Periodontal Status and Endothelial Dysfunction in Patients With Polycystic Ovary Syndrome
Condition: Periodontal Diseases
MeSH terms: Polycystic Ovary Syndrome; Periodontal Diseases
Study type: Observational

Summary: 
      Emerging research indicates a link between polycystic ovary syndrome (PCOS) and periodontal
      diseases, revealing the intricate relationship between oral health and systemic conditions.
      PCOS, a hormonal disorder in women of reproductive age, often associates with obesity,
      dyslipidemia, and insulin resistance, heightening the risk for type 2 diabetes (T2D) and
      cardiovascular disease (CVD). The pathogenesis of PCOS involves an inflammatory response
      marked by increased CRP, inflammatory cytokines, elevated blood leukocytes, adhesion molecule
      expression, and oxidative stress markers like myeloperoxidase (MPO).

      Periodontal diseases, bacterial infections affecting gums, ligaments, cement, and bone,
      include gingivitis (gum inflammation) and periodontitis (irreversible tissue destruction).
      Evidence suggests a link between periodontitis and increased CVD risk, while such association
      with gingivitis is limited.

      Potential mechanisms linking periodontal diseases and CVD involve cytokine release, oral
      bacteria toxin production, and direct bloodstream transfer. Recognition of lipopolysaccharide
      (LPS) and TNFα triggers innate immune cells via TLR4 and TNFR, activating NF-κB and JNK
      expression. JNK amplifies inflammatory responses, inducing proinflammatory genes, and TNFα,
      IL-1, IL-6, and IL-8 can invade endothelial layers, promoting adhesion molecule expression.
      Enhanced leukocyte ROS production, especially in periodontitis, contributes to endothelial
      dysfunction and heightened cardiovascular risk.

      The activation of multiple inflammatory pathways likely links PCOS, periodontal disease, and
      increased cardiovascular risk. Thus, the researchers aim to investigate if the presence of
      periodontal diseases, particularly gingivitis, exacerbates oxidative stress, inflammation and
      atherosclerosis surrogate markers in women with PCOS, and explore the underlying molecular
      mechanisms.
    

Detailed description: 
      The population will be identified using the Rotterdam criteria, which will include the
      following characteristics: irregular ovulation (cycles longer than 35 days or less than 26
      days); elevated levels of free testosterone (>0.5 ng/dl; the cut-off level for free
      testosterone will be determined based on the mean ± 2 standard deviations from normal levels
      observed in the control group); hirsutism (total Ferriman-Gallwey score > 7); and the
      presence of polycystic ovaries, which will be identified through transvaginal ultrasonography
      (i.e., the presence of 12 or more small follicles measuring 2 to 9 mm in each ovary). The
      control group will be matched in BMI and age to the intervention group. In both groups, all
      medication, pathologies, and other medical conditions that could affect their reproductive
      physiology will be registered. All patients will receive detailed information on the study,
      highlighting its benefits and drawbacks, and will sign the informed consent and
      confidentiality commitment.

      A comprehensive periodontal examination will be conducted across all study cohorts, adhering
      to established consensus criteria. Each tooth in the oral cavity will be probed at 6 points-3
      buccal and 3 palatal-utilizing a periodontal chart with a Williams-type millimeter probe.
      Factors such as tobacco use, tooth brushing frequency, and alcohol consumption will be
      considered via a questionnaire, as well as through plaque and calculus indices. The
      periodontal examination will be carried out by a single doctor with training in periodontics
      to avoid inter-explorer bias. The researchers will also ask about dental treatments received,
      emphasizing periodontal treatments or those related to generating changes in soft tissues
      such as orthodontic gingival recessions. The periodontal measures will include the percentage
      of bleeding on probing (BOP), millimeters of clinical attachment level (CAL), millimeters of
      probing pockets depth (PPD), percentage of loss of bone, number of teeth with periodontal
      pockets-PPD ≥4mm- and number of teeth with CAL≥4mm-. The researchers will evaluate the plaque
      and calculus level using the Silness and Löe index for plaque and O´Leary index for calculus
      levels. To facilitate the measurement of plaque and calculus indexes, as they will be carried
      out in a hospital with a high flux of patients, these indices will be measured on the
      Ramfjord index teeth (right permanent maxillary first molar, right permanent incisor, left
      permanent first premolar, left mandibular permanent first molar, right permanent mandibular
      incisor, and right permanent mandibular first premolar).

      A peripheral venous blood sample will be taken after 12 hours of fasting, during the
      follicular phase of the menstrual period of the patients (from the 1st to the 5th day of
      bleeding) to avoid biases dependent on the variability of sexual hormones. Neutrophils and
      PBMC will be isolated from 8 mL of EDTA-anticoagulated peripheral blood by means of an
      immunomagnetic method, following the manufacturer's protocol. In addition, LUNA-FL will be
      used to determine cell count and viability (acridine orange and propidium iodide double
      stain). Proteins will be extracted from neutrophils or PBMC on ice. Neutrophils will be lysed
      for 15 minutes with RIPA Lysis Buffer supplemented with protease and phosphatase inhibitors.
      The supernatant will be collected after centrifugation for 15 minutes at 16,000g. The total
      protein concentration will be quantified using a bicinchoninic acid (BCA) protein assay.
      Aliquots of 25µg of protein will be resolved on 8-16% gradient SDS-polyacrylamide gels and
      transferred to nitrocellulose membranes. Target proteins will be detected by incubating the
      membranes with anti-actin, JNK, NFkB, MCP1, GPX-1, NLRP3, ASC, procaspase 1, caspase 1,
      Beclin, ATG5-ATG12, p62, LC3 I, LC3 II, Pink1, GRP78, eIF2alpha, IRE1 alpha, ATF6, CHOP, PGC1
      alpha, mTFA, VDAC, Complex I, II, III, IV and V. The protein signal will be detected by
      chemiluminescence and analyzed by densitometry.

      A parallel plate flow chamber, connected to an inverted microscope, will enable the
      researchers to measure neutrophil-endothelial cell interactions in vitro. Prior to this,
      immortalized endothelial cells from the human umbilical vein will be seeded. One million
      leukocytes in 1 mL of RPMI medium will be drawn across the HUVEC. A video camera connected to
      the microscope will permit a 5 × 25 mm view of the endothelial cells. Various leukocyte
      parameters, including rolling velocity, rolling flux, and adhesion, will be evaluated over a
      period of 5 minutes. Analysis of serum cytokines, cellular adhesion molecules, and
      glutathione will be measured with a Luminex® 200 analyzer system following the Milliplex® MAP
      Kit manufacturer's procedure or ELISA technique.

      This study was designed to achieve a power of 80% and to detect significant differences of 20
      % in relation to the primary efficacy criterion - leukocyte-endothelium interactions
      (measured by adhesion of neutrophils) - assuming that 3 groups existed. Under these premises,
      at least 12 subjects per group were considered. Normally and non-normally distributed data
      will be expressed as mean ± standard deviation (SD) and median (25th-75th percentiles),
      respectively. Qualitative data will be expressed as percentages. The data will be analyzed
      using an unpaired Student's t test or a Mann-Whitney U test for parametric and non-parametric
      data, respectively. When comparing three groups, statistical significance will be assessed by
      one-way ANOVA or the Kruskal Wallis test followed by post hoc test. The strength of the
      association between variables will be measured by means of Pearson's or Spearman correlation
      coefficient, for parametric and non-parametric data, respectively. To predict the value of a
      variable based on another variable, linear regression analysis will be used. Differences will
      be considered significant when p < 0.05, with a confidence interval of 95%. Analysis will be
      performed with SPSS 22.0.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Primary care clinic
      
Keywords: inflammation; atherosclerosis; gingivitis; oxidative stress