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doi: 10.1371/journal.pone.0049248. Epub 2012 Nov 7.

Mid-gestational gene expression profile in placenta and link to pregnancy complications

Affiliations

Mid-gestational gene expression profile in placenta and link to pregnancy complications

Liis Uusküla et al. PLoS One. 2012.

Abstract

Despite the importance of placenta in mediating rapid physiological changes in pregnancy, data on temporal dynamics of placental gene expression are limited. We completed the first transcriptome profiling of human placental gene expression dynamics (GeneChips, Affymetrix®; ~47,000 transcripts) from early to mid-gestation (n = 10; gestational weeks 5-18) and report 154 genes with significant transcriptional changes (ANOVA, FDR P<0.1). TaqMan RT-qPCR analysis (n = 43; gestational weeks 5-41) confirmed a significant (ANOVA and t-test, FDR P<0.05) mid-gestational peak of placental gene expression for BMP5, CCNG2, CDH11, FST, GATM, GPR183, ITGBL1, PLAGL1, SLC16A10 and STC1, followed by sharp decrease in mRNA levels at term (t-test, FDR P<0.05). We hypothesized that normal course of late pregnancy may be affected when genes characteristic to mid-gestation placenta remain highly expressed until term, and analyzed their expression in term placentas from normal and complicated pregnancies [preeclampsia (PE), n = 12; gestational diabetes mellitus (GDM), n = 12; small- and large-for-gestational-age newborns (SGA, LGA), n = 12+12]. STC1 (stanniocalcin 1) exhibited increased mRNA levels in all studied complications, with the most significant effect in PE- and SGA-groups (t-test, FDR P<0.05). In post-partum maternal plasma, the highest STC1 hormone levels (ELISA, n = 129) were found in women who had developed PE and delivered a SGA newborn (median 731 vs 418 pg/ml in controls; ANCOVA, P = 0.00048). Significantly higher expression (t-test, FDR P<0.05) of CCNG2 and LYPD6 accompanied with enhanced immunostaining of the protein was detected in placental sections of PE and GDM cases (n = 15). Our study demonstrates the importance of temporal dynamics of placental transcriptional regulation across three trimesters of gestation. Interestingly, many genes with high expression in mid-gestation placenta have also been implicated in adult complex disease, promoting the discussion on the role of placenta in developmental programming. The discovery of elevated maternal plasma STC1 in pregnancy complications warrants further investigations of its potential as a biomarker.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Placental genes with considerable expressional change in progression from early to mid-pregnancy.
Ten placental tissue samples subjected to dynamic linear transcriptome expression profiling (Affymetrix HG-U133 plus 2.0 GeneChips) represented gestatational weeks of 5 (38 days), 8 (days 55, 56, 56), 11 (81 days), 13 (91 days), 17 (days 120, 121) and 18 (days 126, 132). The heatmap shows 154 genes (180 probe-sets) with significant gradual increase (red) or decrease (blue) in placental transcription in first and second trimester placentas (ANOVA, FDR corrected P<0.1). Color intensity reflects mean log2 fold change in gene expression with one first trimester sample (week 5; 38 gestational days) as reference. For the placental samples collected at close gestational age, log2 fold change calculations represent the median values of 2–3 samples (55/56/56; 120/121; 126/132 gestational days). Hierarchical clustering with Euclidean distance, visualized by the dendrogram on the left, clearly separated genes with increased and decreased expression. Colorstrip on the right of the heatmap highlights genes with strongest statistical significance of differential expression. A subset 24 genes selected for further experiments are labeled on the vertical axis.
Figure 2
Figure 2. Temporal quantitative gene expression changes in discovery samples over gestational weeks 5–18.
Log2 fold changes between Affymetrix GeneChip transcription values were calculated to estimate the direction of gene expression. Gene expression level at week 5 (gestational day 38) were used as a baseline. For the placental samples collected at close gestational age, fold change calculations represent the median values of 2–3 samples (week 8: 55/56/56 gestational days; week: 17; 120/121 gestational days; week 18: 126/132 gestational days). (A) 14 genes with significant change in transcription (ANOVA, FDR corrected P<0.05, n = 10) and (B) additional 10 genes with known effect on pregnancy that showed mildly significant differential expression (FDR corrected P<0.1) were selected for subsequent RT-qPCR analysis (Table S2). Genes are ordered by decreasing P-value.
Figure 3
Figure 3. Genes with significant dynamic increase in expression during early and mid-gestation as experimentally confirmed by RT-qPCR.
Relative mRNA expression levels in the extended sample set of first and second trimester placentas (n = 31; from gestational days 38 to 147) were determined by TaqMan assays. P-values were calculated by ANOVA and subjected to multiple testing correction (FDR). Genes below the significance threshold of P-value>0.02 are shown in Figure S4.
Figure 4
Figure 4. Expressional dynamics of identified mid-pregnancy specific genes in placenta from early to term gestation.
Relative mRNA levels were determined by RT-qPCR TaqMan assays in placental tissues from early- (5–13 gestational weeks; n = 23), mid- (17–21 gestational weeks; n = 8) and term-gestation (36–41 gestational weeks; n = 12) samples of uncomplicated pregnancy cases. Boxplots show mid-gestation marker genes with (A) significantly increased mRNA expression compared to early- or late-gestation placental samples, (B) significantly increased expression levels compared to early gestation placental samples, and (C) gradual increase in expression during pregnancy. P-values were calculated by Student t-test and subjected to multiple testing correction (FDR) (Table S7).
Figure 5
Figure 5. Glycoprotein hormone STC1 protein levels in maternal blood plasma.
STC1 protein levels in maternal plasma from uncomplicated pregnancies (defined as controls) compared to (A) pregnancies resulting in the birth of small-for-gestational-age (SGA) and large-for-gestational-age (LGA) newborns, as well as pregnancies complicated with preeclampsa (PE) or gestational diabetes mellitus (GDM); (B) cases with PE and (C) GDM grouped by newborn birth weight. Median values are indicated by horizontal bars. Plotted values are represented with no adjustment for confounding effects. Statistical differences between controls and each of the patient groups were assessed by accounting for confounding factors with ANCOVA. Statistical tests were adjusted for newborn birth-weight (initial analyses of PE and GDM cases), gestational age (SGA, LGA, PE), mode of delivery and mother’s weight (all groups), height (PE, except when grouped by newborns birth weight) and age (GDM). The adjusted P-values are given above the data point of the respective study group.
Figure 6
Figure 6. The immunostaining of CCNG2 and LYPD6 proteins was assessed in placental sections from term pregnancies with no complications (controls), with preeclampsia (PE) or with gestational diabetes mellitus (GDM).
(A) Hematoxylin-eosin staining was used to describe histopathological findings in analyzed placental samples (100-fold microscope magnification). In term placentae the mature intermediate (IMV) and small terminal villi (STV) were seen. Characteristic to PE, villous agglutination and infarction (IN; intense eosinophilic staining) and increased number of syncytial knots (SK) were detected. GDM presented with degenerative placental lesions such as focal villous fibrinoid necrosis (FN). (B) Diffuse cytoplasmic staining of LYPD6 antibody was detected in syncytiotrophoblast (ST) cells in all villous types (IMV, STV). Additionally LYPD6 antibody strongly stained the cytoplasm and the nucleus of villous stroma Hoffbauer cells (H), fibroblasts (F) and endothelial cells (E) of villous vessels. No localization differences in LYPD6 antibody stain between the groups were found; however strong tendency to higher staining intensity was observed in PE and GDM placentas compared to normal term placenta. (C) CCNG2 antibody showed fine granular cytoplasmic staining of villous stromal Hoffbauer (H) and fibroblast (F) cells. In addition, weak cytoplasmic staining of syncytiotrophoblast (ST) and endothelial cells (E) of vessel wall was found. No localization differences in CCNG2 staining between the normal, PE and GDM groups were detected. Higher tendency to positivity was seen in PE placental sections. (D) Negative control (NC) staining was performed without primary antibody. Scale bar, 100 μm. Microscope magnifications ×ばつ100 and ×ばつ400 were used. Brown color indicates chromogen-labeled antibody and blue color indicates hematoxylin nuclear staining.

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