Gestational diabetes mellitus in women increased the risk of neonatal infection via inflammation and autophagy in the placenta

Abstract

Background: Gestational diabetes mellitus (GDM) produces numerous problems for maternal and fetal outcomes. However, the precise molecular mechanisms of GDM are not clear.

Methods: In our study, we randomly assigned 22 pregnant women with fasting glucose concentrations, 1 hour oral glucose tolerance test (1H-OGTT) and 2 hour oral glucose tolerance test (2H-OGTT), different than 28 normal pregnant women from a sample of 107 pregnant women at the First Affiliated Hospital of Jinan University in China. Lipopolysaccharide (LPS), interleukin 1 alpha (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) were measured from blood plasma of pregnant women and umbilical arteries using ultraviolet spectrophotometry. Hematoxylin & Eosin (H&E), Periodic acid-Schiff (PAS) or Masson staining were performed to examine whether diabetes mellitus altered the morphology of placenta. Quantitative PCR (Q-PCR), western blotting and immunofluorescent staining were performed to examine whether diabetes mellitus and autophagy altered the gene expressions of the placental tissue.

Results: We found that women with GDM exhibited increased placental weight and risk of neonatal infection. The concentrations of IL-6 protein and IL-8 protein in GDM were increased in both maternal and umbilical arterial blood. H&E, Masson and PAS staining results showed an increased number of placental villi and glycogen deposition in patients with GDM, but no placental sclerosis was found. Q-PCR results suggested that the expression levels of HIF-1α and the toll like receptor 4 (TLR4)/ myeloid differential protein-88 (MyD88)/ nuclear factor kappa-B (NF-κB) pathway were increased in the GDM placenta. Through Western Blotting, we found that the expression of NF-kappa-B inhibitor alpha (IKBα) and Nuclear factor-κB p65 (NF-κB p65) in GDM placenta was significantly enhanced. We also showed that the key autophagy-related genes, autophagy-related 7 (ATG7) and microtubule-associated protein 1A/1B-light chain 3 (LC3), were increased in GDM compared with normal pregnant women.

Conclusions: Our results suggest that women with GDM exhibit an increased risk of neonatal infection via inflammation and autophagy in the placenta.

Figure 1

Screening, randomization and analysis of populations.

Figure 2

The comparison of the inflammatory factors in maternal and umbilical artery blood plasma. A: Mean plasma LPS values (ng/ml) from normal and GDM women. B-E: The ELISA data showing the expression of the inflammatory factors: IL-1α (B), IL-6 (C), IL-8 (D) and TNF-α (E) in maternal blood plasma. F-I: The ELISA data showing the expression of the inflammatory factors: IL-1α (F), IL-6 (G), IL-8 (H) and TNF-α (I) in umbilical artery blood plasma. ^∗ P < .05.

Figure 3

Comparison of the placental morphology in normal and GDM women. A-F: Representative micrographs of normal and GDM placental sections stained with H&E (A-B), Masson (C-D) and PAS (E-F). The top right corner panels in C-F are higher magnification images of the regions highlighted by the dotted squares, respectively. G-H: Bar charts comparing the number of placental villi (G) and percentage of Masson area (H) in normal and GDM women. I: The quantitative PCR data showing the expression levels of HIF-1α in normal and GDM placentas. ^∗∗ P < .01, ^∗∗∗ P < .001. Scale bar = 50 μm in A-B and 50 μm in C-F.

Figure 4

Analysis of inflammatory factors in normal and GDM placentas. A-D: The quantitative PCR data show the gene expression levels of NF-κB p65 (A), TNF-α (B), TLR4 (C) and MyD88 (D) in normal and GDM placentas. E-F: Western blotting data show the gene expression levels of IKBα (E) and NF-κB p65 (F). ^∗ P < .05, ^∗∗ P < .01.

Figure 5

Comparisons of autophagy-related genes in placental villi of normal and GDM groups. A-F: Representative transverse sections of DAPI staining (A and D), ATG7 staining (B and E), and merged images (C and F) that were taken in normal and GDM placentas. G: Western blotting data show the protein expression level of ATG7. H-M: The representative transverse sections of DAPI staining (H and K), LC3II staining (I and L), and merged images (J and M) that were taken in normal and GDM placentas. The white arrows in E and L indicate highly expressing ATG7 and LC3II cells. N: Western blotting data show the protein expression levels of LC3I/II. ^∗ P < .05, ^∗∗ P < .01. Scale bar = 50 μm in A-F and H-M.