Volume 85 • Number 10
Periodontal Status and High-Sensitivity C-Reactive Protein Levels in Polycystic Ovary Syndrome With and Without Medical Treatment Surya Porwal,* Shikha Tewari,* Rajinder K. Sharma,* Savita Rani Singhal,† and Satish C. Narula*
Background: Recently, some studies have revealed the effect of polycystic ovary syndrome (PCOS) on gingival inflammation. This cross-sectional study attempts to assess the periodontal status and systemic inflammation of women receiving medical treatment for PCOS and women newly diagnosed with PCOS. Methods: A total of 126 participants comprising 41 newly diagnosed patients with PCOS (PCOS-N), 45 patients with PCOS on medical treatment (PCOS-MT), and 40 systemically healthy controls (control group [CG]) were examined. Periodontal parameters, anthropometric parameters, and serum levels of high-sensitivity C-reactive protein (hsCRP) were recorded. Results: Women with newly diagnosed PCOS had increased sites with bleeding on probing (BOP), probing depth, clinical attachment level (CAL), waist circumference (WC), hsCRP, and prevalence of periodontitis compared with control and PCOS-MT groups (P £0.05). On partial correlation analysis after controlling for confounders, BOP and CAL correlated positively and significantly with hsCRP (P = 0.01 and P = 0.005). Multivariate linear regression analysis revealed that BOP and CAL (dependent variable) (P = 0.009/R2 = 0.05 and P = 0.005/R2 = 0.07, respectively) had significant association with hsCRP. Furthermore, hsCRP, when considered as outcome, also exhibited association with CAL and WC (P = 0.002/R2 = 0.07 and P = 0.04/R2 = 0.106). Logistic regression analysis demonstrated that the PCOS-N group had 2.88 times increased likelihood of having moderate periodontitis (adjusted odds ratio 2.88, 95% confidence interval 1.18 to 6.98). Conclusions: Women with newly diagnosed PCOS may have increased prevalence and likelihood for periodontitis, with higher measures of periodontal inflammation and breakdown than those on medical treatment for PCOS and systemically healthy females. Furthermore, periodontal breakdown might depend on systemic inflammation and vice versa. J Periodontol 2014;85:1380-1389. KEY WORDS C-reactive protein; inflammation; insulin resistance; periodontitis; polycystic ovary syndrome; waist circumference. * Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences, Rohtak, Haryana, India. † Department of Obstetrics and Gynecology, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India.
doi: 10.1902/jop.2014.130756
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P
olycystic ovary syndrome (PCOS) is a genetically complex endocrine disorder of uncertain etiology and a common cause of anovulatory infertility, menstrual dysfunction, and hirsutism.1,2 PCOS is one of the most common reproductive abnormalities, affecting 5% to 10% of the population of reproductive age in the southeastern part of the United States.3 PCOS appears to be associated with an increased risk of metabolic aberrations, including type 2 diabetes mellitus, dyslipidemia, cardiovascular disease, and endometrial carcinoma.4-7 In its typical form, it includes insulin resistance, hyperinsulinemia, obesity (predominantly the abdominal phenotype), and altered lipid profile.8,9 Insulin resistance and hyperinsulinemia play a critical role in the syndrome’s pathogenesis.10 Both hyperinsulinemia and obesity may be intimately related to the development and maintenance of hyperandrogenism.11,12 Increased androgen levels may directly affect insulin sensitivity in the target tissues, particularly muscles, therefore contributing to the development of the insulin-resistant state.13 The raised C-reactive protein (CRP) levels associated with the syndrome are connected to the low-grade chronic inflammation linked to insulin resistance.14 Periodontitis is a chronic inflammatory disease triggered by bacterial products, such as lipopolysaccharides, and the subsequent exuberant inflammatory response leads to loss of alveolar bone.15 Periodontal disease has been implicated as a risk factor in the onset and development of cardiovascular diseases,16 rheumatoid arthritis,17 diabetes mellitus,18 and occlusive respiratory diseases.19 Chronic low-grade inflammation is emerging as a conceivable etiologic mechanism linking periodontal disease and the conditions cited above as well as other systemic diseases.20 CRP is a serologic marker of systemic inflammation that has been associated with increased risk for various systemic diseases.21,22 Periodontitis has also been linked to elevated CRP levels. It is thus postulated that CRP might be a possible mediator of the association between periodontitis and these systemic conditions.23 Patients with periodontitis also have a higher concentration of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1 in serum and/or gingival crevicular fluid.24,25 Chronic stimulation and secretion of proinflammatory cytokines associated with periodontal infection contribute to insulin resistance.26 This pathognomonic state of chronic systemic inflammation and insulin resistance in both periodontitis and PCOS can be hypothesized as a converging channel associating the disorders. Earlier studies have warranted a possible relationship between PCOS and periodontitis, suggesting
Porwal, Tewari, Sharma, Singhal, Narula
impact of PCOS on gingival inflammation.27-29 However, they have not taken into account the medical treatment status, duration, or type of medications used for the treatment of the syndrome. The present study attempts to investigate periodontal status and systemic inflammation levels through high-sensitivity (hs)CRP in women with PCOS, considering the dichotomous state of medical treatment (whether treatment has been undertaken or not). Therefore, the aim of the study is trifold: 1) to assess and compare the periodontal status, anthropometric parameters, and systemic inflammatory burden among systemically healthy females, patients newly diagnosed with PCOS, and patients receiving medical treatment for PCOS; 2) to observe the association among clinical periodontal parameters, anthropometric parameters, and serum levels of systemic inflammatory markers in the study population; and 3) to explore the contribution of systemic inflammation caused by PCOS toward periodontal inflammation and breakdown and vice versa. MATERIALS AND METHODS Study Population This cross-sectional, single-masked study was executed in the Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences (PGIDS), Rohtak, India, with successful collaboration of the Department of Obstetrics and Gynecology, Post Graduate Institute of Medical Sciences (PGIMS), Rohtak, India. This study followed the standard protocols outlined in the 1964 Declaration of Helsinki, as revised in 2008.30 The study design was reviewed and approved by the Institutional Review Board, Pandit Bhagwat Dayal Sharma University of Health Sciences, Rohtak, and ethical acceptance was obtained from the ethics committee of PGIDS. Enrollment was from March 2012 to September 2013. The study population consisted of 41 patients (aged 15 to 36 years, mean age: 26.28 years) newly diagnosed with PCOS (PCOS-N) who had not started any medical treatment for the syndrome and 45 women with PCOS (aged 16 to 31 years, mean age: 22.68 years) undergoing medical treatment (PCOS-MT) (oral contraceptives, metformin, and lifestyle modification) for at least 6 months. The participants were recruited from the regular outpatient Department of Obstetrics and Gynecology, PGIMS. Forty systemically healthy controls (control group [CG]) matched for age (17 to 32 years, mean age: 23.50 years) and body mass index (BMI) were recruited from the patients attending the regular outpatient Department of Periodontics and Oral Diagnosis, Radiology and Medicine, PGIDS. Enrollment criteria in PCOS groups were females of reproductive age diagnosed with PCOS, with ‡16 natural teeth. The diagnosis of PCOS 1381
Periodontal Status and hsCRP in Polycystic Ovary Syndrome
was made according to Rotterdam criteria,31 in the presence of ‡2 of the following: 1) clinical and/or biochemical hyperandrogenism; 2) chronic oligo- or anovulation; and 3) polycystic ovaries on ultrasound (presence of ‡12 follicles in each ovary measuring 2 to 9 mm in diameter and/or increased ovarian volume >10 mL). Inclusion criteria for systemically healthy controls were regularly menstruating females with no clinical or biochemical sign of hyperandrogenism and ultrasound exclusion of PCOS. The recruitment excluded patients with any history of thyroid dysfunction, hyperprolactinemia, and androgen-secreting tumors to avoid misdiagnosis of PCOS. Patients with chronic inflammatory disease such as nephrotic syndrome, chronic renal failure, significant cardiovascular disease, established type 1 or type 2 diabetes mellitus, or active cancer within the past 5 years were excluded to control the confounders. Additional exclusion criteria included: 1) smoking and alcohol consumption; 2) history of systemic antibiotics within 3 months; 3) periodontal treatment within 6 months; and 4) aggressive periodontitis. An elaborate medical history supported with investigations and consultation with the referring physician was relied on to rule out the presence of any of the above conditions and for the confirmed diagnosis of PCOS. Written informed consent was received from each patient after explaining the procedure along with the risks and benefits in her own language. All the participants in the study had to undergo examination of the various anthropometric parameters, periodontal parameters, and estimation of hsCRP levels. Allotment of patients to different groups was done by one investigator (ST), whereas examination of all parameters was recorded and carried out by another investigator (SP) to ensure investigator masking. Analyses of Anthropometric Parameters Waist circumference32 (WC) was measured in centimeters, at the midpoint between the lower margin of the lowest palpable rib and the top of the iliac crest. For waist-to-hip ratio32 (WHR), hip circumference was measured around the widest portion of the buttocks. BMI was recorded as weight in kilograms divided by height in meters squared. Blood Sampling Blood samples for the investigation of serum hsCRP were collected after overnight fasting. Venous blood from the antecubital vein was collected after applying a tourniquet in plain tubes without additive. Serum hsCRP levels were assessed using a kit‡ with highsensitivity methodology in an auto-analyzer§ according to the manufacturer’s instructions. The test principle was particle-enhanced immune-turbidimetric assay, in which human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. 1382
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The turbidity induced by the formation of immune complexes was measured at 546 nm. The lower detection limit for the assay was 0.15 mg/L. Periodontal Parameters The clinical protocol included registration of the following periodontal parameters: plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL), which were recorded by a single trained examiner (SP) on each tooth (except third molars) with a periodontal probei and mirror. BOP, PD, and CAL were recorded at six sites per tooth: mesio-buccal, mid-buccal, distobuccal, mesio-lingual/palatal, mid-lingual/palatal, and disto-lingual/palatal. PI33 and GI34 were recorded at four sites per tooth: mesio-buccal, mid-buccal, distobuccal, and mid-lingual/palatal. The number of sites with BOP was recorded as a percentage, and the assessment was done dichotomously with 0 as absence and 1 as presence. Assessment of moderate chronic periodontitis (CP) was defined by ‡2 interproximal sites with CAL ‡4 mm or ‡2 interproximal sites with PD ‡5 mm, not on the same tooth.35 Mild CP was defined as ‡2 interproximal sites with CAL ‡3 mm and ‡2 interproximal sites with PD ‡4 mm (not on the same tooth) or one site with PD ‡5 mm.36 Diagnosis of gingivitis was assigned when BOP was present at >50% of all sites and PD was 4 mm and CAL £1 mm and no clinical and/or radiographic sign of periodontitis was evident. Reproducibility of clinical measurements was verified by the same investigator (SP) by carrying out double clinical periodontal data recording for PD and CAL on 10% of the sample. Intraexaminer reproducibility was determined for each site by calculating the percentage of sites examined where the scores were matching or within 1 mm. Assessment of mean difference in the scores (with 90% accuracy, k value 0.84 for PD and 0.80 for CAL) indicated that there was no systemic bias in the measurements. Statistical Analyses The sample size was calculated by power calculations using statistical software,¶ based on an overall significance level of 0.05, and the assumption of normally distributed data indicated that the statistical power exceeded 80% with a fixed effect size of 0.70 for a total 126 participants, divided in comparison groups of PCOS population with allocation ratio 1:1 and control group. The sample size could ‡ C-Reactive Protein (Latex) High-Sensitivity Assay, Roche Diagnostics, Indianapolis, IN. § Konelab Clinical Chemistry Analyzer, Thermo Fisher Scientific, Waltham, MA. i Williams periodontal probe, Hu-Friedy, Chicago, IL. ¶ G-power 3.0.10, Heinrich-Heine University Du ¨ sseldorf, Du ¨ sseldorf, Germany.
J Periodontol • October 2014
not be estimated on the basis of proportion of PCOS population having periodontitis because of the lack of evidence. Post hoc statistical power calculations with 126 participants comparing six parameters indicated that for correlation coefficient (r) r2 value ‡ 0.0645, the statistical power exceeded 80%. The distribution of the data for all groups was analyzed by Kolmogorov–Smirnov test. Following nonnormal distribution of the data, differences among the groups for continuous variables were assessed using Kruskal–Wallis followed by Mann–Whitney U test and for categoric variables using x2 test. Partial correlation among variables was assessed after controlling for potential confounders BMI, WC, and WHR. Further multiple linear stepwise regression analysis was used to develop models of predictor variables associated with the dependent variable. The evaluation of the association between CP and PCOS was estimated by odds ratio (OR) and 95% confidence intervals (CIs), using logistic regression analysis. Models were constructed with presence/absence of periodontitis as the outcome variable and PCOS disease status and medical treatment status for PCOS as a categoric predictor variable. The Hosmer–Lemeshow statistic was applied to verify the diagnosis of the goodness-of-fit regression model used. All statistical analyses were two-tailed with significance level at 0.05 as calculated using software.# RESULTS Of the 168 patients examined, 132 were eligible for the study and met the inclusion and exclusion criteria; six participants eligible for the PCOS-N group refused to take part in the study. So, the total study population comprised 126 women divided into 40 systemically healthy controls, 41 newly diagnosed PCOS, and 45 PCOS on medical treatment. Demographic data along with serum hsCRP, anthropometric, and clinical measurements are outlined in Table 1. According to the Kruskal–Wallis analysis, there was no significant difference among the ages, PI, and BMI scores of participants in the study groups. Mann–Whitney U test was applied to assess the differences among the groups for the remaining parameters. Serum levels of hsCRP were significantly higher in PCOS-N than systemically healthy and PCOSMT groups (P = 0.000 and 0.001, respectively), whereas an apparent increase in hsCRP levels in PCOS-MT was non-significant compared with the control group (P = 0.14). On intergroup comparisons of the anthropometric parameters, PCOS-N had higher WC compared with CG and PCOS-MT (P = 0.000 and 0.05, respectively), and a non-significant difference was noted between PCOS-MT and CG (P = 0.07). PCOS-N and PCOS-MT had significantly higher WHRs than CG (P = 0.01 and 0.02, respectively), albeit no
Porwal, Tewari, Sharma, Singhal, Narula
significant difference was noted in WHR between the two PCOS groups (P = 0.24). The PCOS-N group had significantly higher PD (P = 0.000 and 0.002, respectively), CAL (P = 0.000 and 0.001), and BOP (P = 0.000 and 0.01) than CG and PCOS-MT, and these variables were also significantly higher in PCOS-MT compared with CG (PD, P = 0.001; CAL, P = 0.002; BOP, P = 0.008). The PCOS-N group had significantly higher GI than CG (P = 0.03), and it was similar between PCOS-N and PCOS-MT (P = 0.10) and PCOS-MT and CG (P = 0.11) (Table 1). The results of different variables assessing the periodontal condition and frequency of patients having periodontal disease in PCOS and control groups are presented in Table 2. The mean number of sites with CAL 3 to 4 mm was significantly higher in PCOS-N compared with PCOS-MT and CG (P = 0.03 and 0.00, respectively). The number of sites with CAL 1 to 2 mm differed non-significantly between the two PCOS groups. Furthermore, PCOS-MT showed a significantly higher number of sites with CAL 3 to 4 mm and 1 to 2 mm than CG (P = 0.01 and 0.00, respectively). The mean number of sites with PD ‡4 mm was significantly higher in PCOS-N than PCOSMT (P = 0.008), and PCOS groups differed nonsignificantly compared with CG. The different groups were comparable with regard to number of sites with PD ‡5 mm. The frequency of moderate periodontitis, as assessed using x2 test, was higher in PCOS-N compared with CG and PCOS-MT (P = 0.001 and 0.004, respectively), whereas the frequency differed nonsignificantly between the CG and PCOS-MT. The prevalence of patients having mild periodontitis revealed no significant difference among the three groups. The frequency of gingivitis was significantly higher in CG compared with PCOS-N (P = 0.02) and comparable in PCOS-N and PCOS-MT. Periodontally healthy individuals were more numerous in CG compared with PCOS-N and PCOS- MT (P = 0.001 and 0.05, respectively). Nevertheless, no patient having severe CP was part of the study population (Table 2). Table 3 depicts partial correlation analysis among periodontal parameters and hsCRP of the total population, after adjusting for potential confounders (BMI, WC, and WHR). The result showed BOP (P = 0.01) and CAL (P = 0.005) to be positively and significantly correlated with hsCRP. Table 4 displays the positive association of BOP (P = 0.009/R2 = 0.05) and CAL (P = 0.002/R2 = 0.07) with hsCRP. Table 5 exhibits the positive association of hsCRP when considered as a dependent variable # SPSS, v.19, IBM, Chicago, IL.
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Table 1.
Comparative Evaluation of Age, hsCRP Levels, and Anthropometric and Periodontal Parameters of CG, PCOS-N, and PCOS-MT Groups CG (n = 40)
PCOS-N (n = 41)
Parameter
Mean – SD
95% CI
Age (years)
23.50 – 2.67
22.64 to 24.35
1.38 – 1.48
0.95 to 1.88
BMI (kg/m )
23.76 – 4.82
22.22 to 25.31
WC (cm)
72.02 – 8.09
WHR
Mean – SD
PCOS-MT (n = 45) Mean – SD
95% CI 21.54 to 24.64
22.68 – 4.55
21.31 to 24.05
2.80 to 5.01
1.77 – 1.83
1.30 to 2.33
25.01 – 3.61
23.87 to 26.15
24.57 – 4.38
23.25 to 25.89
69.43 to 74.61
81.69 – 12.45*†
77.76 to 85.62
76.65 – 11.07
73.32 to 79.98
0.79 – 0.09
0.76 to 0.82
0.83 – 0.08*
0.80 to 0.85
0.81 – 0.04*
0.80 to 0.82
PI
0.97 – 0.83
0.70 to 1.23
1.01 – 0.56
0.83 to 1.19
1.03 – 0.80
0.79 to 1.27
GI
1.34 – 1.09
0.98 to 1.69
1.99 – 0.47*
1.84 to 2.14
1.71 – 0.77
1.48 to 1.94
BOP (%)
45.12 – 37.46
33.14 to 57.10
82.94 – 13.59*†
78.65 to 87.23
66.2 – 27.81*
57.93 to 74.65
PD (mm)
1.78 – 0.72
1.55 to 2.01
2.34 – 0.31*
†
2.24 to 2.44
2.13 – 0.40*
2.01 to 2.26
CAL (mm)
0.77 – 1.26
0.37 to 1.18
1.26 – 0.67*†
1.04 to 1.47
0.83 – 0.55*
0.67 to 1.00
hsCRP (mg/L) 2
23.09 – 4.90
95% CI
3.76 – 3.60*†
* P £0.05 compared with CG. † P £0.05 compared with PCOS-MT.
Table 2.
Distribution of Periodontal Condition Variables (mean 6 SD or n [%]) of the Study Population PCOS-N (n = 41; [%])
PCOS-MT P, PCOS-N P, PCOS-MT P, PCOS-N (n = 45; [%]) versus CG versus CG versus PCOS-MT
Parameter
CG (n = 40; [%])
Number of sites with CAL 1 to 2 mm
44.57 – 66.09 98.02 – 29.65 90.80 – 45.49
0.00*
0.00*
NS
Number of sites with CAL 3 to 4 mm
10.90 – 14.89 26.48 – 26.06 11.62 – 11.41
0.00*
0.01*
0.03*
Number of sites with PD ‡4 mm
9.50 – 12.04 13.14 – 10.52
7.06 – 9.19
NS
NS
0.008*
Number of sites with PD ‡5 mm
9.50 – 11.17
9.33 – 7.99
8.00 – 7.94
NS
NS
NS
09 (22.5)
00 (0)
03 (6.67)
0.001*
0.05*
NS
12 (30)
04 (9.75)
10 (22.22)
0.027*
NS
NS
11 (27.5)
13 (31.70)
20 (44.44)
NS
NS
NS
08 (20)
24 (58.55)
12 (26.67)
0.001*
NS
0.004*
Frequency of periodontally healthy patients† Frequency of gingivitis
†
Frequency of mild periodontitis† Frequency of moderate periodontitis
†
NS = not significant. * P £0.05. 2 † x test.
with models of CAL (P = 0.002/R2 = 0.07) and CAL/WC (P = 0.005/0.04/R2 = 0.10). The results of logistic regression analysis (Table 6) revealed that the chances of having moderate periodontitis were >five times higher in PCOS-N compared with PCOS-MT and CG (unadjusted OR = 5.64, 95% CI 2.09 to 15.24), and this difference was statistically significant. Adjustment for these variables 1384
produced a slight decrease in the magnitude of the association, but reaffirmed that PCOS had an independent effect on periodontitis (adjusted OR = 2.88, 95% CI: 1.18 to 6.98). DISCUSSION PCOS is a complex endocrinopathy having a wide range of variations and clinical manifestations, with
Porwal, Tewari, Sharma, Singhal, Narula
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Table 3.
Correlation of Different Variables Using Applying Partial Correlation Analysis After Controlling for Confounders (BMI, WC, and WHR) Parameter hsCRP Correlation Significance (two-tailed)
Age
PI
GI
BOP
PD
CAL
0.085 0.353
0.168 0.063
0.085 0.349
0.227 0.012*
0.132 0.146
0.254 0.005*
0.146 0.108
0.056 0.540
0.046 0.613
0.075 0.411
0.039 0.665
0.629 0.000*
0.519 0.000*
0.475 0.000*
0.334 0.000*
0.829 0.000*
0.649 0.000*
0.286 0.001*
0.671 0.000*
0.400 0.000*
Age Correlation Significance (two-tailed) PI Correlation Significance (two-tailed) GI Correlation Significance (two-tailed) BOP Correlation Significance (two-tailed) PD Correlation Significance (two-tailed)
0.558 0.000*
* P £0.05.
Table 4.
Significant Associations Among Periodontal Parameters (dependent variable) and Biochemical Parameter hsCRP (independent variable) Using Multiple Stepwise Regression Analysis Dependent Variable BOP CAL
Model Predictors
b Unstandardized
Standard Error
b Standardized
P
R2
95% CI
Constant hsCRP
58.62 27.69
3.64 10.37
0.233
0.000 0.009*
0.05
51.41 to 65.83 7.15 to 48.23
0.278
0.000 0.002*
0.07
Constant hsCRP
0.742 0.933
0.102 0.289
0.541 to 0.943 0.360 to 1.50
* P £0.05.
no single etiologic factor that fully accounts for the whole spectrum of the abnormalities of the condition.37 Since 1935, when Stein and Leventhal38 incipiently described the syndrome, exhaustive research has been published associating PCOS and systemic conditions such as cardiovascular diseases and type 2 diabetes mellitus.39 The insulin resistance and hyperinsulinemia associated with PCOS leads to development of a chronic inflammatory state. Moreover, IL-6 and TNF-a levels have been shown to be elevated in females with PCOS compared with an obese control group of females.40 In periodontitis, chronic stimulation and secretion of higher levels of pro-inflammatory cytokines such as
TNF-a and IL-1 also contribute to insulin resistance and a chronic inflammatory state.26 In addition, increased concentrations of inflammatory biomarkers such as CRP and IL-6 in both gingival tissue and serum have been reported in patients with periodontitis.24 Recently, some evidence has shown the plausibility of PCOS being associated with increased susceptibility for periodontal disease, illustrating gingival inflammation as a common finding in these patients,27 and thus paving the way for further research to determine the association of PCOS with periodontal disease. Because chronic systemic inflammation colligates the pathophysiology of both PCOS and periodontal disease, the present study is strategized accordingly 1385
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Table 5.
Significant Associations Among Periodontal and Anthropometric Parameters (independent variable) and Biochemical Parameter hsCRP (dependent variable) Using Multiple Stepwise Regression Analysis Model Predictors
b Unstandardized
Standard Error
b Standardized
P
R2
95% CI
hsCRP
Constant CAL
0.151 0.083
0.034 0.026
0.278
0.000 0.002*
0.077
0.084 to 0.217 0.032 to 0.134
hsCRP
Constant CAL WC
-0.153 0.74 0.004
0.156 0.026 0.002
Dependent Variable
0.247 0.173
0.329 0.005* 0.04*
0.106
-0.461 to 0.155 0.023 to 0.125 0.000 to 0.008
2
Table 5 shows regression coefficient values (R ) of hsCRP (dependent variable) and periodontal and anthropometric parameters (independent variable) in two different models of regression analysis. * P £0.05.
Table 6.
Association Measurements by Logistic Regression Analysis Among PCOS Groups and CP Models
OR
95% CI
P
Unadjusted
5.64
2.09 to 15.24
0.001
Adjusted*
2.88
1.18 to 6.98
0.01
* Adjusted for medical treatment for PCOS.
to investigate the bilateral relationship between them, using the systemic inflammatory serologic marker hsCRP. The study is cross-sectional, designed to assess and compare the inflammatory periodontal status and systemic inflammatory burden among systemically healthy females, females newly diagnosed with PCOS, and females receiving medical treatment for PCOS. Medical treatment for PCOS aims to reduce insulin resistance and systemic inflammation, which in turn improves the clinical and biochemical manifestations of the syndrome. Dursun et al.27 did not consider the medical treatment status of patients; however, medical treatment significantly exerts a role in reduction of systemic inflammation. Therefore, in this study, segregation of the PCOS study population was made according to the status of medical treatment, to evaluate the effect of medical treatment on systemic inflammation along with periodontal status. The overall scenario of a patient with PCOS reveals a centripetal obese state with truncal abdominal fat distribution, which is reflected in this study by elevated WC and WHR ratio compared with healthy controls (Table 1). Studies have revealed raised prevalence of increased abdominal fat in a large proportion of the PCOS population, which is responsible for hyperinsulinemia and insulin resistance compared with 1386
weight-matched controls.41 Furthermore, this fat results in increased macrophage infiltration and enhanced production of pro-inflammatory cytokines such as TNF-a and IL-6,42 thus enhancing the systemic inflammatory burden, which in chronic inflammatory diseases can account for increased predisposition, severity, and progression of periodontitis. In general, data indicate that increased WC, serum lipid levels, and percentage of subcutaneous fat are associated with increased risk for periodontitis.43,44 A similar trend is perceived in this study, where the PCOS-N group displayed notably increased periodontal parameters GI, BOP, PD, mean CAL, sites with CAL 3 to 4 mm and PD ‡4 mm, and elevated abdominal fat levels in terms of WC and WHR (P £0.05) compared with CG (Tables 1 and 2). These results are partially in accordance with those of Dursun et al.,27 who reported significantly higher levels of periodontal parameters in women with PCOS compared with healthy women. However, in their study, the mean plaque score differed significantly at baseline among the groups, which acted as a major confounder in comparative analysis. The patients with PCOS on medical treatment (oral contraceptives, metformin, and lifestyle modification) demonstrated significant differences and decreased WC and hsCRP (P £0.05) compared with newly diagnosed patients with PCOS (Table 1). Studies45,46 have reported significant reduction in BMI, WHR, and WC as effects of medical treatment in women with PCOS. An extensive review by Nickalas et al.47 provided evidence for reduction in CRP, IL-6, IL-8, and TNF-a by long-term dietary weight reduction and physical exercise, thus supporting the present results (reduction in hsCRP) which could be the effect of lifestyle modification. Furthermore, Morin-Papunen et al.48 also revealed a significant effect of metformin on reduction in hsCRP, WC, and WHR at 6 months and suggested
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that CRP could be used as a marker of treatment efficiency in women with PCOS. The lower levels of systemic inflammation and lower indices of periodontal breakdown in PCOS-MT compared with PCOS-N in this study could be a result of medical treatment, a possibility that needs to be further ascertained (Table 1). The literature reveals raised CRP levels in the PCOS population, indicating an increased risk for the development of early-onset cardiovascular diseases.14,49 In the present study, women newly diagnosed with PCOS displayed substantially increased hsCRP (P £0.05) compared with other groups (Table 1). This increased score of systemic inflammatory burden points toward a synergistic etiologic factor which could be hypothesized to be periodontally mediated, as the periodontal parameters are adjunctively higher with anthropometric parameters in PCOS-N compared with CG and PCOS-MT. Marcaccini et al.50 demonstrated that patients with periodontal disease who are otherwise healthy have increased circulating levels of CRP. On partial correlation analysis after controlling for confounders, hsCRP was found to be significantly and positively correlated with BOP and CAL (P £0.05) (Table 3). Linear regression analysis was performed to observe the influence of systemic inflammation (hsCRP) on periodontal parameters, which showed a strong association of hsCRP with periodontal inflammation and breakdown (BOP and CAL), suggesting that increased systemic inflammation due to PCOS may influence the periodontal condition ¨ zcxaka et al. have also shown that PCOS (Table 4). O and gingival inflammation appear to act in a synergistic manner to increase expression of the proinflammatory cytokines IL-6 and TNF-a and stated that PCOS may have an impact on gingival inflammation and vice versa.28 To ascertain the bilateral relationship between these two disease entities, the present authors also analyzed the influence of periodontal and anthropometric parameters on systemic inflammation. CAL and WC were significant predictors positively associated with hsCRP. A statistically significant interaction term between CAL and WC in one of the two models also indicated that the influence of CAL as exposure on hsCRP levels may also depend on WC or varied with WC (Table 5). The comparative perusal of the data also revealed that PCOS-N had a statistically higher number of patients with PCOS associated with CP (P £0.05) compared with CG and PCOS-MT (Table 2). Analysis of the data using logistic regression (Table 6) depicted the influence of PCOS on the periodontal tissue. It was shown that the patients in PCOS-N had a greater probability of developing periodontitis compared with CG and PCOS-MT. After adjusting for medical treatment in PCOS, a decrease in the as-
Porwal, Tewari, Sharma, Singhal, Narula
sociation was noticed. Still the association was found to be strong (P £0.05), with PCOS-N 2.88 times more likely to develop periodontitis. The study encountered certain limitations, as the assessment of the metabolic parameters and duration of medical treatment were used only for inclusion criteria of women with PCOS and not in comparative analysis. The study population was limited due to confined availability of patients (in the outpatient department of Obstetrics and Gynecology) and stringent inclusion and exclusion criteria. The enduring design of the study was based on various observations. The stringent inclusion and exclusion criteria have curbed the confounders. The availability of data on the previous medical treatment status, duration, and type of treatment modality complements the study in confounding the bias of medical treatment. The inclusion of age- and BMI-matched controls creates a better comparative analysis with the test groups. The study population was of the same ethnic background. Using WHR and WC along with BMI better predicts adiposity because BMI alone has pitfalls in measuring it. CONCLUSIONS Within the limitations of the study, the following conclusions can be drawn: 1) females with newly diagnosed PCOS had increased PD, CAL, sites with BOP and CAL 3 to 4 mm, WC, and serum levels of hsCRP compared with systemically healthy controls and females on medical treatment for PCOS; 2) women receiving medical treatment for PCOS had significantly lower measures of periodontal breakdown, WC, and systemic inflammation compared with women newly diagnosed with PCOS, and measures of gingival inflammation and WHR differed nonsignificantly in the two PCOS groups; 3) the analysis of the total population pointed toward correlation of hsCRP with BOP and CAL, and periodontal inflammation and breakdown were associated with systemic inflammation and vice versa; and 4) a higher prevalence of periodontitis found in patients newly diagnosed with PCOS may indicate greater likelihood for development of periodontitis in these patients. The future assessment of periodontal status in patients with PCOS should focus on different drug therapies, as well as on the duration of treatment and the effect of periodontal therapy on the PCOS population. In addition, the association will benefit from a longitudinal, multicenter, randomized, controlled trial with long follow-up to further ameliorate the current literature. ACKNOWLEDGMENT The authors report no conflicts of interest related to this study. 1387
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REFERENCES 1. Hull MG. Epidemiology of infertility and polycystic ovarian disease: Endocrinological and demographic studies. Gynecol Endocrinol 1987;1:235-245. 2. Azziz R. The evaluation and management of hirsutism. Obstet Gynecol 2003;101:995-1007. 3. Konchenhauer ES, Key TI, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: A prospective study. J Clin Endocrinol Metab 1998;83:3078-3082. 4. Ovalle F, Azziz R. Insulin resistance, polycystic ovary syndrome, and type 2 diabetes mellitus. Fertil Steril 2002;77:1095-1105. 5. Wild RA. Long-term health consequences of PCOS. Hum Reprod Update 2002;8:231-241. 6. Legro RS. Polycystic ovary syndrome and cardiovascular disease: A premature association? Endocr Rev 2003;24:302-312. 7. Hardiman P, Pillay OC, Atiomo W. Polycystic ovary syndrome and endometrial carcinoma. Lancet 2003; 361:1810-1812. 8. Pasquali R, Casimirri F. The impact of obesity on hyperandrogenism and polycystic ovary syndrome in premenopausal women. Clin Endocrinol (Oxf) 1993; 39:1-16. 9. Holte J. Disturbances in insulin secretion and sensitivity in women with the polycystic ovary syndrome. Baillieres Clin Endocrinol Metab 1996;10:221-247. 10. Baillargeon JP, Iuorno MJ, Nestler JE. Insulin sensitizers for polycystic ovary syndrome. Clin Obstet Gynecol 2003;46:325-340. 11. Franks S. Polycystic ovary syndrome. N Engl J Med 1995;333:853-861. 12. Poretsky L. On the paradox of insulin-induced hyperandrogenism in insulin-resistant states. Endocr Rev 1991;12:3-13. ¨ rntorp P. Hyperandrogenicity in women — A pre13. Bjo diabetic condition? J Intern Med 1993;234:579-583. 14. Kelly CC, Lyall H, Petrie JR, Gould GW, Connell JM, Sattar N. Low grade chronic inflammation in women with polycystic ovarian syndrome. J Clin Endocrinol Metab 2001;86:2453-2455. 15. Watanabe K, Petro BJ, Shlimon AE, Unterman TG. Effect of periodontitis on insulin resistance and the onset of type 2 diabetes mellitus in Zucker diabetic fatty rats. J Periodontol 2008;79:1208-1216. 16. Demmer RT, Desvarieux M. Periodontal infections and cardiovascular disease: The heart of the matter. J Am Dent Assoc 2006;137(Suppl.):14S-20S, quiz 38S. 17. Ribeiro J, Lea˜o A, Novaes AB. Periodontal infection as a possible severity factor for rheumatoid arthritis. J Clin Periodontol 2005;32:412-416. 18. Grossi SG, Genco RJ. Periodontal disease and diabetes mellitus: A two-way relationship. Ann Periodontol 1998; 3:51-61. 19. Hayes C, Sparrow D, Cohen M, Vokonas PS, Garcia RI. The association between alveolar bone loss and pulmonary function: The VA Dental Longitudinal Study. Ann Periodontol 1998;3:257-261. 20. Moutsopoulos NM, Madianos PN. Low-grade inflammation in chronic infectious diseases: Paradigm of periodontal infections. Ann N Y Acad Sci 2006;1088: 251-264. 1388
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21. Slade GD, Offenbacher S, Beck JD, Heiss G, Pankow JS. Acute-phase inflammatory response to periodontal disease in the US population. J Dent Res 2000;79:49-57. 22. Joshipura KJ, Wand HC, Merchant AT, Rimm EB. Periodontal disease and biomarkers related to cardiovascular disease. J Dent Res 2004;83:151-155. 23. Pitiphat W, Savetsilp W, Wara-Aswapati N. C-reactive protein associated with periodontitis in a Thai population. J Clin Periodontol 2008;35:120-125. 24. Loos BG, Craandijk J, Hoek FJ, Wertheim-van Dillen PM, van der Velden U. Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J Periodontol 2000;71: 1528-1534. 25. Engebretson S, Chertog R, Nichols A, Hey-Hadavi J, Celenti R, Grbic J. Plasma levels of tumour necrosis factor-alpha in patients with chronic periodontitis and type 2 diabetes. J Clin Periodontol 2007;34:18-24. 26. Reaven GM. Insulin resistance: The link between obesity and cardiovascular disease. Med Clin North Am 2011;95:875-892. 27. Dursun E, Akalın FA, Gu ¨ ncu ¨ GN, et al. Periodontal disease in polycystic ovary syndrome. Fertil Steril 2011; 95:320-323. ¨ zcxaka O, Ceyhan BO, Akcali A, Bicxakci N, Lappin DF, 28. O Buduneli N. Is there an interaction between polycystic ovary syndrome and gingival inflammation? J Periodontol 2012;83:1529-1537. ¨ zc 29. O xaka O, Buduneli N, Ceyhan BO, et al. Is interleukin17 involved in the interaction between polycystic ovary syndrome and gingival inflammation? J Periodontol 2013;84:1827-1837. 30. WHO. Ethical principles for medical research involving human subjects. Seoul: World Medical Association: 2008;1-5. 31. Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19:41-47. 32. WHO. Waist circumference and waist-hip ratio: Report of a WHO expert consultation. Geneva: World Health Organization: 2008;5-7. ¨ e H. Periodontal disease in pregnancy. II. 33. Silness J, Lo Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121-135. ¨ e H, Silness J. Periodontal disease in pregnancy. I. 34. Lo Prevalence and severity. Acta Odontol Scand 1963;21: 533-551. 35. Page RC, Eke PI. Case definitions for use in population-based surveillance of periodontitis. J Periodontol 2007;78(Suppl. 7):1387-1399. 36. Eke PI, Page RC, Wei L, Thornton-Evans G, Genco RJ. Update of the case definitions for population-based surveillance of periodontitis. J Periodontol 2012;83: 1449-1454. 37. Daftary SN. ECAB clinical update information. Polycystic ovary syndrome, vol. 1. New Delhi: Reed Elsevier India; 2010:ii-iii. 38. Stein I, Leventhal M. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1935;28:181-185. 39. Mukherjee GG. Long-term health sequelae of polycystic ovary syndrome. Polycystic ovary syndrome, vol. 1. New Delhi: Reed Elsevier India; 2010:96-97. 40. Knebel B, Janssen OE, Hahn S, et al. Increased low grade inflammatory serum markers in patients with polycystic ovary syndrome (PCOS) and their
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41.
42. 43.
44.
45.
46.
relationship to PPARgamma gene variants. Exp Clin Endocrinol Diabetes 2008;116:481-486. Carmina E, Bucchieri S, Esposito A, et al. Abdominal fat quantity and distribution in women with polycystic ovary syndrome and extent of its relation to insulin resistance. J Clin Endocrinol Metab 2007;92:25002505. Ruan H, Lodish HF. Insulin resistance in adipose tissue: Direct and indirect effects of tumor necrosis factoralpha. Cytokine Growth Factor Rev 2003;14:447-455. Bullon P, Morillo JM, Ramirez-Tortosa MC, Quiles JL, Newman HN, Battino M. Metabolic syndrome and periodontitis: Is oxidative stress a common link? J Dent Res 2009;88:503-518. Chaffee BW, Weston SJ. Association between chronic periodontal disease and obesity: A systematic review and meta-analysis. J Periodontol 2010;81:17081724. Elter K, Imir G, Durmusoglu F. Clinical, endocrine and metabolic effects of metformin added to ethinyl estradiol-cyproterone acetate in non-obese women with polycystic ovarian syndrome: A randomized controlled study. Hum Reprod 2002;17:1729-1737. Hoeger K, Davidson K, Kochman L, Cherry T, Kopin L, Guzick DS. The impact of metformin, oral contraceptives, and lifestyle modification on polycystic ovary
47.
48.
49.
50.
syndrome in obese adolescent women in two randomized, placebo-controlled clinical trials. J Clin Endocrinol Metab 2008;93:4299-4306. Nickalas BJ, You T, Pahor M. Behavourial treatments for chronic systemic inflammation: Effects of dietary weight loss and exercise training. Can Med Assoc J 2005;172:1199-1209. Morin-Papunen L, Rautio K, Ruokonen A, Hedberg P, Puukka M, Tapanainen JS. Metformin reduces serum C-reactive protein levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003;88:4649-4654. Boulman N, Levy Y, Leiba R, et al. Increased C-reactive protein levels in the polycystic ovary syndrome: A marker of cardiovascular disease. J Clin Endocrinol Metab 2004;89:2160-2165. Marcaccini AM, Meschiari CA, Sorgi CA, et al. Circulating interleukin-6 and high-sensitivity C-reactive protein decrease after periodontal therapy in otherwise healthy subjects. J Periodontol 2009;80:594-602.
Correspondence: Dr. Shikha Tewari, 6/6-J, Medical Enclave, Rohtak, Haryana, India, Pin- 124001. Fax: 01262- 213876; e-mail: [email protected]. Submitted December 17, 2013; accepted for publication February 22, 2014.
1389
Periodontal Status and High-Sensitivity C-Reactive Protein Levels in Polycystic Ovary Syndrome With and Without Medical Treatment Surya Porwal,* Shikha Tewari,* Rajinder K. Sharma,* Savita Rani Singhal,† and Satish C. Narula*
Background: Recently, some studies have revealed the effect of polycystic ovary syndrome (PCOS) on gingival inflammation. This cross-sectional study attempts to assess the periodontal status and systemic inflammation of women receiving medical treatment for PCOS and women newly diagnosed with PCOS. Methods: A total of 126 participants comprising 41 newly diagnosed patients with PCOS (PCOS-N), 45 patients with PCOS on medical treatment (PCOS-MT), and 40 systemically healthy controls (control group [CG]) were examined. Periodontal parameters, anthropometric parameters, and serum levels of high-sensitivity C-reactive protein (hsCRP) were recorded. Results: Women with newly diagnosed PCOS had increased sites with bleeding on probing (BOP), probing depth, clinical attachment level (CAL), waist circumference (WC), hsCRP, and prevalence of periodontitis compared with control and PCOS-MT groups (P £0.05). On partial correlation analysis after controlling for confounders, BOP and CAL correlated positively and significantly with hsCRP (P = 0.01 and P = 0.005). Multivariate linear regression analysis revealed that BOP and CAL (dependent variable) (P = 0.009/R2 = 0.05 and P = 0.005/R2 = 0.07, respectively) had significant association with hsCRP. Furthermore, hsCRP, when considered as outcome, also exhibited association with CAL and WC (P = 0.002/R2 = 0.07 and P = 0.04/R2 = 0.106). Logistic regression analysis demonstrated that the PCOS-N group had 2.88 times increased likelihood of having moderate periodontitis (adjusted odds ratio 2.88, 95% confidence interval 1.18 to 6.98). Conclusions: Women with newly diagnosed PCOS may have increased prevalence and likelihood for periodontitis, with higher measures of periodontal inflammation and breakdown than those on medical treatment for PCOS and systemically healthy females. Furthermore, periodontal breakdown might depend on systemic inflammation and vice versa. J Periodontol 2014;85:1380-1389. KEY WORDS C-reactive protein; inflammation; insulin resistance; periodontitis; polycystic ovary syndrome; waist circumference. * Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences, Rohtak, Haryana, India. † Department of Obstetrics and Gynecology, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India.
doi: 10.1902/jop.2014.130756
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P
olycystic ovary syndrome (PCOS) is a genetically complex endocrine disorder of uncertain etiology and a common cause of anovulatory infertility, menstrual dysfunction, and hirsutism.1,2 PCOS is one of the most common reproductive abnormalities, affecting 5% to 10% of the population of reproductive age in the southeastern part of the United States.3 PCOS appears to be associated with an increased risk of metabolic aberrations, including type 2 diabetes mellitus, dyslipidemia, cardiovascular disease, and endometrial carcinoma.4-7 In its typical form, it includes insulin resistance, hyperinsulinemia, obesity (predominantly the abdominal phenotype), and altered lipid profile.8,9 Insulin resistance and hyperinsulinemia play a critical role in the syndrome’s pathogenesis.10 Both hyperinsulinemia and obesity may be intimately related to the development and maintenance of hyperandrogenism.11,12 Increased androgen levels may directly affect insulin sensitivity in the target tissues, particularly muscles, therefore contributing to the development of the insulin-resistant state.13 The raised C-reactive protein (CRP) levels associated with the syndrome are connected to the low-grade chronic inflammation linked to insulin resistance.14 Periodontitis is a chronic inflammatory disease triggered by bacterial products, such as lipopolysaccharides, and the subsequent exuberant inflammatory response leads to loss of alveolar bone.15 Periodontal disease has been implicated as a risk factor in the onset and development of cardiovascular diseases,16 rheumatoid arthritis,17 diabetes mellitus,18 and occlusive respiratory diseases.19 Chronic low-grade inflammation is emerging as a conceivable etiologic mechanism linking periodontal disease and the conditions cited above as well as other systemic diseases.20 CRP is a serologic marker of systemic inflammation that has been associated with increased risk for various systemic diseases.21,22 Periodontitis has also been linked to elevated CRP levels. It is thus postulated that CRP might be a possible mediator of the association between periodontitis and these systemic conditions.23 Patients with periodontitis also have a higher concentration of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1 in serum and/or gingival crevicular fluid.24,25 Chronic stimulation and secretion of proinflammatory cytokines associated with periodontal infection contribute to insulin resistance.26 This pathognomonic state of chronic systemic inflammation and insulin resistance in both periodontitis and PCOS can be hypothesized as a converging channel associating the disorders. Earlier studies have warranted a possible relationship between PCOS and periodontitis, suggesting
Porwal, Tewari, Sharma, Singhal, Narula
impact of PCOS on gingival inflammation.27-29 However, they have not taken into account the medical treatment status, duration, or type of medications used for the treatment of the syndrome. The present study attempts to investigate periodontal status and systemic inflammation levels through high-sensitivity (hs)CRP in women with PCOS, considering the dichotomous state of medical treatment (whether treatment has been undertaken or not). Therefore, the aim of the study is trifold: 1) to assess and compare the periodontal status, anthropometric parameters, and systemic inflammatory burden among systemically healthy females, patients newly diagnosed with PCOS, and patients receiving medical treatment for PCOS; 2) to observe the association among clinical periodontal parameters, anthropometric parameters, and serum levels of systemic inflammatory markers in the study population; and 3) to explore the contribution of systemic inflammation caused by PCOS toward periodontal inflammation and breakdown and vice versa. MATERIALS AND METHODS Study Population This cross-sectional, single-masked study was executed in the Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences (PGIDS), Rohtak, India, with successful collaboration of the Department of Obstetrics and Gynecology, Post Graduate Institute of Medical Sciences (PGIMS), Rohtak, India. This study followed the standard protocols outlined in the 1964 Declaration of Helsinki, as revised in 2008.30 The study design was reviewed and approved by the Institutional Review Board, Pandit Bhagwat Dayal Sharma University of Health Sciences, Rohtak, and ethical acceptance was obtained from the ethics committee of PGIDS. Enrollment was from March 2012 to September 2013. The study population consisted of 41 patients (aged 15 to 36 years, mean age: 26.28 years) newly diagnosed with PCOS (PCOS-N) who had not started any medical treatment for the syndrome and 45 women with PCOS (aged 16 to 31 years, mean age: 22.68 years) undergoing medical treatment (PCOS-MT) (oral contraceptives, metformin, and lifestyle modification) for at least 6 months. The participants were recruited from the regular outpatient Department of Obstetrics and Gynecology, PGIMS. Forty systemically healthy controls (control group [CG]) matched for age (17 to 32 years, mean age: 23.50 years) and body mass index (BMI) were recruited from the patients attending the regular outpatient Department of Periodontics and Oral Diagnosis, Radiology and Medicine, PGIDS. Enrollment criteria in PCOS groups were females of reproductive age diagnosed with PCOS, with ‡16 natural teeth. The diagnosis of PCOS 1381
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was made according to Rotterdam criteria,31 in the presence of ‡2 of the following: 1) clinical and/or biochemical hyperandrogenism; 2) chronic oligo- or anovulation; and 3) polycystic ovaries on ultrasound (presence of ‡12 follicles in each ovary measuring 2 to 9 mm in diameter and/or increased ovarian volume >10 mL). Inclusion criteria for systemically healthy controls were regularly menstruating females with no clinical or biochemical sign of hyperandrogenism and ultrasound exclusion of PCOS. The recruitment excluded patients with any history of thyroid dysfunction, hyperprolactinemia, and androgen-secreting tumors to avoid misdiagnosis of PCOS. Patients with chronic inflammatory disease such as nephrotic syndrome, chronic renal failure, significant cardiovascular disease, established type 1 or type 2 diabetes mellitus, or active cancer within the past 5 years were excluded to control the confounders. Additional exclusion criteria included: 1) smoking and alcohol consumption; 2) history of systemic antibiotics within 3 months; 3) periodontal treatment within 6 months; and 4) aggressive periodontitis. An elaborate medical history supported with investigations and consultation with the referring physician was relied on to rule out the presence of any of the above conditions and for the confirmed diagnosis of PCOS. Written informed consent was received from each patient after explaining the procedure along with the risks and benefits in her own language. All the participants in the study had to undergo examination of the various anthropometric parameters, periodontal parameters, and estimation of hsCRP levels. Allotment of patients to different groups was done by one investigator (ST), whereas examination of all parameters was recorded and carried out by another investigator (SP) to ensure investigator masking. Analyses of Anthropometric Parameters Waist circumference32 (WC) was measured in centimeters, at the midpoint between the lower margin of the lowest palpable rib and the top of the iliac crest. For waist-to-hip ratio32 (WHR), hip circumference was measured around the widest portion of the buttocks. BMI was recorded as weight in kilograms divided by height in meters squared. Blood Sampling Blood samples for the investigation of serum hsCRP were collected after overnight fasting. Venous blood from the antecubital vein was collected after applying a tourniquet in plain tubes without additive. Serum hsCRP levels were assessed using a kit‡ with highsensitivity methodology in an auto-analyzer§ according to the manufacturer’s instructions. The test principle was particle-enhanced immune-turbidimetric assay, in which human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. 1382
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The turbidity induced by the formation of immune complexes was measured at 546 nm. The lower detection limit for the assay was 0.15 mg/L. Periodontal Parameters The clinical protocol included registration of the following periodontal parameters: plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL), which were recorded by a single trained examiner (SP) on each tooth (except third molars) with a periodontal probei and mirror. BOP, PD, and CAL were recorded at six sites per tooth: mesio-buccal, mid-buccal, distobuccal, mesio-lingual/palatal, mid-lingual/palatal, and disto-lingual/palatal. PI33 and GI34 were recorded at four sites per tooth: mesio-buccal, mid-buccal, distobuccal, and mid-lingual/palatal. The number of sites with BOP was recorded as a percentage, and the assessment was done dichotomously with 0 as absence and 1 as presence. Assessment of moderate chronic periodontitis (CP) was defined by ‡2 interproximal sites with CAL ‡4 mm or ‡2 interproximal sites with PD ‡5 mm, not on the same tooth.35 Mild CP was defined as ‡2 interproximal sites with CAL ‡3 mm and ‡2 interproximal sites with PD ‡4 mm (not on the same tooth) or one site with PD ‡5 mm.36 Diagnosis of gingivitis was assigned when BOP was present at >50% of all sites and PD was 4 mm and CAL £1 mm and no clinical and/or radiographic sign of periodontitis was evident. Reproducibility of clinical measurements was verified by the same investigator (SP) by carrying out double clinical periodontal data recording for PD and CAL on 10% of the sample. Intraexaminer reproducibility was determined for each site by calculating the percentage of sites examined where the scores were matching or within 1 mm. Assessment of mean difference in the scores (with 90% accuracy, k value 0.84 for PD and 0.80 for CAL) indicated that there was no systemic bias in the measurements. Statistical Analyses The sample size was calculated by power calculations using statistical software,¶ based on an overall significance level of 0.05, and the assumption of normally distributed data indicated that the statistical power exceeded 80% with a fixed effect size of 0.70 for a total 126 participants, divided in comparison groups of PCOS population with allocation ratio 1:1 and control group. The sample size could ‡ C-Reactive Protein (Latex) High-Sensitivity Assay, Roche Diagnostics, Indianapolis, IN. § Konelab Clinical Chemistry Analyzer, Thermo Fisher Scientific, Waltham, MA. i Williams periodontal probe, Hu-Friedy, Chicago, IL. ¶ G-power 3.0.10, Heinrich-Heine University Du ¨ sseldorf, Du ¨ sseldorf, Germany.
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not be estimated on the basis of proportion of PCOS population having periodontitis because of the lack of evidence. Post hoc statistical power calculations with 126 participants comparing six parameters indicated that for correlation coefficient (r) r2 value ‡ 0.0645, the statistical power exceeded 80%. The distribution of the data for all groups was analyzed by Kolmogorov–Smirnov test. Following nonnormal distribution of the data, differences among the groups for continuous variables were assessed using Kruskal–Wallis followed by Mann–Whitney U test and for categoric variables using x2 test. Partial correlation among variables was assessed after controlling for potential confounders BMI, WC, and WHR. Further multiple linear stepwise regression analysis was used to develop models of predictor variables associated with the dependent variable. The evaluation of the association between CP and PCOS was estimated by odds ratio (OR) and 95% confidence intervals (CIs), using logistic regression analysis. Models were constructed with presence/absence of periodontitis as the outcome variable and PCOS disease status and medical treatment status for PCOS as a categoric predictor variable. The Hosmer–Lemeshow statistic was applied to verify the diagnosis of the goodness-of-fit regression model used. All statistical analyses were two-tailed with significance level at 0.05 as calculated using software.# RESULTS Of the 168 patients examined, 132 were eligible for the study and met the inclusion and exclusion criteria; six participants eligible for the PCOS-N group refused to take part in the study. So, the total study population comprised 126 women divided into 40 systemically healthy controls, 41 newly diagnosed PCOS, and 45 PCOS on medical treatment. Demographic data along with serum hsCRP, anthropometric, and clinical measurements are outlined in Table 1. According to the Kruskal–Wallis analysis, there was no significant difference among the ages, PI, and BMI scores of participants in the study groups. Mann–Whitney U test was applied to assess the differences among the groups for the remaining parameters. Serum levels of hsCRP were significantly higher in PCOS-N than systemically healthy and PCOSMT groups (P = 0.000 and 0.001, respectively), whereas an apparent increase in hsCRP levels in PCOS-MT was non-significant compared with the control group (P = 0.14). On intergroup comparisons of the anthropometric parameters, PCOS-N had higher WC compared with CG and PCOS-MT (P = 0.000 and 0.05, respectively), and a non-significant difference was noted between PCOS-MT and CG (P = 0.07). PCOS-N and PCOS-MT had significantly higher WHRs than CG (P = 0.01 and 0.02, respectively), albeit no
Porwal, Tewari, Sharma, Singhal, Narula
significant difference was noted in WHR between the two PCOS groups (P = 0.24). The PCOS-N group had significantly higher PD (P = 0.000 and 0.002, respectively), CAL (P = 0.000 and 0.001), and BOP (P = 0.000 and 0.01) than CG and PCOS-MT, and these variables were also significantly higher in PCOS-MT compared with CG (PD, P = 0.001; CAL, P = 0.002; BOP, P = 0.008). The PCOS-N group had significantly higher GI than CG (P = 0.03), and it was similar between PCOS-N and PCOS-MT (P = 0.10) and PCOS-MT and CG (P = 0.11) (Table 1). The results of different variables assessing the periodontal condition and frequency of patients having periodontal disease in PCOS and control groups are presented in Table 2. The mean number of sites with CAL 3 to 4 mm was significantly higher in PCOS-N compared with PCOS-MT and CG (P = 0.03 and 0.00, respectively). The number of sites with CAL 1 to 2 mm differed non-significantly between the two PCOS groups. Furthermore, PCOS-MT showed a significantly higher number of sites with CAL 3 to 4 mm and 1 to 2 mm than CG (P = 0.01 and 0.00, respectively). The mean number of sites with PD ‡4 mm was significantly higher in PCOS-N than PCOSMT (P = 0.008), and PCOS groups differed nonsignificantly compared with CG. The different groups were comparable with regard to number of sites with PD ‡5 mm. The frequency of moderate periodontitis, as assessed using x2 test, was higher in PCOS-N compared with CG and PCOS-MT (P = 0.001 and 0.004, respectively), whereas the frequency differed nonsignificantly between the CG and PCOS-MT. The prevalence of patients having mild periodontitis revealed no significant difference among the three groups. The frequency of gingivitis was significantly higher in CG compared with PCOS-N (P = 0.02) and comparable in PCOS-N and PCOS-MT. Periodontally healthy individuals were more numerous in CG compared with PCOS-N and PCOS- MT (P = 0.001 and 0.05, respectively). Nevertheless, no patient having severe CP was part of the study population (Table 2). Table 3 depicts partial correlation analysis among periodontal parameters and hsCRP of the total population, after adjusting for potential confounders (BMI, WC, and WHR). The result showed BOP (P = 0.01) and CAL (P = 0.005) to be positively and significantly correlated with hsCRP. Table 4 displays the positive association of BOP (P = 0.009/R2 = 0.05) and CAL (P = 0.002/R2 = 0.07) with hsCRP. Table 5 exhibits the positive association of hsCRP when considered as a dependent variable # SPSS, v.19, IBM, Chicago, IL.
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Table 1.
Comparative Evaluation of Age, hsCRP Levels, and Anthropometric and Periodontal Parameters of CG, PCOS-N, and PCOS-MT Groups CG (n = 40)
PCOS-N (n = 41)
Parameter
Mean – SD
95% CI
Age (years)
23.50 – 2.67
22.64 to 24.35
1.38 – 1.48
0.95 to 1.88
BMI (kg/m )
23.76 – 4.82
22.22 to 25.31
WC (cm)
72.02 – 8.09
WHR
Mean – SD
PCOS-MT (n = 45) Mean – SD
95% CI 21.54 to 24.64
22.68 – 4.55
21.31 to 24.05
2.80 to 5.01
1.77 – 1.83
1.30 to 2.33
25.01 – 3.61
23.87 to 26.15
24.57 – 4.38
23.25 to 25.89
69.43 to 74.61
81.69 – 12.45*†
77.76 to 85.62
76.65 – 11.07
73.32 to 79.98
0.79 – 0.09
0.76 to 0.82
0.83 – 0.08*
0.80 to 0.85
0.81 – 0.04*
0.80 to 0.82
PI
0.97 – 0.83
0.70 to 1.23
1.01 – 0.56
0.83 to 1.19
1.03 – 0.80
0.79 to 1.27
GI
1.34 – 1.09
0.98 to 1.69
1.99 – 0.47*
1.84 to 2.14
1.71 – 0.77
1.48 to 1.94
BOP (%)
45.12 – 37.46
33.14 to 57.10
82.94 – 13.59*†
78.65 to 87.23
66.2 – 27.81*
57.93 to 74.65
PD (mm)
1.78 – 0.72
1.55 to 2.01
2.34 – 0.31*
†
2.24 to 2.44
2.13 – 0.40*
2.01 to 2.26
CAL (mm)
0.77 – 1.26
0.37 to 1.18
1.26 – 0.67*†
1.04 to 1.47
0.83 – 0.55*
0.67 to 1.00
hsCRP (mg/L) 2
23.09 – 4.90
95% CI
3.76 – 3.60*†
* P £0.05 compared with CG. † P £0.05 compared with PCOS-MT.
Table 2.
Distribution of Periodontal Condition Variables (mean 6 SD or n [%]) of the Study Population PCOS-N (n = 41; [%])
PCOS-MT P, PCOS-N P, PCOS-MT P, PCOS-N (n = 45; [%]) versus CG versus CG versus PCOS-MT
Parameter
CG (n = 40; [%])
Number of sites with CAL 1 to 2 mm
44.57 – 66.09 98.02 – 29.65 90.80 – 45.49
0.00*
0.00*
NS
Number of sites with CAL 3 to 4 mm
10.90 – 14.89 26.48 – 26.06 11.62 – 11.41
0.00*
0.01*
0.03*
Number of sites with PD ‡4 mm
9.50 – 12.04 13.14 – 10.52
7.06 – 9.19
NS
NS
0.008*
Number of sites with PD ‡5 mm
9.50 – 11.17
9.33 – 7.99
8.00 – 7.94
NS
NS
NS
09 (22.5)
00 (0)
03 (6.67)
0.001*
0.05*
NS
12 (30)
04 (9.75)
10 (22.22)
0.027*
NS
NS
11 (27.5)
13 (31.70)
20 (44.44)
NS
NS
NS
08 (20)
24 (58.55)
12 (26.67)
0.001*
NS
0.004*
Frequency of periodontally healthy patients† Frequency of gingivitis
†
Frequency of mild periodontitis† Frequency of moderate periodontitis
†
NS = not significant. * P £0.05. 2 † x test.
with models of CAL (P = 0.002/R2 = 0.07) and CAL/WC (P = 0.005/0.04/R2 = 0.10). The results of logistic regression analysis (Table 6) revealed that the chances of having moderate periodontitis were >five times higher in PCOS-N compared with PCOS-MT and CG (unadjusted OR = 5.64, 95% CI 2.09 to 15.24), and this difference was statistically significant. Adjustment for these variables 1384
produced a slight decrease in the magnitude of the association, but reaffirmed that PCOS had an independent effect on periodontitis (adjusted OR = 2.88, 95% CI: 1.18 to 6.98). DISCUSSION PCOS is a complex endocrinopathy having a wide range of variations and clinical manifestations, with
Porwal, Tewari, Sharma, Singhal, Narula
J Periodontol • October 2014
Table 3.
Correlation of Different Variables Using Applying Partial Correlation Analysis After Controlling for Confounders (BMI, WC, and WHR) Parameter hsCRP Correlation Significance (two-tailed)
Age
PI
GI
BOP
PD
CAL
0.085 0.353
0.168 0.063
0.085 0.349
0.227 0.012*
0.132 0.146
0.254 0.005*
0.146 0.108
0.056 0.540
0.046 0.613
0.075 0.411
0.039 0.665
0.629 0.000*
0.519 0.000*
0.475 0.000*
0.334 0.000*
0.829 0.000*
0.649 0.000*
0.286 0.001*
0.671 0.000*
0.400 0.000*
Age Correlation Significance (two-tailed) PI Correlation Significance (two-tailed) GI Correlation Significance (two-tailed) BOP Correlation Significance (two-tailed) PD Correlation Significance (two-tailed)
0.558 0.000*
* P £0.05.
Table 4.
Significant Associations Among Periodontal Parameters (dependent variable) and Biochemical Parameter hsCRP (independent variable) Using Multiple Stepwise Regression Analysis Dependent Variable BOP CAL
Model Predictors
b Unstandardized
Standard Error
b Standardized
P
R2
95% CI
Constant hsCRP
58.62 27.69
3.64 10.37
0.233
0.000 0.009*
0.05
51.41 to 65.83 7.15 to 48.23
0.278
0.000 0.002*
0.07
Constant hsCRP
0.742 0.933
0.102 0.289
0.541 to 0.943 0.360 to 1.50
* P £0.05.
no single etiologic factor that fully accounts for the whole spectrum of the abnormalities of the condition.37 Since 1935, when Stein and Leventhal38 incipiently described the syndrome, exhaustive research has been published associating PCOS and systemic conditions such as cardiovascular diseases and type 2 diabetes mellitus.39 The insulin resistance and hyperinsulinemia associated with PCOS leads to development of a chronic inflammatory state. Moreover, IL-6 and TNF-a levels have been shown to be elevated in females with PCOS compared with an obese control group of females.40 In periodontitis, chronic stimulation and secretion of higher levels of pro-inflammatory cytokines such as
TNF-a and IL-1 also contribute to insulin resistance and a chronic inflammatory state.26 In addition, increased concentrations of inflammatory biomarkers such as CRP and IL-6 in both gingival tissue and serum have been reported in patients with periodontitis.24 Recently, some evidence has shown the plausibility of PCOS being associated with increased susceptibility for periodontal disease, illustrating gingival inflammation as a common finding in these patients,27 and thus paving the way for further research to determine the association of PCOS with periodontal disease. Because chronic systemic inflammation colligates the pathophysiology of both PCOS and periodontal disease, the present study is strategized accordingly 1385
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Table 5.
Significant Associations Among Periodontal and Anthropometric Parameters (independent variable) and Biochemical Parameter hsCRP (dependent variable) Using Multiple Stepwise Regression Analysis Model Predictors
b Unstandardized
Standard Error
b Standardized
P
R2
95% CI
hsCRP
Constant CAL
0.151 0.083
0.034 0.026
0.278
0.000 0.002*
0.077
0.084 to 0.217 0.032 to 0.134
hsCRP
Constant CAL WC
-0.153 0.74 0.004
0.156 0.026 0.002
Dependent Variable
0.247 0.173
0.329 0.005* 0.04*
0.106
-0.461 to 0.155 0.023 to 0.125 0.000 to 0.008
2
Table 5 shows regression coefficient values (R ) of hsCRP (dependent variable) and periodontal and anthropometric parameters (independent variable) in two different models of regression analysis. * P £0.05.
Table 6.
Association Measurements by Logistic Regression Analysis Among PCOS Groups and CP Models
OR
95% CI
P
Unadjusted
5.64
2.09 to 15.24
0.001
Adjusted*
2.88
1.18 to 6.98
0.01
* Adjusted for medical treatment for PCOS.
to investigate the bilateral relationship between them, using the systemic inflammatory serologic marker hsCRP. The study is cross-sectional, designed to assess and compare the inflammatory periodontal status and systemic inflammatory burden among systemically healthy females, females newly diagnosed with PCOS, and females receiving medical treatment for PCOS. Medical treatment for PCOS aims to reduce insulin resistance and systemic inflammation, which in turn improves the clinical and biochemical manifestations of the syndrome. Dursun et al.27 did not consider the medical treatment status of patients; however, medical treatment significantly exerts a role in reduction of systemic inflammation. Therefore, in this study, segregation of the PCOS study population was made according to the status of medical treatment, to evaluate the effect of medical treatment on systemic inflammation along with periodontal status. The overall scenario of a patient with PCOS reveals a centripetal obese state with truncal abdominal fat distribution, which is reflected in this study by elevated WC and WHR ratio compared with healthy controls (Table 1). Studies have revealed raised prevalence of increased abdominal fat in a large proportion of the PCOS population, which is responsible for hyperinsulinemia and insulin resistance compared with 1386
weight-matched controls.41 Furthermore, this fat results in increased macrophage infiltration and enhanced production of pro-inflammatory cytokines such as TNF-a and IL-6,42 thus enhancing the systemic inflammatory burden, which in chronic inflammatory diseases can account for increased predisposition, severity, and progression of periodontitis. In general, data indicate that increased WC, serum lipid levels, and percentage of subcutaneous fat are associated with increased risk for periodontitis.43,44 A similar trend is perceived in this study, where the PCOS-N group displayed notably increased periodontal parameters GI, BOP, PD, mean CAL, sites with CAL 3 to 4 mm and PD ‡4 mm, and elevated abdominal fat levels in terms of WC and WHR (P £0.05) compared with CG (Tables 1 and 2). These results are partially in accordance with those of Dursun et al.,27 who reported significantly higher levels of periodontal parameters in women with PCOS compared with healthy women. However, in their study, the mean plaque score differed significantly at baseline among the groups, which acted as a major confounder in comparative analysis. The patients with PCOS on medical treatment (oral contraceptives, metformin, and lifestyle modification) demonstrated significant differences and decreased WC and hsCRP (P £0.05) compared with newly diagnosed patients with PCOS (Table 1). Studies45,46 have reported significant reduction in BMI, WHR, and WC as effects of medical treatment in women with PCOS. An extensive review by Nickalas et al.47 provided evidence for reduction in CRP, IL-6, IL-8, and TNF-a by long-term dietary weight reduction and physical exercise, thus supporting the present results (reduction in hsCRP) which could be the effect of lifestyle modification. Furthermore, Morin-Papunen et al.48 also revealed a significant effect of metformin on reduction in hsCRP, WC, and WHR at 6 months and suggested
J Periodontol • October 2014
that CRP could be used as a marker of treatment efficiency in women with PCOS. The lower levels of systemic inflammation and lower indices of periodontal breakdown in PCOS-MT compared with PCOS-N in this study could be a result of medical treatment, a possibility that needs to be further ascertained (Table 1). The literature reveals raised CRP levels in the PCOS population, indicating an increased risk for the development of early-onset cardiovascular diseases.14,49 In the present study, women newly diagnosed with PCOS displayed substantially increased hsCRP (P £0.05) compared with other groups (Table 1). This increased score of systemic inflammatory burden points toward a synergistic etiologic factor which could be hypothesized to be periodontally mediated, as the periodontal parameters are adjunctively higher with anthropometric parameters in PCOS-N compared with CG and PCOS-MT. Marcaccini et al.50 demonstrated that patients with periodontal disease who are otherwise healthy have increased circulating levels of CRP. On partial correlation analysis after controlling for confounders, hsCRP was found to be significantly and positively correlated with BOP and CAL (P £0.05) (Table 3). Linear regression analysis was performed to observe the influence of systemic inflammation (hsCRP) on periodontal parameters, which showed a strong association of hsCRP with periodontal inflammation and breakdown (BOP and CAL), suggesting that increased systemic inflammation due to PCOS may influence the periodontal condition ¨ zcxaka et al. have also shown that PCOS (Table 4). O and gingival inflammation appear to act in a synergistic manner to increase expression of the proinflammatory cytokines IL-6 and TNF-a and stated that PCOS may have an impact on gingival inflammation and vice versa.28 To ascertain the bilateral relationship between these two disease entities, the present authors also analyzed the influence of periodontal and anthropometric parameters on systemic inflammation. CAL and WC were significant predictors positively associated with hsCRP. A statistically significant interaction term between CAL and WC in one of the two models also indicated that the influence of CAL as exposure on hsCRP levels may also depend on WC or varied with WC (Table 5). The comparative perusal of the data also revealed that PCOS-N had a statistically higher number of patients with PCOS associated with CP (P £0.05) compared with CG and PCOS-MT (Table 2). Analysis of the data using logistic regression (Table 6) depicted the influence of PCOS on the periodontal tissue. It was shown that the patients in PCOS-N had a greater probability of developing periodontitis compared with CG and PCOS-MT. After adjusting for medical treatment in PCOS, a decrease in the as-
Porwal, Tewari, Sharma, Singhal, Narula
sociation was noticed. Still the association was found to be strong (P £0.05), with PCOS-N 2.88 times more likely to develop periodontitis. The study encountered certain limitations, as the assessment of the metabolic parameters and duration of medical treatment were used only for inclusion criteria of women with PCOS and not in comparative analysis. The study population was limited due to confined availability of patients (in the outpatient department of Obstetrics and Gynecology) and stringent inclusion and exclusion criteria. The enduring design of the study was based on various observations. The stringent inclusion and exclusion criteria have curbed the confounders. The availability of data on the previous medical treatment status, duration, and type of treatment modality complements the study in confounding the bias of medical treatment. The inclusion of age- and BMI-matched controls creates a better comparative analysis with the test groups. The study population was of the same ethnic background. Using WHR and WC along with BMI better predicts adiposity because BMI alone has pitfalls in measuring it. CONCLUSIONS Within the limitations of the study, the following conclusions can be drawn: 1) females with newly diagnosed PCOS had increased PD, CAL, sites with BOP and CAL 3 to 4 mm, WC, and serum levels of hsCRP compared with systemically healthy controls and females on medical treatment for PCOS; 2) women receiving medical treatment for PCOS had significantly lower measures of periodontal breakdown, WC, and systemic inflammation compared with women newly diagnosed with PCOS, and measures of gingival inflammation and WHR differed nonsignificantly in the two PCOS groups; 3) the analysis of the total population pointed toward correlation of hsCRP with BOP and CAL, and periodontal inflammation and breakdown were associated with systemic inflammation and vice versa; and 4) a higher prevalence of periodontitis found in patients newly diagnosed with PCOS may indicate greater likelihood for development of periodontitis in these patients. The future assessment of periodontal status in patients with PCOS should focus on different drug therapies, as well as on the duration of treatment and the effect of periodontal therapy on the PCOS population. In addition, the association will benefit from a longitudinal, multicenter, randomized, controlled trial with long follow-up to further ameliorate the current literature. ACKNOWLEDGMENT The authors report no conflicts of interest related to this study. 1387
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Correspondence: Dr. Shikha Tewari, 6/6-J, Medical Enclave, Rohtak, Haryana, India, Pin- 124001. Fax: 01262- 213876; e-mail: [email protected]. Submitted December 17, 2013; accepted for publication February 22, 2014.
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