to develop upon implantation of the blastocyst (fertilized egg) as it attaches to the endometrium. The outer layer of the blastocyst consists of trophoblasts, which also form the outer layers of the placenta. The trophoblast layer is further divided into two layers: the cytotrophoblast and the overlying syncytiotrophoblast layer. Examples of proteins expressed in cytotrophoblasts include the highly conserved PEG10, involved in cell proliferation, differentiation and apoptosis, and PAGE4, a member of the GAGE gene-family.

Proteins specifically expressed in syncytiotrophoblasts

Syncytiotrophoblasts form when undifferentiated and highly proliferative cytotrophoblast fuse together. Syncytiotrophoblasts are specialized epithelial cells that cover the floating placental villi and are involved in maintaining pregnancy through the production of growth factors and hormones. Since the syncytiotrophoblasts are in direct contact with the maternal blood these cells are closely involved in exchange of gas, nutrients and waste between the mother and the fetus. Examples of proteins expressed in syncytiotrophoblasts include CSH1, a member of the somatotropin/prolactin family of hormones, KISS1, a stimulator of gonadotropin secretion, and transcription factor GCM1, glial cells missing homolog 1.

Proteins specifically expressed in extravillous trophoblasts

Extravillous trophoblasts are formed when cytotrophoblasts proliferate to form anchoring villi that attach to the uterine wall. From the anchoring villi, extravillous trophoblasts can form by detaching from the placental villi and migrate into the decidua. Examples of proteins expressed in extravillous trophoblast cells include PAPPA2, a metalloproteinase thought to be a local regulator of insulin-like growth factor (IGF), and PRG2, a predominant constituent in the eosinophil granule and present in placenta where it exists as a complex with several other proteins including PAPPA.

Proteins specifically expressed in placental blood vessels and stroma

The placenta is highly vascularized with capillaries and blood vessels composed of endothelial cells, pericytes and vascular smooth muscle cells. Examples of proteins expressed in endothelial cells in the placenta include FCGR2B, a low affinity receptor for the Fc region of immunoglobulin gamma complexes involved in the phagocytosis of immune complexes and LIN28B, a suppressor of microRNA biogenesis. FBN2, a component of connective tissue microfibrils potentially involved in elastic fiber assembly, shows expression in the stroma of placental villi.

Proteins specifically expressed in decidual cells

The decidua is the maternal part of the placenta. The decidua is derived from the maternal endometrium and includes specifically altered endometrial stroma cells that are formed in a response to progesterone in a process called decidualization. IGFBP1,expressed in decidual cells, is a secreted protein that binds both insulin-like growth factors I and II, and is also expressed in the uterus and liver.

Genes shared between placenta and other tissues

There are 75 group enriched genes expressed in the placenta. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including placenta, compared to all other tissues.

In order to illustrate the relation of placenta tissue to other tissue types, a network plot was generated, displaying the number of commonly expressed genes between different tissue types.

Figure 2. An interactive network plot of the�placenta�enriched and group enriched genes connected to their respective enriched tissues (grey circles).�Red�nodes represent the number of�placenta enriched genes and�orange�nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to�4�tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

The placenta shares of a few genes with most other tissue types but has most shared group enriched genes with testis (25 genes). Examples of group enriched genes shared between placenta and other tissues are CXorf67, previously uncharacterized, also expressed in testis and DLK1, a transmembrane protein involved in differentiation of several cell types including adipocytes and neuroendocrine cells, also expressed in adrenal gland.

Placenta function

The placenta is a temporary organ formed during pregnancy. It connects the developing fetus to the uterine wall to allow nutrient uptake, waste elimination, and gas exchange via the mother's blood supply. The placenta has two components: the fetal placenta which develops from the same blastocyst that forms the fetus, and the maternal placenta, the decidua, which develops from the maternal uterine tissue.

Besides supplying nutrients and oxygen to the fetus the placenta also has an endocrine function where it produces several pregnancy-associated hormones and growth factors including human placental lactogen (hPL), human chorionic gonadotropin (hCG), estrogen and progesterone.

Placenta histology

The normal term placenta measures 15-20 cm in diameter, 1.5-3 cm in thickness and weighs approximately 450-600 grams. The main components are the villous parenchyma and maternal decidual tissue, umbilical cord (54-61 cm in length, containing two umbilical arteries and one umbilical vein) and placental membranes. The placental membranes include the amnion (the innermost lining of the amniotic cavity composed of a single layer of flat epithelial cells) and the chorion (connective tissue that carries the fetal vasculature).

The main structure of the placenta is the villous "trees". The placenta villi can float freely in the intervillous space or be attached to the decidua which provides structural stability for the placenta.

Cell types of the placenta

Cell types of the placenta villi include:

• Cytotrophoblasts and syncytiotrophoblasts which cover the surface of the villous tree and are surrounded by maternal blood.
• Mesenchymal cells, Hofbauer cells and fibroblasts which are located within the villous core stroma between the trophoblasts.
• Fetal vessels and fetal blood vessels, including vascular smooth muscle cells, pericytes and endothelial cells.

The other part of the placenta, the decidua, includes decidua cells and maternal vascular cells.

The histology of human placenta including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.

Background

Here, the protein-coding genes expressed in the placenta are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the placenta.

Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 8 fresh frozen tissues representing normal placenta was compared to 164 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in placenta. A tissue-specific score, defined as the ratio between mRNA levels in placenta compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression. These categories include: genes with elevated expression in placenta, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in placenta, and genes not expressed in any tissue. Genes with elevated expression in placenta were further sub-categorized as i) genes with enriched expression in placenta, ii) genes with group enriched expression including placenta and iii) genes with enhanced expression in placenta.

Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.

Relevant links and publications

Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed: 25613900 DOI: 10.1126/science.1260419

Yu NY et al, 2015. Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res.
PubMed: 26117540 DOI: 10.1093/nar/gkv608

Fagerberg L et al, 2014. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics.
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600

Histology dictionary - the placenta