Salivary Free Testosterone and Gingival Health Condition among a Group of Women with Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women in their reproductive age.It is characterized by anovulation or oligo-ovulation and hyperandrogensim.Androgen excess is the central defect in polycystic ovary syndrome. It is a complex disorder affects general health in addition to oral health.This study aimed to assess the gingival health status among a group of women with polycystic ovary syndrome as well as to estimate the levels of salivaryfree testosterone in unstimulated saliva in relation to gingival health condition. Materials and methods: Sixty two women with an age range 20-25 years old and with a body mass index range18.5-24.9 (normal weight) were included in this study. They were divided into two groups; the study group which included thirty one women with polycystic ovary syndrome, those women. nattended Babylon Teaching Hospital/ Infertility Center and the control group which included thirty one women with regular menstrual cycles,without clinical or biochemical features of hyperandrogenism and ultrasound exclusion of polycystic ovary (without polycystic ovary syndrome). Collection of unstimulated salivary samples was carried out under standardrizedconditions, salivary free testosterone levels were estimated. Plaque index of Silness and Lӧe (1964) was used to assess dental plaque. While gingival index of Lӧe and Silness (1963) was used to assess gingival inflammation. Data analysis was conducted through the application of the (SPSS version 18). The analysis of data included: Mean, Standard deviation, t-test, Pearson Correlation (r), Receiver Operative Characteristics Curve (ROC curve). Results:Results showed that the mean value of plaque index was found to be higher in the control group (1.18±0.03) than that in the study group (1.00±0.08) with statistically highly significant difference (p<0.01).While the mean value of gingival indexwas higher among the study group (1.52±0.06)than that in the control group (1.31±0.05)with statistically highly significant difference (p<0.01).Salivary levels of free testosterone among women with polycystic ovary syndrome (44.12±1.37 pg/ml) were found to be higher than that of control (18.50±0.87) with statistically highly significant difference (p<0.01).The results in this study showed that the correlations between salivary free testosterone with the oral variables (plaque index, gingival index) were statistically not significant (P>0.05). In order to assess the specificity and sensitivity of the use of salivary free testosterone as a diagnostic tool, statistical analysis of Receiver Operative Characteristics Curve (ROC) was used and predictive Value Measurmentswere done in this study. The results showed that the area under the curve for salivary free testosterone test was (1.000) with highly significant p-value (p<0.01). The best optimal cut off point for salivary free testosterone was (30.86) with (100%) sensitivity and specificity, indicating a very good predictive capacity for using salivary free testosterone as a marker for the diagnosis of polycystic ovary syndrome. Conclusions : It was concluded that gingival inflammation was high among women with polycystic ovary syndrome. Furthermore, salivary levels of free testosterone were found to be high among women with polycystic ovary syndrome.


INTRODUCTION
Polycystic ovary syndrome (PCOS) is now recognized as a common, heterogenous, heritable disorder affecting women throughout lifetime (1) and it is the leading cause of female anovulatory infertility (2) .The exact cause of PCOS is unknown, however, it results from a combination of genetic and environmental factors (3) .The precise prevalence of PCOS is unknown as the syndrome depends on the diagnostic criteria used, however, World Health Organization (WHO) estimates that it affected 116 million women worldwide in 2012 (3.4% of women) (4) .PCOS is chronic condition of anovulation or oligovulation with clinical or biochemical hyperandrogenism which occurs in the absence of other underlying condition (5) .It was found that the majority of women with PCOS demonstrated elevated circulating androgen levels (6) .Serum level of free testosterone, and not total testosterone, are more frequently elevated in women with PCOS (7) .
It has become evident over the past 30 years that PCOS is more than a reproductive disorder (8) .PCOS shows a link with oral health in addition to its effect on general health (9) .Periodontal diseases and PCOS are the most common disorders in women with significant public health impact (10) .This syndrome have an impact on gingival inflammation or vice versa (9) .It was found that the salivary free testosterone level was high in gingivitis patients (11) .
Salivary diagnostic approaches have been developed to monitor oral diseases such as periodontal diseases (12,14) .Saliva is said to be a "mirror of the body" because it provides vital clues to systemic health (14) ; therefore, saliva has become

Vol. 29(1), March 2017
Salivary Free Pedodontics, Orthodontics and Preventive Dentistry 166 useful as an alternative for blood in medical diagnosis and research (15) .Besides, the development of new technologies may promote a wider use of salivary assay in the near future (16) .
As far as it is known, there was no previous Iraqi study concerning the estimation of the salivary levels of free testosterone and their relation with gingival health condition among women with PCOS.Furthermore, in order to gain knowledge regarding the gingival health status and salivary free testosterone for this target group in which saliva may provide a simple, inexpensive and noninvasive measure, therefore, this study was designed and conducted.

MATERIALS AND METHODS
The total sample composed of sixty two females aged (20-25) years with normal weight (body mass index range of 18.5-24.9).Those were divided into two groups: thirty one women newly diagnosed with PCOS who referred to Babylon Teaching Hospital/ Clinic as a study group and thirty one women with regular menses,without clinical or biochemical features of PCOS and ultrasound exclusion of polycystic ovary syndrome from the relatives of those women as a control group.This study was carried out during the period from the end of December (2014) till the end of May (2015).The study group was diagnosed according to Rotterdam criteria (17) .Exclusion criteria included: smocking,pregnancy,previous diagnosis of condition with hormonal disturbance,presence of systemic disease(diabetes,hypertention,cardiovascurdisease),m edication affecting periodontium (antiepeliptic,immunosuppresent, antihypertensive, corticosteroid), confounding medications(contraceptive pills, steroid hormone), use of antibiotic and /or inflammatory drugs within the last months,the presence of less than 20 natural teeth (less representive for periodontal condition).Collection of unstimulated salivary samples was carried out under standardrized conditions following the instruction cited by (Navazesh and Kumer,) (18) ,the salivary samples were collected and stored in -20C until analysis, salivary free testosterone level (pg/dl) was measured by ELISA method using DEMEDITEC ELISA kit (19) .Plaque index of Silness and Lӧe (20) was used to assess dental plaque.While gingival inflammation was evaluated using gingival index of Lӧe and Silness (21)   .Data analysis was conducted through the application of the (SPSS version 18).The analysis of data included:Mean, Standard deviation, t-test, Pearson Correlation (r), Receiver Operative Characteristics Curve (ROC curve).

RESULTS
Table (1) shows the mean values of plaque index and gingival index among the study and control groups.It was found that the mean value of plaque index was higher in the control group than that in the study group with statistically highly significant difference (p<0.01).While the mean value of gingival index was higher among the study group than the control group with statistically highly significant difference (p<0.01).
Table (2) shows the mean values of salivary free testosterone for both the study and control groups.The result revealed that the salivary level of free testosterone was higher among women with PCOS than their level in the control group with statistically highly significant difference (p<0.01).Table (3) shows the correlation between salivary free testosterone with oral variables (PlI and GI).It was found that all correlation between salivary free testosterone and oral variables were statistically not significant(P>0.05).
Table (4) shows the best optimal cut off point for salivary free testosterone as a diagnostic tool.It was found that the optimal cut off point for salivary free testosterone was (30.86) with (100%) sensitivity and specificity and the area under the curve was (1.00).

DISCUSSION
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women in their reproductive years (22) .It affects general health as well as oral health (9) .The results of the current study showed that the mean value of the plaque index was found to be higher in the control group compared to that in the study group with statistically highly significant difference (p˂0.01).This may be due to oral health negligence in the control group and this could be attributed to lack of motivation about dental plaque control.This resultwas inconsistent with the result reported by previous studies (9,10,23) which found that the mean value of dental plaque was lower in the control group.The result of present study showed a highly significant increased gingival inflammation represented by higher gingival index among PCOS group.This result was also reported by other researchers (8,23) who found thatgingivitis was higher in the PCOS group than that in the control group.This could be explained by the fact that gingival inflammation may be linked to systemic inflammation (24) .It was reported that PCOS is considered as a state of a low grade chronic inflammation (25) .Furthermore, PCOS is one of the endocrine disorder characterized by hormonal imbalance and among these, steroid hormones (26) which have been mostly linked with periodontal pathogenesis (27) .It was found that the gingival tissue is one of the targets for steroid hormones which can exacerbate gingivitis during period of hormonal flactuation (28) .A previous study reported an increase in the level of estrogen hormone among women with PCOS (26) , the gingival tissue respond to increased level of estrogen hormone by undergoing vasodilatation and increased capillary permeability also there is increased migration of fluid and white blood cells out of blood vessels.Cyclic increase in the production of sex steroid hormones often alter the biology of gingival tissue and vasculature, and recognition by effector cells of the local immune system (29,30) .The current study revealed that salivary free testosterone level was higher in the study group than that in the control group with statistically highly significant difference (p˂0.01).This result was also reported by previous study (31) .The highly significant increase in salivary free testosterone among women with PCOS could be explained by the fact that androgen excess is the central defect in PCOS women and hyperandrogenism is the most common characteristic feature of this syndrome, the level of serum free testosterone is increased in women with PCOS (5) due to decrease sex hormone binding globulin (SHBG) production (7) .It was reported that there is a correlation between serum free testosterone and salivary free testosterone (32) ,so, the increase in the level of free testosterone in serum could explain the increase in salivary free testosterone level among women with PCOS.In this study, it was found that salivary free testosterone is considered as the most specific and sensitive biochemical marker that used as a diagnostic tool for women with PCOS, this is supported by the result of Receiver Operative Characteristics Curve (ROC) that was used to assess the specificity and sensitivity of the use of salivary free testosterone as a biomarker for PCOS.In medicine, the ROC analysis had been extensively used in the evaluation of diagnostic tests (33) .It was found that the area under the curve for free salivary testosterone was (1.00) and (100%) sensitivity and (100%) specificity with cut off point of (30.86) indicating very good predictive capacity for the use of salivary free testosterone as a biomarker for the diagnosis of PCOS.
In current study, the correlations between salivary free testosterone with oral variables (PlI, GI) were found to be statistically not significant (p˃0.05).The result of this study was inconsistent with previous study (11) which found that GI, PlI were significantly correlated with increased salivary free testosterone.On the other hand, another study (34) found that the level of salivary free testosterone decreased in gingivitis patients.It was reported that testosterone has an anti-inflammatory effect on periodotium (35,36) .It has an inhibitory effects in the cyclooxygenasepathway of arachidonic acid metabolism in the gingiva by inhibiting prostaglandin secretion (37) ,and stimulates bone cell proliferation and differentiation and therefore has a positive effect on bone metabolism (38) .One must keep in mind the contrast in the result among studies could be attributed to differences in diagnostic criteria employed,sample size, differences in biochemical procedures, even differences in statistical methods employed and difference in the way of saliva collecting, type of saliva collected (stimulated or unstimulated),in addition to differences in inclusion criteria used for selection of women, in this study, only women with a normal weight were included in the study while in previous studies, there was no restriction in the criteria used for selection of women with PCOS.
From the results of this study, it was concluded that women with PCOS had high gingival inflammation, thus, an organized, comprehensive oral health preventive and educational programs in addition to the intense oral hygiene programs are essential to improve the gingivalhealth condition of women with polycystic ovary syndrome.Frthermore, salivary levels of free testosterone were found to be high among women with polycystic ovary syndrome.
An interesting result in this study that salivary free testosterone can be used as a marker for diagnosis of women with polycystic ovary syndrome.

( 1 )
Master student , Department of Pedodontic and Preventive Dentistry, College of Dentistry, University Baghdad.(2) Assistant Professor, Department of Pedodontic and Preventive Dentistry, College of Dentistry, University Baghdad.

Table 2 : Salivary free testosterone (mean±SD) among the study and control groups.
**=Highly significant at P<0.

Table 3 : Correlation coffecient between salivary free testosterone with plaque index, gingival index among the study and control groups.
# 0.