Periodontal Status Assessment, Molecular Mechanisms Underlying Inflammatory Response, and Endothelial Dysfunction Evaluation in Patients With Polycystic Ovary Syndrome

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.