J Pathol Transl Med.  2017 May;51(3):264-283. 10.4132/jptm.2016.12.20.

Characteristic Changes in Decidual Gene Expression Signature in Spontaneous Term Parturition

• El-Azzamy
• Balogh A
• Romero R
• Xu Y
• LaJeunesse C
• Plazyo O
• Xu Z
• Price TG
• Dong Z
• Tarca AL
• Papp Z
• Hassan SS
• Chaiworapongsa T
• Kim CJ
• Gomez-Lopez N
• Than NG

• KMID: 2392594
• DOI: http://doi.org/10.4132/jptm.2016.12.20

Abstract

BACKGROUND
The decidua has been implicated in the "terminal pathway" of human term parturition, which is characterized by the activation of pro-inflammatory pathways in gestational tissues. However, the transcriptomic changes in the decidua leading to terminal pathway activation have not been systematically explored. This study aimed to compare the decidual expression of developmental signaling and inflammation-related genes before and after spontaneous term labor in order to reveal their involvement in this process.
METHODS
Chorioamniotic membranes were obtained from normal pregnant women who delivered at term with spontaneous labor (TIL, n = 14) or without labor (TNL, n = 15). Decidual cells were isolated from snap-frozen chorioamniotic membranes with laser microdissection. The expression of 46 genes involved in decidual development, sex steroid and prostaglandin signaling, as well as pro- and anti-inflammatory pathways, was analyzed using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR). Chorioamniotic membrane sections were immunostained and then semi-quantified for five proteins, and immunoassays for three chemokines were performed on maternal plasma samples.
RESULTS
The genes with the highest expression in the decidua at term gestation included insulin-like growth factor-binding protein 1 (IGFBP1), galectin-1 (LGALS1), and progestogen-associated endometrial protein (PAEP); the expression of estrogen receptor 1 (ESR1), homeobox A11 (HOXA11), interleukin 1β (IL1B), IL8, progesterone receptor membrane component 2 (PGRMC2), and prostaglandin E synthase (PTGES) was higher in TIL than in TNL cases; the expression of chemokine C-C motif ligand 2 (CCL2), CCL5, LGALS1, LGALS3, and PAEP was lower in TIL than in TNL cases; immunostaining confirmed qRT-PCR data for IL-8, CCL2, galectin-1, galectin-3, and PAEP; and no correlations between the decidual gene expression and the maternal plasma protein concentrations of CCL2, CCL5, and IL-8 were found.
CONCLUSIONS
Our data suggests that with the initiation of parturition, the decidual expression of anti-inflammatory mediators decreases, while the expression of pro-inflammatory mediators and steroid receptors increases. This shift may affect downstream signaling pathways that can lead to parturition.

Keyword

Chemokines; Cytokines; Estrogens; Galectins; Leukocytes; Progesterone

MeSH Terms

Chemokines
Cytokines
Decidua
Estrogen Receptor alpha
Estrogens
Female
Galectin 1
Galectin 3
Galectins
Gene Expression*
Genes, Homeobox
Humans
Immunoassay
Interleukin-8
Interleukins
Leukocytes
Membranes
Microdissection
Parturition*
Plasma
Pregnancy
Pregnant Women
Progesterone
Real-Time Polymerase Chain Reaction
Receptors, Progesterone
Receptors, Steroid
Sexual Development
Transcriptome*
Chemokines
Cytokines
Estrogen Receptor alpha
Estrogens
Galectin 1
Galectin 3
Galectins
Interleukin-8
Interleukins
Progesterone
Receptors, Progesterone
Receptors, Steroid

Figure

• 

  Fig. 1. Decidual gene expression in healthy term gestation. (A) The heatmap depicts the mean gene expression levels in the decidua of women with no labor at term (TNL, n=15) or those in term labor (TIL, n=14). Bar denotes color coding for gene expression levels (–∆Ct). Out of all genes sorted by their expression levels, the expression of insulin-like growth factor-binding protein 1 (IGFBP1) was the highest and interleukin 17A (IL17A) expression could not be detected with our method. Stars depict the differentially expressed genes in the decidua between TIL and TNL cases. White areas represent nondetectable gene expression. (B) The relative expression of IGFBP1 was 28-fold higher in the decidua compared to the chorioamnion as visualized in box-plots (p=.01). (C) A representative micrograph shows that IGFBP1 immunostaining was strong in the decidua, while it was weak in the chorion and amnion layers of the membrane.

• 

  Fig. 2. Differential expression of genes involved in sex steroid signaling and decidual development. Box-plots represent gene expression levels (–∆Ct) in the decidua of women with no labor at term (TNL, n = 15) or those in term labor (TIL, n = 14). Estrogen receptor 1 (ESR1) (A), progesterone receptor membrane component 2 (PGRMC2) (B), and homeobox A11 (HOXA11) (C) expression was higher in TIL cases compared to TNL cases (8.1-fold, false discovery rate–adjusted p-value [pFDR]=.032; 1.7-fold, pFDR=.096; 4-fold, pFDR=.032, respectively).

• 

  Fig. 3. Differential expression of chemokines, cytokines, and prostaglandin signaling genes. Box-plots represent gene expression levels (–∆Ct), while immunohistochemical (IHC) staining and immunoscoring show protein abundance in the decidua of women with no labor at term (TNL, n =15) or those in term labor (TIL, n=14). (A) Relative mRNA expression of interleukin 8 (IL8) in the decidua was higher in TIL cases than in TNL cases (7.3-fold, false discovery rate–adjusted p-value [pFDR]=.004; left panel). IHC staining confirmed higher expression of IL-8 in TIL cases than in TNL cases (middle panels), also quantified by immunoscoring (right panel; TIL 1.5 vs TNL 0.9; p=.027). (B, C) Relative mRNA expression of chemokine C-C motif ligand 2 (CCL2) and CCL5 in the decidua was lower in TIL cases than in TNL cases (8.7-fold, pFDR=.013; 3.5-fold, pFDR=.052, respectively; left panel). IHC staining confirmed lower CCL2 protein expression in TIL cases than in TNL cases (middle panels), also quantified by immunoscoring (right panel; TIL 1.9 vs TNL 2.7; p<.0001). (D, E) Relative decidual mRNA expression of IL1B and PTGES was higher in TIL cases than in TNL cases (2.8-fold, pFDR=.037; 6.7-fold, pFDR=.032, respectively).

• 

  Fig. 4. Differential expression of anti-inflammatory mediators in the decidua. Box-plots represent gene expression levels (–∆Ct), while immunohistochemical (IHC) staining and immunoscoring show protein abundance in the decidua of women with no labor at term (TNL, n =15) or those in term labor (TIL, n=14). Galectin-1 (LGALS1) (A), galectin-3 (LGALS3) (B), and progestagen-associated endometrial protein (PAEP) (C) gene expression was lower in TIL cases than in TNL cases (5.2-fold, false discovery rate–adjusted p-value [pFDR]=.039; 4.1-fold, pFDR= .075; 12.6-fold, pFDR=.056, respectively). IHC staining confirmed lower galectin-1 (TIL 2.1 vs TNL 2.6, p=.016) (A), galectin-3 (B) (TIL 1.4 vs TNL 2.0, p=.041), and PAEP (C) (TIL 1.0 vs TNL 2.0, p=.003) expression in TIL cases than in TNL cases.

• 

  Fig. 5. Correlation analyses between decidual gene expression and maternal plasma concentrations of chemokines. (A–C) The scatterplots demonstrate the absence of correlation between the decidual gene expression and maternal plasma concentrations of secreted chemokines chemokine C-C motif ligand 2 (CCL2) (r=.10, p=.66), CCL5 (r=.27, p=.21), and interleukin 8 (IL-8) (r=.03, p=.88) in women with no labor at term (TNL, n=15) or those in term labor (TIL, n=14). ELISA, enzyme-linked immunosorbent assay; qRT-PCR, quantitative real-time polymerase chain reaction.

• 

  Fig. 6. Conceptual framework. The increased decidual expression of a signaling factor responsible for decidual maturation and development (HOXA11) is preceded by the increased expression of chemokines (CCL2 and CCL5), which may stimulate the early recruitment of monocytes into the decidua as the onset of labor approaches. These immune cells will be activated by the local microenvironment and contribute to the orchestration of inflammation. With the initiation of parturition, the decidual expression of anti-inflammatory mediators (LGALS1, LGALS3, and PAEP) decreases, while the expression of pro-inflammatory mediators (IL1B and PTGES) and steroid receptors (ESR1 and PGRMC2) increases, contributing to “functional progesterone withdrawal” and heightened inflammation, eventually leading to uterine contractions, cervical ripening, and membrane rupture. The relatively late increase of IL8 expression will be followed by the recruitment of neutrophils, which plays a key role in tissue repair. These results strengthen earlier findings on the decidua being the earliest among gestational tissues that get primed during parturition. CCL2, chemokine C-C motif ligand 2; CCL5, chemokine C-C motif ligand 5; DSCs, decidual stromal cells; ESR1, estrogen receptor 1; HOXA11, homeobox A11; IL1B, interleukin-1β; IL8, interleukin 8; LGALS1, galectin-1; LGALS3, galectin-3; Mo, monocytes; MΦ, macrophages; PAEP, progestogen-associated endometrial protein; PGRMC2, progesterone receptor membrane component 2; PMNs, neutrophil granulocytes; PTGES, prostaglandin E synthase.

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