Maternal Periodontal Inflammation and Pregnancy Outcomes: Salivary and Amniotic Biomarkers

This prospective observational cohort study will be conducted at the Department of Obstetrics and Gynecology and the Department of Periodontology, Gülhane Training and Research Hospital, Health Sciences University, Ankara, Türkiye. The study is designed to investigate the association between maternal periodontal inflammation and inflammatory and oxidative stress biomarkers measured in saliva and amniotic fluid, and to evaluate their relationship with pregnancy outcomes.

A total of 80 pregnant women between 28 and 32 weeks of gestation will be recruited consecutively from the Obstetrics and Gynecology outpatient clinic. The required sample size was determined using G*Power version 3.1 based on the primary outcome variable (difference in amniotic fluid 8-hydroxy-2'-deoxyguanosine [8-OHdG] levels between women with and without periodontitis). Assuming an effect size (Cohen's d) of 0.70, a two-sided significance level (α = 0.05), and a statistical power of 85% (1-β = 0.85), the minimum required sample size was calculated as 76 participants. To account for potential dropouts, incomplete biological samples, and missing clinical data, the final recruitment target was increased to 80 participants.

Eligible participants will be women aged 18-40 years with singleton pregnancies between 28 and 32 gestational weeks, who are systemically healthy, have undergone routine gestational diabetes screening with normal results, are not receiving medications other than routine prenatal vitamin and iron supplementation, have not received periodontal therapy within the previous six months, and are classified as low-risk pregnancies. Women with multiple pregnancies, smoking or alcohol consumption, acute infections, antibiotic or anti-inflammatory drug use during pregnancy, placental pathology, major fetal anomalies, previous chorioamnionitis, or other obstetric high-risk conditions will be excluded.

All participants will receive detailed verbal and written information regarding the study, and written informed consent will be obtained before enrollment. The study involves no experimental intervention, and all clinical procedures will be performed in accordance with routine clinical practice.

Following enrollment, participants will be referred to the Department of Periodontology for a comprehensive periodontal examination. Clinical periodontal parameters will be recorded by a calibrated periodontist using a manual periodontal probe and will include Plaque Index (PI), Gingival Index (GI), Bleeding on Probing (BOP), Probing Pocket Depth (PPD), and Clinical Attachment Level (CAL). The diagnosis of periodontitis will be established according to the 2017 World Workshop Classification of Periodontal and Peri-Implant Diseases and Conditions.

Participants will then be categorized into two cohorts according to periodontal status: pregnant women without periodontitis (including periodontally healthy participants and those with gingivitis) and pregnant women with periodontitis.

After completion of the periodontal examination, unstimulated whole saliva samples will be collected. Participants will be instructed not to eat, drink, brush their teeth, or use mouthwash for at least two hours before sample collection. Saliva will be allowed to accumulate naturally and will be collected into sterile collection tubes over approximately 15 minutes. Samples will immediately be transferred into sterile tubes and stored at -80°C until laboratory analysis. After saliva collection, all participants will receive standardized oral hygiene instructions. No periodontal treatment will be performed as part of the study protocol before delivery.

Amniotic fluid samples will be collected only during routine delivery procedures. During vaginal delivery, samples will be obtained immediately after spontaneous or artificial rupture of the fetal membranes. During cesarean delivery, amniotic fluid will be collected immediately after entering the uterine cavity. Sample collection will not require any additional invasive procedures beyond standard obstetric care. The collected amniotic fluid specimens will be transferred into sterile tubes and stored at -80°C until biochemical analysis.

Pregnancy outcome data will be obtained from hospital medical records after delivery. Recorded variables will include gestational age at delivery, pregnancy outcome (term [≥37 completed weeks] or preterm [\<37 completed weeks]), birth weight, low birth weight (\<2500 g), mode of delivery, and the occurrence of pregnancy complications, including preeclampsia.

Inflammatory and oxidative stress biomarkers will be measured in both saliva and amniotic fluid using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' protocols. The biomarkers to be analyzed include:

8-Hydroxy-2'-deoxyguanosine (8-OHdG) Malondialdehyde (MDA) Interleukin-6 (IL-6) Matrix Metalloproteinase-8 (MMP-8)

All saliva and amniotic fluid samples will be assigned unique identification codes before laboratory analysis. The investigator performing the ELISA analyses will be blinded to the participants' periodontal status, pregnancy outcomes, and clinical data until completion of all laboratory measurements.

Statistical Analysis

Statistical analyses will be performed using IBM SPSS Statistics Version 21.0 (IBM Corp., Armonk, NY, USA). Continuous variables will first be evaluated for normality using the Shapiro-Wilk test (and confirmed visually by histograms and Q-Q plots when appropriate). Descriptive statistics will be presented as mean ± standard deviation (SD) for normally distributed variables and median (interquartile range) for non-normally distributed variables. Categorical variables will be summarized as frequencies and percentages.

The primary outcome is the difference in amniotic fluid 8-OHdG concentration between pregnant women with and without periodontitis.

Comparisons between the two periodontal cohorts will be performed using the Independent Samples t-test for normally distributed variables or the Mann-Whitney U test for non-normally distributed variables.

Following delivery, pregnancy outcomes will be classified as term (≥37 completed weeks of gestation) or preterm (\<37 completed weeks of gestation). Secondary subgroup analyses will evaluate biomarker levels and clinical outcomes according to pregnancy outcome within each periodontal cohort, when sample size permits. Depending on data distribution, comparisons will be performed using the Independent Samples t-test, Mann-Whitney U test, One-Way Analysis of Variance (ANOVA), or Kruskal-Wallis test, as appropriate. When significant overall differences are detected, appropriate post hoc analyses (Tukey's HSD, Dunnett's T3, or Bonferroni-adjusted Mann-Whitney U tests) will be applied.

Associations between categorical variables will be evaluated using the Chi-square test or Fisher's exact test, where appropriate.

Correlations between periodontal clinical parameters and biomarker concentrations measured in saliva and amniotic fluid will be assessed using Pearson correlation coefficients for normally distributed variables or Spearman's rank correlation coefficients for non-normally distributed variables.

If clinically relevant baseline variables (such as maternal age, body mass index, or parity) differ significantly between the two periodontal cohorts, multivariable linear regression analyses will be performed to adjust for potential confounding factors and to evaluate the independent association between periodontal disease and biomarker levels.

All statistical tests will be two-tailed, and a p-value \<0.05 will be considered statistically significant.