BACKGROUND: ß -estradiol is a steroid sex hormone that plays important role in oral physiology. The aim of this study

was to determin the association of the menstrual cycle and salivary ß-estradiol with the production of volatile sulfur

compounds (VSC).

METHODS: In this cross-sectional study, twenty female dental students in good oral and general health were recruited for

evaluation of VSC, salivary flow, ß-estradiol concentration, oral dryness feeling and dysmenorrhea. Data were analyzed

by marginal homogeneity test and xtgee model.

RESULTS: The only covariates that significantly related to VSC were being at the follicular phase (B = -0.21, P = 0.02),

being at the pre-menstrual phase (B = -0.25, P = 0.01), stressfulness (B = 0.02, P = 0.01) and oral dryness feeling

(B = 0.34, P = 0.02). Salivary ß-estradiol was at the level of 59.7  31.2 in those having halitosis (VSC ≥ 75) while in

the others (VSC < 75) it was about 40.8  18.0 (P < 0.009). This difference was not statistically significant in multivariate

analysis.

CONCLUSIONS: The menstrual cycle, stress and oral dryness were the most important determinants of halitosis. The

salivary ß-estradiol level could not explain such relationship as its effect was eliminated considering the other factors.

KEY WORDS: Halitosis, menstrual cycle, ß-estradiol, volatile sulfur compound

Barkhori, Fahimeh

Gholamhoseinian, Ahmad

Kakoei, Shahla

Mirzazadeh, Ali

Mohammadi, Mohammad

Simorgh Research Repository

Received: 25 Oct. 2011 Accepted: 24 Apr. 2012  1 Associate Professor, Kerman Oral and Dental Diseases Research Center, Department of Oral Medicine, School of Dentistry, Kerman, Iran. 2 Dentist, Kerman, Iran. 3 MD, MPH, Kerman Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran. 4 Assistant Professor, Department of Periodontist, School of Dentistry, Kerman, Iran.  5 Professor, Kerman Physiology Research Center, Department of Biochemistry, School of Medicine, Kerman, Iran. Correspondence to: Ali Mirzazadeh MD, MPH, PhD. Email: ali.mirzazadeh@hivhub.ir  JOHOE/Spring 2012; Vol. 1, No. 1 41 Original Article Influence of menstrual cycle and salivary ß-estradiol on volatile  sulfur compound  Shahla Kakoei DDS, MSc1, Fahimeh Barkhori DDS2,  Ali Mirzazadeh MD, MPH, PhD3, Mohammad Mohammadi DDS, MSc4,  Ahmad Gholamhoseinian PhD5  Abstract BACKGROUND: ß -estradiol is a steroid sex hormone that plays important role in oral physiology. The aim of this study was to determin the association of the menstrual cycle and salivary ß-estradiol with the production of volatile sulfur compounds (VSC). METHODS: In this cross-sectional study, twenty female dental students in good oral and general health were recruited for evaluation of VSC, salivary flow, ß-estradiol concentration, oral dryness feeling and dysmenorrhea. Data were analyzed by marginal homogeneity test and xtgee model. RESULTS: The only covariates that significantly related to VSC were being at the follicular phase (B = -0.21, P = 0.02), being at the pre-menstrual phase (B = -0.25, P = 0.01), stressfulness (B = 0.02, P = 0.01) and oral dryness feeling  (B = 0.34, P = 0.02). Salivary ß-estradiol was at the level of 59.7  31.2 in those having halitosis (VSC ≥ 75) while in the others (VSC < 75) it was about 40.8  18.0 (P < 0.009). This difference was not statistically significant in multiva-riate analysis. CONCLUSIONS: The menstrual cycle, stress and oral dryness were the most important determinants of halitosis. The  salivary ß-estradiol level could not explain such relationship as its effect was eliminated considering the other factors. KEY WORDS: Halitosis, menstrual cycle, ß-estradiol, volatile sulfur compound.  J Oral Health Oral Epidemiol 2012; 1(1): 41-45  alitosis is a term defined as offensive mouth odor that can have intra or extra-oral sources.1 Volatile sulfur compounds (VSC) caused by activation of anaerobic gram negative micro-organisms on sulfur containing substrates in oral cavity such as food, saliva, blood and epithelial cells can have effect on patient’s social health2. This activation mainly happens with gingival bleeding, periodontitis, tongue coating, dental caries, periodontitis around the implantation, dental necrosis, oral ulcers, food debris and other factors.3 There are many extra-oral factors that cause  oral mouth odor such as respiratory distur-bances like sinusitis, nasal discharge, infection, lung carcinoma and other systemic disease in-cluding digestion disease, diabetes mellitus and uremia.3,4 Endocrine system affects oral homeostasis. Previous studies showed that sex hormones have influence on periodontal tis-sue.5,6 For instance, some reports indicated that women in menopause phase feel more oral dryness and discomfort.7 ß-estradiol is a steroid sex hormone that plays important role in oral physiology. It seems that the decrease of this hormone during HMenstrual cycle, salivary ß-estradiol and volatile sulfur compound Kakoei et al. 42 JOHOE/Spring 2012; Vol. 1, No. 1 menopause influences on oral epithelium and causes discomfort during this period.7 To the best of our knowledge, there are few articles about the association between steroid sex hor-mones and halitosis.2 The aim of this study was to evaluate the influence of menstrual cycle and estradiol hormone on production of VSC in healthy young women volunteers. Methods This study was approved by the ethic commit-tee of Kerman University of Medical Sciences (No. K/89/10). In this cross-sectional survey we recruited the study subjects using conveni-ence sampling method. Based on the findings from Calil et al. 2 that recruited 14 women in their study, a power analysis was done and it was found that with a sample of 20, we would have a power of 98% (considering Alpha as the level of 5%). Therefore, twenty undergraduate female dental students between 20 to 26 years old were voluntarily participated in this study. Participants were signed a consent form after that the aim and method of the study were ful-ly explained for them.   Inclusion and exclusion criteria All participants should have been healthy and free of systemic disease, had at least 20 teeth, no dental caries, calculus and no defective res-toration. All subjects who had either smoking habit or were on medicine consumption were excluded from the study. All subjects com-pleted an information form including demo-graphic data, history of smoking and habits. All subject received periodontal examination by a periodontist for approving periodontal sta-tus of the subjects. They could be included in this study if they had plaque index and bleeding in-dex under 10%, probing depth equal or less than 3 mm and without tongue coating.8 Women could participate in this study if they had regular menstruation cycle, did not have pregnancy and did not use contraceptive or hormonal therapy during previous 6 months.   Procedure The subjects were asked to detect the different  times of their menstrual cycles for scheduling. These times were including 1/ days 1-4 for menstrual phase, 2/ days 7-11 for follicular phase, 3/ days 24-27 for the pre-menstrual phase.2 Some rules for the day of sampling were explained for subjects. They were prohi-bited using any spicy foods like onion and gar-lic and smelling cosmetics for 24 hours before the experiment. They should avoid using breath freshener or mouth rinse for 1 week and eating or drinking at least 8 hours before the experiment but they were allowed to drink wa-ter up to 3 hours before it.2 Participants were asked to complete the Beck Anxiety Inventory (BAI) questionnaire. BAI is a 21-item questionnaire that the degree of severity of anxiety is scored using Likert scale (0-7 indicating no anxiety, 8-15 mild,  16-25 moderate and 26-63 sever anxiety). To prevent possible anxiety, we avoided doing experiment during examination time or other stressful situations.2,9 Saliva was collected by spitting method for 5 minutes in the plates that were weighted  before. Salivary flow rates were assayed in ml/min after weighting by digital scale  (AND-GF). The mean salivary flow less than 0.1 ml/min was defined as dry mouth.10 Then, the VSC measurements were done by sulfide monitor (Halimeter, Interscan, Ca, USA). The subjects were requested to avoid opening mouth one minute before measurement and deep breathing during that time. A plastic straw (sampler) were kept on the posterior dorsal of the tongue by subjects while not touching the oral mucosa. They were asked to keep opening their mouth about 1.5 cm during sampling. The device were recorded the peak of VSC after 3 times measurements. The mean of VSC were evaluated as without halitosis for values less than 75 ppb, physiologic or mild halitosis for 75-110 ppb and sever halitosis for values equal or more than 110 ppb.11 Saliva samples were kept in freezer at  -20°C. The mean of ß-estradiol hormone was evaluated by radioimmunoassay (RIA) method using commercial kit (Diasorin, Italy) after cen-trifuging 5000 rpm for 5 minutes and filtering  Menstrual cycle, salivary ß-estradiol and volatile sulfur compound Kakoei et al. JOHOE/Spring 2012; Vol. 1, No. 1 43 by Elisa method.  Statistical analysis The relation of halitosis and different phases of menstruation cycle was determined by the marginal homogeneity test. The VSC score (outcome variable) has been transferred to normal distribution by square root transforma-tion. Xtgee model (normal distribution for the dependent variable and the log as the link function) was applied for estimating the effects of menstrual cycle considering different cova-riates on the VSC score. P value < 0.05 was used as the level of significance. Stata (version 10 for windows, TX, USA) was the statistical software used for the analysis. Results Finally, the data of 20 subjects were analyzed. Overall, the percentage of sever and mild hali-tosis was about 16.7% and 43.3%, respectively. At the menstrual phase, about 50% of the sub-jects had severe halitosis while it was reduced to less that 1% in both follicular phase and pre-menstrual phases (P < 0.001) (Figure 1).  Sever Halitosis was reported among 58.3% of those with dysmenorrhea, and it was about 27.5% among the others (P = 0.8). Almost all with oral dryness feeling presented halitosis (VSC ≥ 75) compared to others with only 55.5% halitosis (P = 0.035) (Table 1).  Multivariate analysis (xtgee model) showed   Figure 1. The percentage of halitosis by menstural cycle  Table 1. Halitosis in relation to oral dryness feeling and dysmenorrhea Covariates VSC ≥ 75 VSC < 75 P-value Oral Dryness feeling (%) 6 (100%) 0 0.035 Dysmenorrhea at menstrual phase (%) 8 (66.7%) 4 (33.3%) 0.81 Dysmenorrhea at Pre-menstrual phase (%) 7 (100%) 0 0.015 Saliva ß - estradiol pg/ml (Mean + SD) 59.7 + 31.2 40.8 + 18.0 0.009 Saliva Weight gr/5min (Mean + SD) 1.68 + 1.3 1.9 + 0.6 0.32  Table 2. Multiple xtgee modeled the relationship of different covariates against sqoure root of volatile sulfur compounds  Covariates Beta Se P-value Follicular phase -0.21 0.09 0.02 Pre-menstrual phase -0.25 0.08 0.001 Stress 0.02 0.003 0.001 Oral dryness feeling 0.34 0.15 0.02 Dysmenorrhea 0.04 0.09 0.64 Saliva ß-estradiol (pg/ml) -0.003 0.001 0.06 Age -0.01 0.03 0.88  Menstrual cycle, salivary ß-estradiol and volatile sulfur compound Kakoei et al. 44 JOHOE/Spring 2012; Vol. 1, No. 1  the covariates that were significantly related to square root of VSC were follicular phase, pre-menstrual phase, stress and oral dryness  (Table 2). Discussion The results of this study showed that mouth odor was influenced by different phases of menstruation. It was associated with other co-variates like stress and oral dryness feeling. The salivary ß-estradiol Level could not ex-plain the relationship between menstrual cycle and the halitosis and its effect was ruled out considering the other factors. The present study showed that the level of VSC was higher in menstruation in comparison with follicular and pre-menstruation phases. This result is in accordance with previous studies.2,9,12 In the unadjusted analysis, it was shown that there was significant relationship between ß-estradiol and halitosis. It seems that steroid hormones have effect on oral mucosa in wom-en. There are few studies about the effect of sex hormones on oral mucosa in women.13 Study of Agha-Hosseini et al. showed that there was a negative correlation between the concentra-tion of ß-estradiol hormone and oral dryness feeling in menopause women.7 Although ß-estradiol level has fluctuation in menstrual cycle and is reduced in pre-menstrual and menstrual phases, we found that the different level of this hormone did not have significant effect on halitosis when the association was adjusted. In addition, it has been shown that estradiol hormone can cause increase in the number of anaerobes such as Prevotella inter-media,14 but Calil et al. did not see this effect in their study.2 Fischer et al. could not find any cyclic variation of microbial flora during men-struation cycle too.15 A decreased salivary flow was not observed significantly during different phases of the menstrual cycle. Calil et al. had the same result in their research.2 There was significant corre-lation between sensation of oral dryness feel-ing with halitosis but not salivary flow rate. Although the exact mechanism of influence of oral dryness feeling on production of VSC has been not understood, it seems that the sensa-tion of oral dryness is associated with the psy-chological aspects and anxiety on mouth odor. All women who suffered from pre-menstrual syndrome had higher VSC. Previous studies had similar results.9,16 This might be caused by the pain and anxiety in this special period of time in women life. In adjusted anal-ysis this was not statistically significant. Anxie-ty and stress could be influenced by fluctua-tion of hormonal changes during different phases of menstrual cycle and affect psychia-tric symptoms such as oral dryness feeling and eventually can increase the VSC. Anxiety and stress could have effect on sympathetic system activation and then the composition of saliva,17 but its mechanism has not been fully unders-tood so far. Conclusion In conclusion, this study showed the possibili-ty of influence of sex hormones on production of VSC. Halitosis is becoming one major prob-lem for society and more research on it can pave the way for its solution. It seems that dif-ferent sex hormones are involved on mouth odor and this subject needs more research for discovering the possible causes. Conflict of Interest Authors have no Conflict of Interest. Acknowledgement The authors wish to thank the volunteers for their assistance in all periods of our study. This study was supported by grant proposal No. K/89/10 of Kerman University of Medical Science. The authors declare that they have no competing interests.  References 1. Rayman S, Almas K. Halitosis among racially diverse populations: an update. Int J Dent Hyg 2008; 6(1): 2-7. 2. Calil CM, Lima PO, Bernardes CF, Groppo FC, Bado F, Marcondes FK. Influence of gender and menstrual cycle on volatile sulphur compounds production. Arch Oral Biol 2008; 53(12): 1107-12. Menstrual cycle, salivary ß-estradiol and volatile sulfur compound Kakoei et al. JOHOE/Spring 2012; Vol. 1, No. 1 45 3. Van den Broek AM, Feenstra L, de BC. A review of the current literature on aetiology and measurement methods of halitosis. J Dent 2007; 35(8): 627-35. 4. Cortelli JR, Barbosa MD, Westphal MA. Halitosis: a review of associated factors and therapeutic approach.  Braz Oral Res 2008; 22(Suppl 1): 44-54. 5. Guncu GN, Tozum TF, Caglayan F. Effects of endogenous sex hormones on the periodontium--review of literature. Aust Dent J 2005; 50(3): 138-45. 6. Machtei EE, Mahler D, Sanduri H, Peled M. The effect of menstrual cycle on periodontal health. J Periodontol 2004; 75(3): 408-12. 7. Agha-Hosseini F, Mirzaii-Dizgah I, Mansourian A, Khayamzadeh M. Relationship of stimulated saliva 17beta-estradiol and oral dryness feeling in menopause. Maturitas 2009; 62(2): 197-9. 8. Muhlemann HR, Son S. Gingival sulcus bleeding--a leading symptom in initial gingivitis. Helv Odontol Acta 1971; 15(2): 107-13. 9. Queiroz CS, Hayacibara MF, Tabchoury CP, Marcondes FK, Cury JA. Relationship between stressful situations, salivary flow rate and oral volatile sulfur-containing compounds. Eur J Oral Sci 2002; 110(5): 337-40. 10. Liu XN, Shinada K, Chen XC, Zhang BX, Yaegaki K, Kawaguchi Y. Oral malodor-related parameters in the  Chinese general population. J Clin Periodontol 2006; 33(1): 31-6. 11. Murata T, Yamaga T, Iida T, Miyazaki H, Yaegaki K. Classification and examination of halitosis. Int Dent J 2002; 52(Suppl 3): 181-6. 12. Tonzetich J, Preti G, Huggins GR. Changes in concentration of volatile sulphur compounds of mouth air during the menstrual cycle. J Int Med Res 1978; 6(3): 245-54. 13. Meurman JH, Tarkkila L, Tiitinen A. The menopause and oral health. Maturitas 2009; 63(1): 56-62. 14. Kornman KS, Loesche WJ. The subgingival microbial flora during pregnancy. J Periodontal Res 1980; 15(2): 111-22. 15. Fischer CC, Persson RE, Persson GR. Influence of the menstrual cycle on the oral microbial flora in women: a case-control study including men as control subjects. J Periodontol 2008; 79(10): 1966-73. 16. Calil CM, Marcondes FK. Influence of anxiety on the production of oral volatile sulfur compounds. Life Sci 2006; 79(7): 660-4. 17. Garrett JR, Ekström J, Anderson LC. Glandular Mechanisms of Salivary Secretion. 1st ed. Basle: S. Karger  Publishing; 1998. 

Influence of menstrual cycle and salivary ß-estradiol on volatile

sulfur compound

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Influence of menstrual cycle and salivary ß-estradiol on volatile sulfur compound

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