Periodontitis and gestational diabetes

Background + Aims

• Periodontitis affects over 60% of pregnant women and is linked to adverse pregnancy outcomes including gestational diabetes mellitus (GDM).
• Systemic inflammation is a shared feature, but the mechanistic connection remains unclear.
• EVs, released by both host cells and gram-negative bacteria (as outer membrane vesicles, OMVs), enable cell-to-cell communication and have been implicated in insulin signalling and glucose metabolism. The authors previously showed higher gingival crevicular fluid (GCF) EV concentrations in women who later developed GDM, but the proteomic makeup of these EVs had not been characterised.
• This study aimed to:
  • Characterise GCF and plasma EVs and their proteomic cargo in healthy pregnant women compared with women who have periodontitis alone or periodontitis with GDM.

Materials + Methods

• Cross-sectional study of 104 pregnant women at 24–30 weeks’ gestation (in Santiago, Chile).
• 3 groups:
  • Gingivally healthy (n = 18; 17.3%)
  • Periodontitis alone (n = 52; 50%)
  • Periodontitis + GDM (n = 34; 32.7%).
• GDM diagnosis was determined via 75 g oral glucose tolerance test (IADPSG criteria)
• Periodontitis diagnosis was determined via 2018 classification (probing depth, clinical attachment loss, bleeding on probing, plaque index).
• GCF collected on paper strips
• Fasting plasma also sampled.
• EV isolation
  • Precipitation (ExoQuick) plus ultracentrifugation. Characterisation by nanoparticle tracking analysis (NTA – measures particle size and concentration), transmission electron microscopy (TEM), and a multiplex immunoassay for EV surface markers (CD9, CD63, CD81 – tetraspanins that sit in the EV membrane; syntenin-1, VLA-4, cytochrome c).
• Proteomics
  • Label-free liquid chromatography–tandem mass spectrometry (LC-MS/MS) on GCF-EVs pooled per group
  • peptides matched against the human proteome and the Human Oral Microbiome Database.
  • Enrichment based on Gene Ontology (GO) terms.
• Statistics: Kruskal–Wallis with Dunn’s post-hoc tests.

Results

• Clinical:
  • The 3 groups were similar in age, BMI, nutritional status and smoking.
  • Women with both GDM and periodontitis had the highest blood-sugar readings (fasting glycaemia p = 0.036; 1-hour OGTT p < 0.0001) and more severe indices (all p < 0.0001). They also had more severe (Stage IV) periodontitis and slightly fewer teeth.
• Gingival-crevice EVs:
  • Nearly x3 more EVs were released into the gum crevice in the GDM + periodontitis group than in healthy women (p = 0.0015), with both small and large subtypes raised.
  • Surface markers CD9 and CD81 (proteins that sit on the EV membrane) were reduced, and the CD81/CD63 ratio was lower – a pattern linked in other studies to more inflammatory EV activity.
  • EVs in the bloodstream did not differ between groups, suggesting the changes were local to the gums.
• Protein cargo
  • Almost 2,000 proteins were identified inside the gingival-crevice EV – around 80% matched known EV proteins, confirming their identity.
  • Cargo pointed to immune inflammation (neutrophil activity, complement, reactive oxygen species) and, notably, to glucose metabolism and insulin response – including insulin-signalling proteins (PIK3R1, MEAK7) and inflammatory mediators (IL-18, MMP-8, MMP-9).
  • EVs also carried bacterial proteins, with different bacterial mixes in each group. The GDM + periodontitis group showed more proteins from known periodontal pathogens (Fusobacterium, Porphyromonas, Prevotella), including virulence factors such as gingipains and fimbrial proteins.

Limitations