J Periodontol May 2010

Effects of Menstrual Cycle on Periodontal Health and Gingival Crevicular Fluid Markers
zgu zc Sema Becerik,* O xaka,* Ays xe Nalbantsoy, Gu n O l Atilla,* Peter Celec,i Michal Behuliak, and Gu lnur Emingil,*
Background: Fluctuations in sex steroid hormones, which are also noticeable through the menstrual cycle of women, may impact periodontal health. The aim of this study is to evaluate the effect of hormonal changes occurring in the menstrual cycle on gingival inammation and the gingival crevicular uid (GCF) levels of interleukin 6 (IL-6), prostaglandin E2 (PGE2), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-2 (PAI-2). Methods: Twenty-ve gingivitis patients and 25 periodontally healthy subjects having regular menstrual cycles were seen at menstruation (ME) (1 to 2 days of menstruation), ovulation (OV) (12 to 14 days), and premenstrual phases (PM) (22 to 24 days). GCF and saliva samples were collected and clinical parameters including plaque index and bleeding on probing were recorded at each menstrual phase. Salivary estrogen and progesterone levels were analyzed to determine exact menstrual cycle days. GCF levels of IL-6, PGE2, t-PA, and PAI-2 were measured by enzyme-linked immunosorbent assay. Results: The percentages of sites with bleeding on probing were signicantly higher in ME (60.85 18.36) and OV (58.92 25.04) than in the PM (40.12 20.10) phase in the gingivitis group (P <0.001; repeated measures analysis of variance), whereas it was similar for all phases in the healthy group (P >0.05; repeated measures analysis of variance). GCF levels of IL-6 were signicantly elevated in gingivitis patients compared to healthy subjects in all phases (P = 0.004, P = 0.041, and P = 0.046 for ME, OV, and PM, respectively; Mann-Whitney U test). GCF levels of IL-6, PGE2, t-PA, and PAI-2 were unchanged in different menstrual phases in both groups (P >0.05; Friedman test). Conclusion: The present study suggests that changes in the sex steroid hormones during menstrual cycles might have a limited effect on the inammatory status of gingiva, but GCF cytokine levels were not affected. J Periodontol 2010;81:673-681. KEY WORDS Gingivitis; interleukin-6; menstrual cycle; plasminogen activator inhibitor-2; prostaglandin E2; tissue plasminogen activator.
* i Department of Periodontology, School of Dentistry, Ege University, Izmir, Turkey. Department of Bioengineering, School of Engineering, Ege University. Institute of Pathophysiology, Comenius University, Bratislava, Slovakia. Institute of Molecular Biomedicine, Comenius University. Department of Molecular Biology, Comenius University.

he biologic changes that occur in tissues of the periodontium during puberty, pregnancy, menopause, and oral contraceptive use have heightened interest in the relationship among sex steroid hormones and periodontal health. Gingival tissues contain receptors for androgens, estrogen, and progesterone1-3 and it was documented that these hormones have effects on the oral mucosa and the periodontium.4 Estrogen and progesterone changes seem to modify the gingival tissues, leading to a higher vascular permeability and decreased keratinization of the gingival epithelium. Gingival inammation is greater during pregnancy than postpartum and its onset is correlated with an increase in circulating levels of estrogen and progesterone.5,6 The changes in the circulating levels of female sex hormones also affect the host response against dental plaque.7 Muhlemann8 clinically and histologically described a case of gingivitis intermenstrualis where bright red hemorrhagic lesions of the interdental papilla developed prior to the menses. It was demonstrated that women with gingivitis experienced increased inammation with an associated increase in crevicular uid exudate during ovulation (OV) compared to healthy controls.9 Recently, Machtei et al.10 showed that the extent of gingivie tis, as dened by mean values of the Lo index,11 differed between the time of menstruation (ME) and OV but there were
doi: 10.1902/jop.2010.090590

673

Menstrual Cycle and Periodontal Health

Volume 81 Number 5

no changes in probing depths or clinical attachment levels. Women also often have different pain complaints during their menstrual cycles.12 Alteration in local immune system components was studied to explain whether sex hormones have an impact on the periodontium. Interleukin (IL)-6, a major mediator in periodontal inammation, was found to be down-regulated by progesterone in gingival broblasts.13 The increase in circulating levels of progesterone during pregnancy stimulates prostaglandin production, possibly generating an increase in gingival inammation.14 Kinnby et al.15 postulated another mechanism concerning the effects of sex hormones on periodontium and pregnancy gingivitis. It was reported that the balance of the brinolytic system may be disturbed as high progesterone levels during pregnancy resulted in lower levels of plasminogen activator inhibitor-2 (PAI-2), an important inhibitor of tissue proteolysis.15 A relationship between female sex hormone levels and periodontal changes during puberty, pregnancy, and menopause has been postulated, but there are a limited number of studies investigating the effects of menstrual cycle on periodontal health.10,16 The changes in gingival tissues occurring during menstrual phases of women could be related to the changes in inammatory markers in gingival crevicular uid (GCF). The aim of this study is to evaluate the effect of hormonal changes occurring in the menstrual cycle on plaque accumulation and gingival inammation, and GCF levels of IL-6, prostaglandin E2 (PGE2), tissue plasminogen activator (t-PA), and PAI-2 in women with gingivitis or healthy gingiva. MATERIALS AND METHODS One hundred and fty-eight women were screened for this study; 65 of them refused to participate in the study or did not meet the inclusion criteria. A total of 93 women were divided into two study groups according to their periodontal health; 21 women from the gingivitis group and 22 from the periodontally healthy group discontinued the study or dropped out from the study because of having early or late ME. Finally, a total of 50 premenopausal women (25 with gingivitis and 25 with healthy periodontium) were included in this study. All consecutive subjects were recruited from the Department of Periodontology, School of Dentistry, Ege University, Izmir, Turkey, over a period of 1 year between 2007 and 2008. The study protocol was approved by the ethics committee of the Ege University School of Medicine. Prior to participation, the purpose and procedures were fully explained to all patients and all participants gave written informed consent in accordance with the Helsinki Declaration. The study was designed, con674

ducted, analyzed, and reported according to the Guideline for Good Clinical Practice.17 Medical and dental histories were taken and patients received clinical and radiographic evaluation at prescreening visits. Inclusion criteria were premenopausal women, age 20 to 40 years, with regular menstrual cycles (i.e., the length of their cycle varied by no more than 28 3 days) for the last 12 months. Exclusion criteria were 1) pregnancy or breast-feeding; 2) use of oral contraceptives or any other drugs that might affect sex hormones; 3) metabolic or systemic condition that might affect the periodontium; 4) use of antibiotic or anti-inammatory or immunosuppressive drugs during a 3-month period prior to the start of the trial; 5) use of tobacco products; 6) periodontal therapy during the last 3 months; and 7) having destructive periodontal disease (clinical attachment loss >3 mm, radiologic bone loss). The subjects were classied as follows:18 gingivitis group, 25 women (mean age, 29.40 4.967 years; age range, 21 to 39 years) having bleeding on probing (BOP) in more than 50% of the probing sites; healthy group, 25 women (mean age, 27.20 5.346 years; age range, 22 to 40 years) having BOP in <10% of the probing sites. Study Protocol All consecutive women were assigned to either the gingivitis or periodontally healthy group based on clinical examination. They were seen at 1 to 2 days of their ME. GCF samples were collected from buccal aspects of the two single-rooted teeth including maxillary incisives and canines. The following clinical parameters were recorded at six sites of each tooth: plaque index (PI),19 dichotomous measurement of presence of BOP, and probing depth (PD). All measurements were performed by a single calibrated examiner using a manual Williams periodontal probe. The patients were seen again at 12 to 14 days on OV and 22 to 24 days on premensturation (PM) of their ME. GCF samples were taken again and clinical parameters including PI and BOP were repeated. The unstimulated saliva samples were also collected in each menstrual phase. Subjects were asked to spit the saliva into a 5-ml tube until a 2.5-ml sample was collected. The specimen was dated and stored at -20C until analysis. Patients started trial during ME, and if a woman had the following ME phase earlier (<25 days) or later than expected (>30 days) she was dropped from the study. GCF Sampling GCF samples were collected with lter paper strips from two BOP-negative sites in the healthy group and from two BOP-positive sites in the gingivitis
PerioPaper, ProFlow, Amityville, NY.

J Periodontol May 2010

zc Becerik, O xaka, Nalbantsoy, et al.

group. Prior to GCF sampling, the supragingival plaque was removed from the interproximal surfaces with a sterile curet; these surfaces were dried gently by an air syringe and were isolated by cotton rolls. Paper strips were carefully inserted approximately 1 mm into the crevice and left there for 30 seconds.20 Care was taken to avoid mechanical injury. Strips contaminated with blood were discarded.21 The absorbed GCF volume of each strip was determined by an electronic device# and placed into a sterile polypropylene tube and kept at -80C until being analyzed. The readings from the electronic device were converted to an actual volume (microliter) by reference to the standard curve. Salivary Estrogen and Progesterone Analysis Salivary estrogen and progesterone were measured by using enzyme-linked immunosorbent assay kits.** Intra-assay and inter-assay variability was under 5% and 10%, respectively. GCF Levels of IL-6, PGE2, PAI-2, and t-PA Analysis Prior to the quantication of the inammatory markers GCF was eluted from the paper strips by placing the strips in 400 ml of phosphate buffered saline/0.1% bovine serum albumin/0.05% thimerosal for 18 hours at 4C.22,23 GCF levels of IL-6, PGE2, t-PA, and PAI-2ii were assayed by using commercially available enzyme-linked immunosorbent assay kits. Procedures were performed according to the instructions in the kit. The minimum detectable limits of IL-6, PGE2, t-PA, and PAI-2 were 1 pg/ml, 1 pg/ml, 4 pg/ml, and 5 pg/ml, respectively. The amounts of IL-6, PGE2, t-PA, and PAI-2 in each sample were calculated based on the dilutions and the results were expressed as the total amount of each mediator in the 30-second GCF sample. Calculation of the concentration data for each mediator was performed by dividing the amount of each mediator by the GCF volume. Statistical Analysis Sample size of the present study is calculated to detect a 0.5 difference in PI and percentage of BOP scores at the 0.05 probability level with a power of 80%. The power calculation analysis revealed that the required sample size was a minimum of 15 subjects for each study group. The primary efcacy variables were whole-mouth mean PI and percentage of sites with BOP. Statistical analysis was performed on data obtained from patients who completed the trial. The decision about whether to use parametric or nonparametric tests was made based on the results of Kolmogorov-Smirnov test for normal distribution. Demographic variables and PD were analyzed using two sample t tests. The mean values of the subject whole-mouth PI, BOP, and GCF volume for both

groups were calculated and were analyzed using repeated measures analysis of variance (ANOVA) and Bonferroni analysis.24 GCF levels of IL-6, PGE2, t-PA, and PAI-2 of the sampling sites were calculated and the patient was regarded as the unit of analysis. For the data of IL-6, PGE2, t-PA, and PAI-2 that were not distributed normally non-parametric analysis was used. Comparisons of the GCF levels of IL-6, PGE2, t-PA, and PAI-2 among different phases in the same study group were tested by the Friedman test. The Mann-Whitney U test was performed to compare the GCF levels of these markers between the gingivitis and healthy groups. The Wilcoxon signed test was used to compare the salivary estrogen and progesterone levels between menstrual phases, and the post hoc Bonferroni test was performed when there was a difference. Spearman correlation analysis was performed between the clinical parameters, GCF markers, and salivary hormone levels. RESULTS The ow of participants through each stage of the study is outlined in Figure 1. A total of 50 patients, 25 from the gingivitis group and 25 from the healthy control group, completed the trial. Intent-to-treat analysis was not performed because data for noncompliant patients included only baseline data. Therefore, statistical analysis was performed on data from patients who completed the trial. Baseline demographic and clinical characteristics of patients completing the trial in gingivitis and healthy groups are presented in Table 1. Gingivitis and healthy groups had similar age (P >0.05; two sample t tests). Clinical Findings Baseline whole-mouth PD of the gingivitis group (2.32 0.30) was signicantly higher compared to those of the control group (1.99 0.22) (P <0.001, two sample t tests). Gingivitis patients had higher percentage of sites with BOP and PI scores than healthy subjects as expected (P <0.001) (Table 2). In the gingivitis group percentages of sites with BOP were signicantly higher in ME (60.85 18.36) and OV (58.92 25.04) phases than in PM (40.12 20.10) (P <0.001; repeated measures ANOVA), whereas there was no difference between different phases in the healthy group (P >0.05; repeated measures ANOVA) (Table 2). PI scores were similar at ME, OV, and PM phases in each group (P >0.05; repeated measures ANOVA). GCF volume was signicantly higher in the gingivitis group compared to the healthy group
# ** ii Periotron 8000, ProFlow. Diametra, Milan, Italy. Invitrogen, Carlsbad, CA. Diametra. Invitrogen. American Diagnostica, Stamford, CT.

675

Menstrual Cycle and Periodontal Health

Volume 81 Number 5

Table 1.

Description of the Study Population (SD and range)

Gingivitis Characteristic Age (years) Mean (SD) Range Duration of cycles (days) Mean (SD) Range n = 25 29.40 4.967 21 to 39 26.72 1.94 25 to 30 Healthy n = 25 27.20 5.346 22 to 40 27.20 2.137 25 to 30

Figure 1.
Flow diagram of study outline.

(P <0.001; repeated measures ANOVA), whereas it was similar among different phases in both groups (P >0.05; repeated measures ANOVA) (Table 2). Salivary Estrogen and Progesterone Levels Salivary estrogen and progesterone levels of the study groups are shown in Figure 2. Estrogen levels were signicantly elevated at OV and PM compared to ME in both groups (P <0.05; Wilcoxon signed test, Bonferroni analysis). Salivary progesterone levels were also increased from ME to OV and peak on PM (P <0.05; Wilcoxon signed test, Bonferroni analysis). GCF Levels of IL-6, PGE2, PAI-2, and t-PA GCF total amounts of IL-6 were signicantly higher in gingivitis patients compared to healthy subjects in all phases (P = 0.004, P = 0.041, and P = 0.046 for ME,
676

OV, and PM, respectively; Mann-Whitney U test). GCF levels of IL-6 were unchanged in different menstrual phases in both study groups (P >0.05; Friedman test) (Fig. 3). Gingivitis and healthy groups had similar GCF total amounts of PGE2 in all phases (P >0.05; Mann-Whitney U test). GCF total amounts of PGE2 were similar in different menstrual phases in both study groups (P >0.05; Friedman test) (Fig. 4). GCF levels of t-PA were slightly higher in the gingivitis group than the healthy group, but this difference did not reach signicance (P >0.05; Mann-Whitney U test). GCF levels of t-PA were similar among different phases of the menstrual cycle in each group (P >0.05; Friedman test) (Fig. 5). GCF levels of PAI-2 were not signicantly different between gingivitis and healthy groups (P >0.05; Mann-Whitney U test) and also between different phases of the menstrual cycle in each group (P >0.05; Friedman test) (Fig. 6). When the data were expressed as concentration, gingivitis patients had higher GCF concentration of IL-6 compared to healthy subjects in all phases ( P <0.05; Mann-Whitney U test). Gingivitis and healthy groups had similar GCF concentration of PGE2, t-PA, and PAI-2 in all phases. GCF concentrations of IL-6, PGE2, t-PA, and PAI-2 did not show any difference between different phases of the menstrual cycle in each group (P >0.05; Friedman test) (Table 3). There was a positive correlation between the salivary progesterone levels and GCF levels of PGE2 (r = 0.435; P = 0.03), t-PA (r = 0.418; P = 0.038), and PAI-2 (r = 0.453; P = 0.023) in the ME phase. The percentage of sites with BOP was also positively correlated with the GCF levels of t-PA (r = 0.584; P = 0.002), and PAI-2 (r = 0.589; P = 0.002) in the ME phase. DISCUSSION The present study describes changes in periodontal parameters and GCF inammatory markers during

J Periodontol May 2010

zc Becerik, O xaka, Nalbantsoy, et al.

Table 2.

Clinical Parameters for the Whole Mouth According to Study Groups (mean SD)
Gingivitis Parameter BOP (%) PI GCF (ml) ME 60.85 18.36* 3.31 0.74* 1.01 0.46* OV 58.92 25.04* 3.20 0.70* 1.08 0.40* PM 40.12 20.10* 3.11 0.77* 1.12 0.46* ME 10.02 8.59 2.53 1.01 0.75 0.44 Healthy OV 8.19 8.23 2.80 0.65 0.79 0.35 PM 8.95 8.96 2.48 0.65 0.76 0.36

* Signicant difference from the healthy group for the same period (P <0.001, repeated measures ANOVA, Bonferroni analysis). Signicant difference from the other periods in the same group (P <0.001, repeated measures ANOVA, Bonferroni analysis).

Figure 2.
Saliva estrogen levels in healthy (A) and gingivitis (B) groups. Saliva progesterone levels in healthy (C) and gingivitis (D) groups.*Signicant difference from the ME period (P <0.05; Wilcoxon signed test, Bonferroni analysis). Box plots show medians, 25th, and 75th percentiles as boxes; 10th and 90th percentiles as whiskers. Outside values are shown as blue dots.

the menstrual cycles of premenopausal women with gingivitis or healthy periodontium. Gingivitis patients had elevated gingival inammation, which was indicated by percentage of sites with BOP in OV and ME compared to PM phases, whereas it was similar in all phases in the healthy subjects. But the levels of inammatory markers in GCF were similar in different phases of the menstrual cycle in both groups. The menstrual cycle is controlled by the secretion of sex hormones over a 25- to 30-day period and is responsible for continued OV until menopause.25,26 Fluctuations in hormonal levels occur throughout

the menstrual cycle of women. Serum estrogen levels peak at OV and drop immediately, with a second peak during PM. Progesterone levels start increasing slightly at OV, continue increasing, and peak at PM. Luteinizing hormone and follicular-stimulating hormone peak at the height of OV.27 In the present study, salivary estrogen and progesterone levels were analyzed to determine the menstrual phases of women included in the study. The salivary steroids reect the free bioactive fraction of plasma levels of the steroids including estrogen and progesterone.28 Saliva sampling was preferred over collecting serum because it
677

Menstrual Cycle and Periodontal Health

Volume 81 Number 5

Figure 5. Figure 3.
GCF total amounts of IL-6 of the study groups in different periods. *Signicant difference from the healthy group in each period (P <0.05; Mann-Whitney U test). Box plots show medians, 25th, and 75th percentiles as boxes; 10th and 90th percentiles as whiskers. Outside values are shown as blue dots. GCF total amounts of t-PA of the study groups in different periods. Box plots show medians, 25th, and 75th percentiles as boxes; 10th and 90th percentiles as whiskers. Outside values are shown as blue dots.

Figure 6. Figure 4.
GCF total amounts of PGE2 of the study groups in different periods. Box plots show medians, 25th, and 75th percentiles as boxes; 10th and 90th percentiles as whiskers. Outside values are shown as blue dots. GCF total amounts of PAI-2 of the study groups in different periods. Box plots show medians, 25th, and 75th percentiles as boxes; 10th and 90th percentiles as whiskers. Outside values are shown as blue dots.

is a feasible and non-invasive method for determining menstrual cycle proles.29 The salivary estrogen levels were higher in the OV and PM phases compared to the ME phase in this study, which is consistent with serum estrogen levels given in other studies.27,28 The results of our study suggest that the salivary progesterone levels were at peak during PM. These results are in harmony with the results of other studies.27,28 In the present study, percentages of sites with BOP were signicantly higher in OV and ME than in PM phases in the gingivitis group, whereas it was similar in all phases in the healthy group. The earlier studies
678

have suggested that gingival bleeding and swollen gingiva were most pronounced in the presence of gingivitis.8,9 Similar to our ndings, Koreeda et al.30 reported periodic exacerbation of gingival inammation during OV and ME phases in a case report. They found higher numbers of bleeding sites in OV than in ME, whereas it was similar in both phases in the present study. The increase in gingival inammation could be caused by the higher estrogen levels, which peak during OV and just before ME phases. There were no differences in GCF volume among different phases in gingivitis and healthy groups. Our ndings are in e,31 who accordance with Holm-Pedersen and Lo found no change in GCF volume during the menstrual

J Periodontol May 2010

zc Becerik, O xaka, Nalbantsoy, et al.

Table 3.

Concentrations of Biochemical Parameters in GCF From Sampling Sites of the Study Groups in Different Periods of Menstruation
Biochemical Parameter (pg/ml) IL-6 PGE2 tPA PAI-2 Gingivitis ME 0.452* (0.132 to 4.38) 15.67 (0.19 to 115.6) OV 0.346* (0.015 to 1.052) 19.913 (0.31 to 147.38) PM 0.428* (0.137 to 4.431) 20.429 (1.08 to 22.178) ME 0.132 (0.009 to 3.340) 19.528 (0.19 to 833) Healthy OV 0.127 (0.006 to 2.490) 25.173 (6.585 to 96.702) PM 0.110 (0.009 to 2.660) 29.877 (8.191 to 271.98)

121.983 76.454 123.459 103.542 40.129 168.707 (1.921 to 586.207) (1.845 to 419.255) (6.878 to 496.667) (5.854 to 401.389) (2.523 to 442.95) (7.333 to 655.891) 1.857 (0.055 to 7.124) 0.189 (0.049 to 9.964) 1.468 (0.086 to 11.466) 0.658 (0.084 to 21.328) 0.213 (0.092 to 10.951) 2.137 (0.072 to 21.217)

Data are given as median (minimum to maximum). * Signicant difference from the healthy group for the same period (P <0.05; Mann-Whitney U test).

m9 described cycle. In contrast, Lindhe and Attstro an increase in GCF volume during OV in a study on patients with preexisting mild gingival inammation. In a recent study, Bas xer et al.32 found the percentage of BOP and GCF volume was signicantly higher in progesterone peak day of periodontally healthy women compared to OV. This discrepancy between the two studies might be caused by the patient selection criteria and the days of clinical sampling. In the present study, healthy subjects who have less bleeding sites were included compared to other studies.32 Expression of GCF data as total amount per standardized sampling time is a more sensitive way than reporting them as concentration.20 Because collecting a standard amount of GCF is essential to express the results as concentration and also because GCF volume is very small and exhibits wide variations, expressing GCF data as total activity is a more appropriate way, rather than reporting them as concentration.20,33 In the present study, we collected GCF samples for the same length of time and reported the data as total amount per sample and concentration.18,34 Our ndings show that the total amount of IL-6, PGE2, t-PA, and PAI-2 levels in GCF samples were decisive enough to reveal the difference among menstrual phases. Pregnancy was found to be associated with signicant increase in GCF levels of IL-6.35 It was suggested that the state of pregnancy, perhaps by altering the hormonal levels and the immune response, might result in the local increase in the periodontal inammatory response to the preexistent ora.35 Cohen-Solal et al.36 observed that peripheral blood monocytes from postmenopausal women had elevated IL-1, tu-

mor necrosis factor-a, and IL-6, which was decreased by estrogen therapy. In an in vitro study, IL-6 production by gingival broblasts was shown to be inhibited by progesterone in a dose-dependent manner.13 In the present study, GCF levels of IL-6 were signicantly higher in the gingivitis group than the healthy group for all menstrual phases, but were similar among different phases of ME in each group. Although other studies have shown associations among sex hormones and IL-6 levels, our data suggest that hormonal changes occurring during the menstrual cycle did not alter the GCF levels of IL-6. On the other hand, hormonal inuences should not be eliminated from consideration because the percentage of sites with BOP was reduced during PM compared to other phases. PGE2 has been implicated as a key inammatory mediator in periodontal disease.37 There are conicting data in GCF levels of PGE2 in gingivitis. Some authors showed signicantly increased PGE2 concentrations in gingivitis,38 whereas others found no or only tentative elevations in gingivitis sites compared to healthy sites.39 In the present study, GCF levels of PGE2 were slightly higher in the gingivitis group, although statistically not signicant. Miyagi et al.40 studied the effect of sex hormones on the function of human monocytes and demonstrated that PGE2 production was enhanced by progesterone. It was also shown that estrogen reduced PGE2 production at 0.4 ng/ml, but showed enhancement at 20 ng/ml.40 In the present study, PGE2 levels are similar in different phases of ME in gingivitis and healthy patients; on the other hand, the salivary progesterone levels and GCF PGE2 levels are positively correlated in the ME phase, which might indicate the limited effects of progesterone on GCF levels of PGE2.
679

Menstrual Cycle and Periodontal Health

Volume 81 Number 5

The plasminogen activating system in GCF has previously been characterized and t-PA and PAI-2 were found in high concentrations and thought to play a signicant role in periodontal tissue destruction.41 In the present study, t-PA levels are slightly higher in the gingivitis group than the healthy group, but this difference was not signicant. Production of PAI-2 in cultured human peripheral blood monocytes was shown to be reduced by estrogen and progesterone and no effect of these hormones was detected on t-PA production.42 Kinnby et al.15 showed a negative correlation between blood progesterone levels and GCF levels of PAI-2 of pregnant woman. Although possible interactions of sex hormones and the brinolytic system have been addressed previously,15,42 GCF levels of t-PA and PAI-2 were found to be similar in different phases of the menstrual cycle. However, a positive correlation was present between the saliva progesterone levels and GCF levels of t-PA and PAI-2 in the ME phase in the present study. CONCLUSIONS The present study suggests that changes in the sex steroid hormones during menstrual cycles might have a limited transient effect on gingival inammation. On the other hand, increases in clinical signs of periodontal inammation were observed during ME and OV phases of the menstrual cycle in the presence of gingival inammation. Our data led us to speculate that studies concerned with the changes in periodontal parameters should consider the menstrual cycle when including women in their study. Within the limitation of the present study we can recommend that researchers record clinical parameters and sampling from women in the same time of their menstrual cycles, preferably between ME and OV in which minimum hormonal changes occur. Further studies to investigate other possible cellular pathways should be undertaken to explore the effects of the menstrual cycle on markers of gingival inammation. ACKNOWLEDGMENTS This study was funded by a research foundation of Ege University, Izmir, Turkey (project no. 08DIS-19). The authors report no conicts of interest related to this study. REFERENCES
1. Parkar MH, Newman HN, Olsen I. Polymerase chain reaction analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissue. Arch Oral Biol 1996;41:979-983. 2. Gornstein RA, Lapp CA, Bustos-Valdes SM, Zamorano P. Androgens modulate interleukin-6 production by gingival broblasts in vitro. J Periodontol 1999;70: 604-609. 680

3. Kawahara K, Shimazu A. Expression and intracellular localization of progesterone receptors in cultured human gingival broblasts. J Periodontal Res 2003; 38:242-246. 4. Mariotti A. Sex steroid hormones and cell dynamics in the periodontium. Crit Rev Oral Biol Med 1994;5:2753. 5. Raber-Durlacher JE, Van Steenbergen TJ, Van der Velden U, De Graaf J, Abraham-Inpijn L. Experimental gingivitis during pregnancy and post-partum: Clinical, endocrinological, and microbiological aspects. J Clin Periodontol 1994;21:549-558. 6. Tilakaratne A, Soory M, Ranasinghe AW, Corea SM, Ekanayake SL, de Silva M. Periodontal disease status during pregnancy and 3 months post-partum, in a rural population of Sri-Lankan women. J Clin Periodontol 2000;27:787-792. 7. Reinhardt RA, Payne JB, Maze CA, Patil KD, Gallagher SJ, Mattson JS. Inuence of estrogen and osteopenia/osteoporosis on clinical periodontitis in postmenopausal women. J Periodontol 1999;70:823828. 8. Muhlemann HR. Gingivitis intermenstrualis (in German). Schweiz Mschr Zahnhelik 1948;58:865-885. 9. Lindhe J, Attstrom R. Gingival exudation during the menstrual cycle. J Periodontal Res 1967;2:194-198. 10. Machtei EE, Mahler D, Sanduri H, Peled M. The effect of menstrual cycle on periodontal health. J Periodontol 2004;75:408-412. e H. The gingival index, plaque index and the 11. Lo retention index system. J Periodontol 1967;38:610616. se T. Effect of the menstrual 12. Ozc xaka O, Bic xakc xi N, Ko cycle on pain experience associated with periodontal therapy: Randomized, pilot study. J Clin Periodontol 2005;32:1170-1174. 13. Lapp CA, Thomas ME, Lewis JB. Modulation by progesterone of interleukin-6 production by gingival broblasts. J Periodontol 1995;66:279-284. 14. Offenbacher S, Jared HL, OReilly PG, et al. Potential pathogenic mechanisms of periodontitis associated pregnancy complications. Ann Periodontol 1998;3: 233-250. 15. Kinnby B, Matsson L, Astedt BJ. Aggravation of gingival inammatory symptoms during pregnancy associated with the concentration of plasminogen activator inhibitor type 2 (PAI-2) in gingival uid. J Periodontal Res 1996;31:271-277. 16. Fischer CC, Persson RE, Persson GR. Inuence of the menstrual cycle on the oral microbial ora in women: A case-control study including men as control subjects. J Periodontol 2008;79:1966-1973. 17. ICH (issued as CPMP/ICH/135/95). Harmonised tripartite guideline for good clinical practice. Available at: http://www.ich.org/LOB/media/MEDIA482.pdf. 18. Armitage GC. Development of a classication system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-7. 19. Quigley GA, Hein JW. Comparative cleansing efciency of manual and power brushing. J Am Dent Assoc 1962;65:26-29. 20. Lamster IB, Oshrain RL, Gordon JM. Enzyme activity in human gingival crevicular uid: Considerations in data reporting based on analysis of individual crevicular sites. J Clin Periodontol 1986;13:799-804. 21. Cimasoni G. Method of Collection. Crevicular Fluid Updated. Basel (Switzerland): Karger; 1983:29-36.

J Periodontol May 2010

zc Becerik, O xaka, Nalbantsoy, et al.

22. Reinhardt RA, Masada MP, Kaldahl WB, et al. Gingival uid IL-1 and IL-6 levels in refractory periodontitis. J Clin Periodontol 1993;20:225-231. 23. Atilla G, Ku xu tu kc ler N. Crevicular uid interleukin1beta, tumor necrosis factor-alpha, and interleukin-6 levels in renal transplant patients receiving cyclosporine A. J Periodontol 1998;69:784-790. 24. Neter J, Kutner MH, Nachtseim CJ, Wasserman W. Applied Linear Statistical Models, 4th ed. Irwin; Chicago, IL; 1996:736-738. 25. Ganong WF. Review of Medical Physiology. Norwalk: Appleton & Lange; 1995:379-417. 26. McCartney CR, Gingrich MB, Hu Y, Evans WS, Marshall JC. Hypothalamic regulation of cyclic ovulation: Evidence that the increase in gonadotropinreleasing hormone pulse frequency during the follicular phase reects the gradual loss of the restraining effects of progesterone. J Clin Endocrinol Metab 2002;87: 2194-2200. 27. Palter FP, Olive DL. Reproductive physiology. In: Berek JS, ed. Novaks Gynecology. Hong Kong: Williams and Wilkins; 1996:158-163. 28. Choe JK, Khan-Dawood FS, Dawood MY. Progesterone and estradiol in the saliva and plasma during the menstrual cycle. Am J Obstet Gynecol 1983;147:557-562. D, Skoknova M, Hodosy J, Putz Z, 29. Celec P, Ostanikova dela M. Salivary sex hormones during the menstrual Ku cycle. Endocr J 2009;56:521-523. 30. Koreeda N, Iwano Y, Kishida M, et al. Periodic exacerbation of gingival inammation during the menstrual cycle. J Oral Sci 2005;47:159-164. e H. Flow of gingival exudate as 31. Holm-Pedersen P, Lo related to menstruation and pregnancy. J Periodontal Res 1967;2:13-20. 32. Baser U, Cekici A, Tanrikulu-Kucuk S, Kantarci A, Ademoglu E, Yalcin F. Gingival inammation and interleukin-1 beta and tumor necrosis factor-alpha levels in gingival crevicular uid during the menstrual cycle. J Periodontol 2009;80:1983-1990. 33. Hanioka T, Takaya K, Matsumori Y, Matsuse R, Shizukuishi S. Relationship of the substance P to indicators of host response in human gingival crevicular uid. J Clin Periodontol 2000;27:262-266. 34. Emingil G, Atilla G, Huseyinov A. Gingival crevicular uid monocyte chemoattractant protein-1 and RANTES

35.

36.

37. 38.

39.

40.

41.

42.

levels in patients with generalized aggressive periodontitis. J Clin Periodontol 2004;31:829-834. Offenbacher S, Lin D, Strauss R, et al. Effects of periodontal therapy during pregnancy on periodontal status, biologic parameters, and pregnancy outcomes: A pilot study. J Periodontol 2006;77:2011-2024. Cohen-Solal ME, Graulet AM, Denne MA, Gueris J, Baylink D, de Vernejoul MC. Peripheral monocyte culture supernatants of menopausal women can induce bone resorption: Involvement of cytokines. J Clin Endocrinol Metab 1993;77:1648-1653. Seymour GJ, Gemmell E. Cytokines in periodontal disease: Where to from here? Acta Odontol Scand 2001;59:167-173. Ohm K, Albers HK, Lisboa BP. Measurement of eight prostaglandins in human gingival and periodontal disease using high pressure liquid chromatography and radioimmunoassay. J Periodontal Res 1984;19: 501-511. Nakashima K, Roehrich N, Cimasoni G. Osteocalcin, prostaglandin E2 and alkaline phosphatase in gingival crevicular uid: Their relations to periodontal status. J Clin Periodontol 1994;21:327-333. Miyagi M, Morishita M, Iwamoto Y. Effects of sex hormones on production of prostaglandin E2 by human peripheral monocytes. J Periodontol 1993;64: 1075-1078. Yin X, Bunn CL, Bartold PM. Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) in gingival crevicular uid from healthy, gingivitis and periodontitis patients. J Clin Periodontol 2000;27:149-156. Kinnby B, Astedt B, Casslen B. Reduction of PAI-2 production in cultured human peripheral blood monocytes by estradiol and progesterone: No effect on t-PA, u-PA and PAI-1. Fibrinolysis and Proteolysis 1995;9: 152-156.

Correspondence: Sema Becerik, Department of Periodontology, School of Dentistry, Ege University, Bornova35100, Izmir, Turkey. Fax: 90-232-3880325; e-mail: sema.cinar@ege.edu.tr. Submitted October 21, 2009; accepted for publication January 19, 2010.

681