UTICAJ ESTROGENA NA ZDRAVLJE GINGIVE KOD DEVOJČICA THE INFLUENCE OF ESTROGEN ON THE GINGIVAL HEALTH OF GIRLS

Uvod. Inflamacija gingive je veoma česta u detinjstvu i pubertetu. Cilj istraživanja bio je odrediti indekse gingive i vrednosti 17β estradiola u pljuvački i serumu i njegov uticaj na zdravlje gingive kod tinejdžerki. Pacijenti i metode. Studija je obuhvatila 30 devojčica (starosti od 11 do 14 godina) sa inflamacijom gingive i 30 devojčica kontrolne grupe koje nemaju inflamaciju gingive. Stepen zdravlja gingive je procenjen kliničkim merenjem indeksa gingive. Koncentracija 17β estradiola u serumu i pljuvački određena je DRG Estradiol ELISA (EIA-2693) i DRG Salivary Estradiol ELISA (SLV-4188) metodom. Rezultati. Vrednosti indeksa gingive jasno ukazuju na prisustvo inflamacije gingive. Analiza vrednosti nivoa estrogena u serumu i pljuvački ukazuju na pozitivnu korelaciju indeksa, posebno ističući uticaj 17β estradiola na inflamaciju i krvarenje gingive. Zaključak. Dobijene vrednosti koncentracije hormona u serumu i pljuvački pokazuju u obe grupe njegov potencijalni uticaj na zdravlje gingive, što ističe ulogu stomatologa u preventivi i terapiji parodontopatije u pubertetu.


Introduction
There is an extensive body of research which seeks to fully elucidate the events associated with fluctuating levels of sex steroid hormones in different physiological conditions and periods of human life, and their impact on oral and periodontal health.Puberty, which is the beginning of sexual maturation of individuals, involves reproductive changes.It is considered that this period may influence the occurrence of gingival inflammation and gingival enlargement in both sexes, with greater prevalence among girls.Pubertal gingivitis is a topic on which conflicting results may be found, some of which clearly confirm the trend of increased signs of gingival disease, and others their absence 1,2 .
In girls, it is considered that changes in levels of estrogen and progesterone are transient and at postpuberty they return to normal levels.Most of the girls who are in good general and gingival health will not develop significant periodontal changes due to puberty or the menstrual cycle, unlike those with initial signs of gingival inflammation with even relatively small accumulation of dental plaque 3 .Gingival enlargement can often be found in the population with puberty gingivitis and represents direct confirmation that endocrine disorders or hormonal variations affect the gingival tissue modifying the tissue response to local factors, which may favor plaque accumulation and progression of disease 4,5 .It is thought that after puberty, with normalization of hormonal variations, there is a spontaneous reduction of gingival increase, but only in the absence of dental plaque and dental calculus 6 .

Aims
Taking into consideration the involvement of sex hormones in numerous physiological processes in the body and maintaining gingival health, the aim of the study was to determine: − serum and salivary levels of 17β estradiol in girls with and without gingival inflammation at puberty; − the gingival status among teenage girls using gingival index values; − the possible influence of estrogen homone on gingival health in girls in puberty.

Patients and methods
The investigation was performed at the Clinic for Oral Pathology and Periodontology in Skopje, including two groups of puberty girls.The first group consisted of 30 female patients, aged 11 to 14 years with diagnosed puberty gingivitis, and the second, control group, consisted of an equal number of girls with no signs of gingival inflammation.All patients met the following inclusion criteria: absence of general diseases, not receiving antibiotic therapy in the previous three months, and not receiving hormone therapy.

Laboratory tests
To determine the levels of steroid hormone 17β-estradiol, 5 ccm of blood was taken from all patients by venepuncture of the cubitalis vein.After spinning at 3000 rpm for 10 minutes, separated serum was dragged into test tubes and frozen at -20°C until the day of analysis.To determine the salivary levels of 17β-estradiol, in the morning, unstimulated saliva was collected by a simple extraction, in all the subjects.Tubes with saliva were frozen at -20 ° C until the day of treatment.Saliva after thawing was undergone to ultracentrifugation and the supernatant extract was analyzed with the appropriate method.Serum and salivary 17β estradiol levels were assessed using the ELISA method, at the Department of Obstetrics and Gynecology, Medical Faculty in Skopje.For the determination of serum 17β estradiol, ELISA DRG Estradiol (EIA-2693) kit was used.The method is based on the principle of competitive binding of a polyclonal rabbit antibody directed to the antigenic sites of the estradiol molecule.Endogenous estradiol from patients' samples is in competition with peroxidase conjugated estradiol for binding to the antibodies.After sa vezanom peroksidazom je obrnuto proporcionalna koncentraciji estradiola u uzorku.Posle dodavanja supstrata rastvoru, intenzitet razvijene boje bio je obrnuto proporcionalan koncentraciji estradiola u uzorku pacijenata.Nivo 17β estradiola u pljuvački određen je uz pomoć DRG pljuvačnog estradiol ELISA (SLV-4188) kita na osnovu kompetitivnosti i razdvajanja uzoraka na mikropločici.Nepoznata količina estradiola u uzorku i poznata količina estradiola u peroksidaznom konjugatu kompetitivna su za mesta vezivanja u estradiolnom poliklonalnom antiserumu.Posle dva sata inkubacije, mikro ploča je isprana kako bi se prekinula kompetitivna reakcija.Posle dodavanja rastvora supstrata, koncentracija 17β estradiola obrnuto je proporcionalna izmerenoj optičkoj gustini.
Pirsonov koeficijent korelacije (r) ukazuje na snažnu pozitivnu korelaciju između serumskih vrednosti 17β estradiola i inflamacije gingive i krvarenja, kao i između pljuvačnih vrednosti estradiola i krvarenja gingive (Tabela 4).incubation, unbound conjugate was washed away.The amount of peroxidase-linked conjugate was inversely proportional to the concentration of estradiol in the sample.After adding the substrate solution, intensity of developed color was inversely proportional to the concentration of estradiol in patient sample.Determination of salivary17β estradiol level was done using DRG Salivary Estradiol ELISA (SLV-4188) kit.It was based on the principle of competition and separation on a microplate.An unknown amount of estradiol present in the sample and a particular amount of estradiol peroxidase conjugate competed for binding sites of estradiol polyclonal antiserum.After two-hour incubation, microplate was washed to stop the competitive reaction.After adding the substrate solution, concentration of 17β estradiol was inversely proportional to the measured optical density.

Statistical method
The collected data were statistically processed by the computer program "Statistika" 6 using descriptive and inferential statistical methods.
Pearson's coefficient of correlation (r) indicates a strong positive correlation between the plaque index and gingival inflammation (0,68), and between the calculus index and gingival bleeding (Table 2).
The average values of 17β estradiol in experimental group range from 4.15 pg / ml in the saliva to 56.73 pg / ml in serum, whereas in girls with no signs of puberty gingivitis they range from 3.31 pg / ml in saliva to 46.20 pg / ml in serum (Table 3).
There is a strong positive correlation (r=0,69) between 17β estradiol values in saliva and gingival bleeding (Figure 3).
Analysis using Mann-Whitney U tests shows that there are statistically significant differences in mean values of serum 17β estradiol values between girls with and without gingivitis (Z=-2.447,p=0.0144) (Figure 4).
Analysis using Mann-Whitney U test shows that there are statistically significant differences in mean values of salivary 17β estradiol between girls with and without gingivitis (Z=-3.152,p=0.0016) (Figure 5).

Discussion
Recent years have dramatically enhanced the perception of action of sex steroid hormones in terms of health.While there is no doubt about the importance of sex hormones in reproductive endocrinology, there is growing evidence that suggests much broader role of gonadal hormones in the human organism 10,11 .It is thought that estrogen hormones are directly or indirectly involved in the regulation of various tissues such as brain, kidney, heart, liver, skin, and periodontal tissue complex.Although the blood circulating sex hormones exist in extremely low concentrations, they are able to regulate differentiation and growth in selected tissues distant from the site of secretion.
The growing body of evidence suggests that periodontal tissue reactions are modulated by the action of androgens, estrogens and progestins.Some of them imply that periodontal tissues are target tissues of sex hormone 12,13 , referring to the clinical manifestations during periods of hormonal changes.These clinical observations confirm the prevalence of gingival disease with fluctuating levels of sex hormones, even in conditions when oral hygiene remains unchanged 14 .
Because the period of starting and duration of puberty shows individual variations, all clinical, microbiological and hormonal parameters should be in line with chronological age and the parameters that describe pubertal maturation.Hence, demonstration of gingival index values which increase during puberty strongly depends on the proper selection of parameters and analytical methods 15,16 .Some authors 17 noticed that all additional factors that could modify the inflammatory response must be eliminated in order to explore the effects of hormone action on gingival health.For these reasons, it is believed that children of teenage group are most adequate, since the concentration of sex hormones and the incidence of gingivitis are increased at that period of life.The onset of gingivitis, which is evident in children and adolescents, may be associated with the composition of dental plaque, inflammatory cell response, hormonal changes, kvantitativnim promenama oralnog biofilma 20- 24 .Mombelli i sar. 25 su u longitudionalnom istraživanju utvrdili da je prosečna vrednost indeksa krvarenja papile (PBI) u vezi sa razvojem sekundarnih polnih karakteristika dece u pubertetu.Vrednosti ovog indeksa su dostigle svoj vrhunac 1,5 godinu od početka puberteta kod 35% osoba, ali ukupan plak indeks u toku godine nije bio u porastu.Nasuprot ovome, druga istraživanja nisu otkrila značajnu povezanost između ranog puberteta i gingivalnih promena kod devojčica 26 .
This disproportion could be due to factors such as status of oral hygiene or the design of the study.The average age at which girls and boys reach the maximum degree of gingival inflammation was 12 years and 10 months and 13 years and 10 months respectively, while according to another more extensive study it is estimated that gingival inflammation starts at 11 years of age in both sexes, whereby the level of plaque remains constant across all age groups 27 .Growth of gingival inflammatory reaction, in part, can be explained by the factors of host defenses 28 .Significant proportion occurs between serum IgG antibodies directed to P. intermedia and the level of sex hormones in both sexes noted in gingival inflammation, while the ratio between IgM antibodies directed to the strains of P. intermedia and sex hormones is significant only in boys with marked gingival reaction 29 .
Kako bi se procenio uticaj 17β estradiola na zdravlje gingive u eksperimentalnoj grupi devojčica, određen je nivo estradiola u serumu i gingivalni indeks i uočena jaka međusobna veza.Prema ovome, odnos vrednosti 17β estradiol u serumu i inflamacije gingive je the findings of several authors who suggest an increased trend of gingival disorder in girls during puberty maturation 2,15,25 , which depends on the fluctuating levels of sex hormones, age, bacterial composition of dental plaque, inflammatory cell response, vascular reactions, morphological differences, etc 30,31 .During puberty, an increased gingival bleeding index significantly grows with development of puberty, while significant downward trend is seen after 14 years of age for girls 15 .
In order to determine the influence of local etiological factors on gingival health, the ratio between the index of dental plaque, index of calculus, index of gingival inflammation, index of gingival enlargement and gingival bleeding was established.The obtained results demonstrated a strong positive correlation between the plaque index and gingival inflammation (r=0.68), while the ratio between dental plaque and gingival enlargement ranged from weak insignificant (r=0.10) to moderately positive ratio (r=0.24), with regard to the index of gingival bleeding.These findings emphasize the supreme role of dental plaque in the development of gingival inflammation and suggest the involvement of additional factors for their expression.
Regarding the impact of dental plaque as a local irritating factor, a strong correla-tion with gingival bleeding index (r=1) was obtained, as well as a moderate positive correlation (r=0.44) with the index of gingival enlargement and slight positive correlation (r=0.16) with gingival inflammation.According to the results from this study, Yanover and Ellen's 32 longitudinal study on girls who normally go through puberty indicates a positive correlation observed between gingival index and index of dental plaque.In a longitudinal study, Nakagawa et al. 29 presented statistically significant increase in gingival inflammation in girls at the age of puberty, unlike the prepubertal age, without noting significant differences in indices of dental plaque and calculus.Contrary, Tianen et al. 33 report statistically high significant correlation between dental plaque and the degree of gingival bleeding at the beginning and at the end of puberty, sugge-sting the dominant importance of oral hygiene on =0,64, uvećanja gingive i krvarenja gingive r=0,52 i r=0,62, za svaki pojedinačno.
gingival condition despite fluctuations in the level of sex hormones.
Besides the evaluation of clinical parameters of gingival status in girls during puberty, in our study, serum and salivary concentrations of ovarian hormone 17β estradiol in experimental group ranged from 4.15pg/ml in saliva to 56.73pg/ml in serum, whereas in girls with no signs of puberty gingivitis ranged from 3.31pg/ml in saliva to 46.20pg/ml in serum.In order to determine the impact of 17β estradiol on gingival health in the experimental group of girls, correlations between serum levels of estradiol and indices of gingival status were established and strong positive correlation with each of them was obtained.Thus, the correlation of serum 17β estradiol with gingival inflammation was r=0.64, while gingival enlargement and gingival bleeding were r=0.52 and r=0.62, respectively.The results from our study are consistent with findings from longitudinal study 34 presenting statistically high significant correlation between increased serum levels of 17β estradiol and gingival index in girls with puberty gingivitis.Other authors 32 observed a positive ratio with the increased plasma levels of estradiol and the presence of black pigmented strains of Bacteroides in girls in early puberty.They suggest that the hormonal events do not unconditionally support the colonization of pathogenic strains prevalent in adult periodontal disease.Serum levels of estradiol in girls at puberty were positively correlated with the level of P. Intermedia and P. Nigrescens while the levels of serum IgG antibodies directed to these strains are significantly increased, which also shows a significant positive correlation with levels of female sex hormones 29 .
Analysis of the ratio between salivary valuesof 17β estradiol and gingival indices in girls with puberty gingivitis reveals a strong positive correlation r=0.69 with the index of gingival bleeding and moderate positive correlative value of gingival inflammation r = 0.44 and gingival enlargement r = 0.49.In our study, despite the determination of serum and salivary concentrations of estradiol in pubertal girls with gingivitis, the level of the appropriate hormone was determined in girls without signs of gingival disease.Comparing Zaključak 1. Vrednosti 17β estradiola u serumu ukazuju na jaku pozitivnu vezu sa gingivalnim indeksom i indeksom krvarenja gingive i umerenu sa indeksom uvećanja gingive.Vrednosti 17β estradiola u pljuvački ukazuju na jaku pozitivnu vezu sa krvarenjem gingive i umerenu sa gingivalnim indeksom i indeksom uvećanjam gingive.Takođe, i u serumu i u pljuvački uočena je jaka pozitivna veza sa koncentracijom 17β estradiola.
3. Dobijene vrednosti koncentracija hormona u oba medijuma, u obe grupe, sugerišu njihov potencijalni uticaj na zdravlje gingive.Ovaj nalaz ističe ulogu stomatologa u preventivi i terapiji gingivitisa kod pacijenata u pubertetu.the obtained values of serum 17β estradiol in both groups of girls, the analysis using the Mann-Whitney U test showed a statistically significant difference in average values of serum estradiol in both groups.Also, the analysis of average salivary values of estradiol, using the Mann-Whitney U test, showed that there are statistically significant differences in the level of estradiol in girls with apparent gingival inflammation and in girls without gingivitis.
Recent findings [34][35][36] are certainly another direct confirmation of involvement of sex hormones in the development of gingival alterations among girls in puberty.

Conclusions
1. Serum 17β estradiol values indicated a strong positive correlation with the index of gingival inflammation and gingival bleeding and moderate positive correlation with the index of gingival enlargement.Salivary 17β estradiol values indicated a strong positive correlation with gingival bleeding, and moderate positive correlation with the index of gingival inflammation and gingival enlargement.Also, in both mediaserum and saliva, a strong positive correlation between the concentrations of 17β estradiol was found.
2. The indices of gingival status in the experimental group of girls at the age of puberty reveal considerable presence of gingival inflammation.
3. The obtained values of the hormone concentrations in both media and in both groups suggest their potential influence on the gingival health.This emphasizes the role of dentists in preventive and treatment modalities in patients with gingivitis in puberty.

Table 2 .
Pearson's coefficient of correlation (r) between the values of gingival indices and indices of oral hygiene in girls with puberty gingivitis Tabela 3. Srednje vrednosti 17β estradiola (E) u serumu i pljuvački kod devojčica sa i bez gingivitisa

Table 3 .
Mean values of17 β estradiol(E) in serum and saliva in female children with and without gingivitis

Table 4 .
Pearson's coefficient of correlation(r) between the values of 17 β estradiol(E) in serum and saliva and gingival indices in female children with puberty gingivitis