Effect of non-surgical periodontal therapy on the degree of gingival inflammation and stress markers related to pregnancy

Abstract Objectives The purpose of this study was to determine the impact of nonsurgical periodontal therapy considering the salivary stress-related hormone and cytokine levels in the gingival crevicular fluid (GCF) on pregnant and nonpregnant women. Material and Methods Thirty non-pregnant (control group) and 30 pregnant women (test group) that met the study inclusion criteria were chosen. Only participants with gingivitis were included. Clinical data and samples of GCF and saliva were collected at baseline and after periodontal therapy. The levels of interleukin-1 beta (Κ-1β) and IL-10, and concentration of salivary chromogranin A (CgA) hormone were analyzed by enzyme-linked immunosorbent assay (ELISA). The repeated measures analysis of variance was used for intragroup and intergroup analyses. Results A major decrease in the gingival inflammation was observed in both groups after periodontal therapy (p<0.05). Periodontal treatment decreased the level of IL-1β in GCF (p<0.05) in control group, but no statistical difference was determined for GCF IL-1β in the test group. However, after periodontal therapy, the CgA hormone concentration was reduced in both groups (p<0.05). However, there was no difference in salivary CgA concentration, GCF IL-10 levels, and perceived stress scale (PSS)-10 between the groups (p>0.05). Conclusions Within the limitations of this study, periodontal therapy significantly improved the periodontal status and stress level. In addition, the severity of the gingival inflammation during pregnancy was related to stress. However, further studies will be needed to substantiate these early findings.


Introduction
Pregnancy gingivitis has been described as a prominent inflammatory reaction of the gingiva to the microbial dental plaque, which typically happens in the second and third trimesters of pregnancy 11 .
The clinical and histological features of pregnancy gingivitis are similar to plaque-induced gingivitis 16 .
Clinically, there is a moderate-to-severe inflammation, which can progress to severe hyperplasia, pain, and bleeding 16 . Different etiological pathways have been suggested in an attempt to understand the intense gingival inflammation. The most important theories to define pregnancy include hormonal effects on the immune system, the subgingival biofilm, the specific cells of the periodontium, and the vasculature.
These potential etiologies have been studied in other articles 7,11 . However, there has been no consensus on the etiology of pregnancy gingivitis since periodontal complication is multifactorial in nature 19 .
According with the immune system theory, immunomodulatory alterations would render tissues in the periodontium more prone to gingival inflammation and release of proinflammatory cytokines during pregnancy 18 . Interleukin-1 beta (IL-1β), which has a major role in regulating the inflammatory reaction, is a multifunctional proinflammatory mediator 5 . Dinarello, et al. 5 (1991) stated that the biological activity of IL-1β is multifunctional, activating acute-phase proteins, prostaglandins, and other cytokines as well as inducing collagen and collagenase production and bone resorption. Therefore, proinflammatory activity stimulates the action and coordinates the course of the immune response 14 .
Physiological or hormonal responses to pregnancy stress and the effect of pregnancy-specific stress are important issues. Physiological and hormonal changes in the body during pregnancy can affect the psychosocial state and increase susceptibility to stress 3 . Increased stress during pregnancy may lead to increased stress-related hormone levels 29 .
Chromogranin A (CgA), one of the stress-related hormones, is released along with a glycoprotein under stress within sympathetic nerve endings and catecholamines from the adrenal medulla 2 . CgA is not only a marker of stress in the central nervous system, but it also participates in bacterial infections 2 . Metz-Boutıgue, et al. 21 (2003) reported that vasostatin-1, the N-terminal fragment of CgA, was released by animal polymorphonuclear (PMNs) lymphocytes during the stress process. Interestingly, PMN is the first step in the natural immune response to bacterial challenge in periodontal disease 10 . For this reason, CgA fragments are likely to be produced locally by human PMNs in periodontal disease 10 . Among these factors, interactions require a multi-dimensional stress concept including psychological, social, and physiological aspects and clarifying the stress research in pregnant women. To date, few researchers have investigated the effect of pregnancy on hormonal changes and the impact of pregnancy-specific stress on periodontal status 16,29 .
Psychosocial stress is known to affect periodontal status. Physiological and psychological changes in the body during pregnancy (weight gain, concerns of the mother about the baby, etc.) may affect the periodontal status. Therefore, the goal of this study is to examine the effect of stress state during pregnancy on periodontal status and the effect of non-surgical periodontal treatment on pregnant and non-pregnant women cytokines levels in gingival crevicular fluid (GCF) and on salivary stress-related hormone. Results were calculated using the standard curves formed by each assay for saliva and GCF volume and were given in pmol/mL and pg/mL, respectively.

Statistical analysis
The clinical data were analyzed using statistical software (IBM SPSS Statistics 19, SPSS Inc., Somers, NY, USA) and were expressed by mean and standard deviation. The difference between the two means for the continuous variables was tested for significance using one-way analysis of variance (ANOVA). Two-way

Results
Demographic and socioeconomic variables of the study as means and standard deviations are indicated in Table 1. There were no differences between groups in terms of all demographic and socioeconomic variables at baseline (p>0.05). In Table 2, the clinical data, including means of the gingival index, plaque index, clinical attachment levels and probing on depth scores, and at baseline and after NPT are shown.
Periodontal clinical parameters decreased statistically after therapy in both groups (p<0.05). In the test group, periodontal probing depth index, gingival index, and plaque index scores were higher than the control group (p<0.05). Table 3 shows the cytokine levels in the GCF throughout the study. There were no differences in levels of GCF IL-10 and IL-1β levels at baseline (p>0.05). Regarding IL-10 levels, there were no differences between groups during the study (p>0.05).
In addition, GCF IL-1β cytokine levels had a statistically significant reduction in the control group. However, there was no difference for GCF IL-1β level in the test group after three weeks. There were no statistically significant differences for IL-1β levels in the test group compared to the control group at baseline, but there was a higher statistically significant difference after three weeks (p<0.05). CgA levels in saliva and PSS-10 results throughout the study are shown in Table   3. There were no differences in the concentration of CgA and in PSS-10 scores between groups throughout the study (p>0.05). Furthermore, the concentration of the CgA hormone significantly reduced among groups after therapy (p<0.05).   groups. These findings suggest that stress levels decreased due to periodontal healing. Furthermore, there was a significant decrease in the control group after periodontal therapy, and no significant decrease in IL-1β levels of the test group. These findings may explain, at least in part, that the reduction of GCF IL-1β levels in pregnant women may not be associated with stress, and the reduction of GCF IL-1β in non-pregnant women may be associated with stress.

Variable
This study has some limitations. Stress is a versatile concept and additional markers that are known to be associated with stress need to be examined in assessing stress. Also, the psychological state of pregnancy is highly variable and the evaluation of acute and chronic stresses will provide more accurate results than assessing the effect of stress.
Additionally, the assessment of gingival disease requires postpartum periodontal status, this study has a limited sample size, and the findings need to be proved in the future by clinical trials.

Conclusions
Within the limitations of this study, periodontal therapy significantly reduced the stress-related hormone level and ensured a significant decrease in clinical periodontal parameters. Also, the severity of the gingival inflammation during pregnancy was believed to be related to stress. This finding should be interpreted with caution since more prospective studies are needed to prove the effect of non-surgical periodontal therapy on gingival inflammation status and on salivary stress-related hormones during pregnancy.