Disrupted placental serotonin synthetic pathway and increased placental serotonin: Potential implications in the pathogenesis of human fetal growth restriction

Abstract

Objectives: Placental insufficiency contributes to altered maternal-fetal amino acid transfer, and thereby to poor fetal growth. An important placental function is the uptake of tryptophan and its metabolism to serotonin (5-HT) and kynurenine metabolites, which are essential for fetal development. We hypothesised that placental 5-HT content will be increased in pregnancies affected with fetal growth restriction (FGR).

Methods: The components of the 5-HT synthetic pathway were determined in chorionic villus samples (CVS) from small-for gestation (SGA) and matched control collected at 10-12 weeks of human pregnancy; and in placentae from third trimester FGR and gestation-matched control pregnancies using the Fluidigm Biomarker array for mRNA expression, the activity of the enzyme TPH and 5-HT concentrations using an ELISA.

Results: Gene expression for the rate limiting enzymes, TPH1 and TPH2; 5-HT transporter, SLC6A4; and 5-HT receptors HTR5A, HTR5B, HTR1D and HTR1E were detected in all CVS and third trimester placentae. No significant difference in mRNA was observed in SGA compared with control. Although there was no significant change in TPH1 mRNA, the mRNA of TPH2 and SLC6A4 was significantly decreased in FGR placentae (p < 0.05), while 5-HT receptor mRNA was significantly increased in FGR compared with control (p < 0.01). Placental TPH enzyme activity was significantly increased with a concomitant increase in the total placental 5-HT concentrations in FGR compared with control.

Conclusion: This study reports differential expression and activity of the key components of the 5-HT synthetic pathway associated with the pathogenesis of FGR. Further studies are required to elucidate the functional consequences of increased placental 5-HT in FGR pregnancies.

Fig. 1.

Gene expression of the components of the 5-HT synthetic pathway in third trimester FGR placentae compared with gestation-matched control placentae. Placental mRNA of TPH1 (Fig. 1A); TPH2 (Fig. 1B); SLC6A4 (Fig. 1C); and 5-HT receptors HTR5A (Fig. 1D), HTR1D (Fig. 1E) and HTR1E (Fig. 1F) relative to the geometrical averaging of the three housekeeping genes 18S rRNA, YWHAZ and TBP were determined using the 2^−ΔΔCT method [32,33] and analysed using the Mann-Whitney U test.

Fig. 2.

Gene expression of the components of the 5-HT synthetic pathway in first trimester SGA and matched control villus tissues. Placental mRNA of TPH1 (Fig. 2A), SLC6A4 (Fig. 2B), HTR5A (2C), HTR5B (2D) and HTR1D (2E) relative to the housekeeping gene 18S rRNA were determined using the 2^−ΔΔCT method [32] and analysed using the Mann-Whitney U test.

Fig. 3.

Quantitation of TPH1/2 enzyme activity. As described in the methods section, the rate limiting enzyme, TPH (1 and 2) activity was measured in the placental tissues (Fig. 3A) and in the cultured trophoblasts (Fig. 3B) obtained from third trimester FGR pregnancies compared with gestation-matched control pregnancies. Fig. 3C depicts the total placental 5-HT content in FGR pregnancies compared with gestation-matched control. Concentrations of 5-HT are represented as nmol 5-HT/μg protein.

Fig. 4.

Immunohistochemical localisation of TPH1, SERT (SLC6A4) and HTR5A protein in first trimester control placental tissues was performed as described in the methods section. Representative images are depicted in Fig. 5. Arrows indicate immunoreactivity for TPH1 (Fig. 4A) in the villous cytotrophoblasts (VCT), syncytiotrophoblast (ST) and in the extravillous cytotrophoblasts (EVCT), while immunoreactivity for SERT (Fig. 4B) and HTR5A (Fig. 4C) localisaed in ST, endothelial cells (EC) and in some stromal cells (Str). Immunoreactivity to IgG was used as a negative control (Fig. 4D).

Fig. 5.

Immunolocalisation for the components of 5-HT synthetic pathway was performed in the third trimester FGR and in gestation-matched control placentae as described in the methods section. Representative images are depicted in Fig. 5. Arrows indicate immunoreactivity for TPH1 (Fig. 5A-Control; Fig. 5B-FGR); SLCA64 (Fig. 5C-Control; Fig. 5D-FGR) and HTR5A (Fig. 5E-Control; Fig. 5F-FGR) in the syncytiotrophoblast (ST), endothelial cells (EC) and in some stromal cells (Str). Immunoreactivity to IgG was used as a negative control (Fig. 5G).