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Neuroendocrinology Letters Volume
Associations between brain-derived neurotrophic factor and estradiol in women's saliva Chisa Matsuki 1#, Masahiro To 1#, Yusuke Kondo 1,2,
Hiroki Sugiyama 1, Yuko Yamamoto 1, Tomoko Shimizu 1,
Yohei Kamata 3, Juri Saruta 1, Keiichi Tsukinoki 1
1 Department of Environmental Pathology, Kanagawa Dental University Graduate School of Dentistry, 2 Departments of Pathology, Tokai University School of Medicine, Isehara, Japan3 Department of Clinical Oral Medicine, Kanagawa Dental University Graduate School of Dentistry, # These authors contributed equally to this work. Correspondence to: Keiichi Tsukinoki, DDS., PhD.
Department of Environmental Pathology, Kanagawa Dental University Graduate School of Dentistry82, Inaoka, Yokosuka, Kanagawa, Japan.
tel/fax: +81 46 822 8866; e-mail: [email protected] Submitted: 2014-06-05 Accepted: 2014-06-12 Published online: 2014-06-27 Key words: BDNF; E2; fertile women; menstruation; saliva
Neuroendocrinol Lett 2014; 35(3):236–241 PMID: 24977975 NEL350314A06 2014 Neuroendocrinology Letters • www.nel.edu
OBJECTIVE: The aim of this study was to assess the relationship between brain-
derived neurotrophic factor (BDNF) and sex hormones (estradiol [E2] and pro-
gesterone), using saliva samples obtained from healthy women.
METHODS: Forty female dental hygienist students were divided into groups
according to being in the follicular phase or luteal phase. Saliva BDNF, E2, and
progesterone levels were measured using a sandwich ELISA system. The correla- tion between these factors was analyzed using Spearman's index, and fluctuations of these levels in the whole menstrual cycle were investigated classifying the subjects by every 4 days according to the phase of their menstrual cycle.
RESULTS: Saliva BDNF variations strongly correlated with saliva E2 levels in
the follicular phase (r=0.721, p=0.000) and luteal phase (r=0.770, p=0.000). The correlation coefficient showing the relationship between progesterone and BDNF levels in the luteal phase (r=0.371, p=0.157) was lower than that in the follicular phase (r=0.631, p=0.001). Moreover, the fluctuation of BDNF levels in the men- strual cycle followed a similar pattern to that of E2.
CONCLUSIONS: We found that saliva BDNF and E2 levels were closely related
in healthy young women. In particular, for first time, that correlation was inves-tigated throughout the menstrual cycle. Monitoring of saliva BDNF may yield insight into women's reproductive and mental health.
To cite this article: Neuroendocrinol Lett 2014; 35(3):236–241
BDNF and E2 in saliva ening (Ahn et al. 2011). In addition, using ELISA and western blotting, we have demonstrated that human Brain-derived neurotrophic factor (BDNF) is a member saliva contains BDNF. Saliva BDNF concentrations in of the neurotrophin family (NT), which includes nerve males tended to be lower than those in females (Saruta growth factor (NGF), NT-3, -4/5, -6, and -7. BDNF et al. 2012), resembling the profile of plasma BDNF is abundantly expressed in the central and peripheral (Lommatzch et al. 2005). To date, there are no studies nervous system, and, in particular, in the hippocam- evaluating the relationship between BDNF and sex hor- pus (Saruta et al. 2010). Hippocampal BDNF plays an mones in saliva. important functional role in learning and memory (Hall In recent years, analysis of saliva has become an et al. 2000). Although it was originally identified in the attractive sampling approach. The collection of saliva is nervous system, BDNF is now known to be expressed easier than venipuncture and can readily be repeated at in a wide variety of non-neural cells, such as the lachry- frequent intervals (Ahn et al. 2011). These advantages mal glands (Ghinelli et al. 2003), heart (Scarisbrick et al. allow for a better assessment of the diurnal rhythm- 1993), and retina (Seki et al. 2003) in rodents, as well as mediated secretion of sex hormones and related factors, in the testis (Muller et al. 2006), lung (Ricci et al. 2004), such as BDNF (Ahn et al. 2011). Thus, saliva testing and endothelial cells (Nakahashi et al. 2000) in humans. may contribute to the monitoring of women's health. We previously reported that BDNF expression in the Consequently, the aim of the present study was to rat submandibular gland is up-regulated by chronic assess correlations between BDNF and E2 as well as stress, and localization of BDNF mRNA and protein progesterone in the saliva of healthy female subjects.
was observed in salivary acini cells in humans (Saruta
et al. 2012). In recent years, immunohistochemical MATERIALS AND METHODS
and in vitro studies have demonstrated BDNF expres-sion in reproductive organs, including the ovaries Subjects(Kawamura et al. 2005), in particular in the granular The study was approved by the Ethics Committee of cells (Domingue et al. 2011), and the endometrium the Kanagawa Dental University, graduate school of (Krizsan-Agbas et al. 2003). Moreover, it was found Dentistry. Forty females, volunteer dental hygienist that the ovaries specifically expressed a BDNF receptor, students, were included in the present study. They were TrkB (Kawamura et al. 2009). These reports suggested divided into the following two groups: fertile women in that there is a close association between BDNF and the the follicular phase (n=24), aged 21.0±3.5 y (mean±SD, reproductive system in women.
range 19–32 y), with a body mass index (BMI) 19.4±1.5 Moreover, changes in plasma BDNF levels have been (range 16.2–21.9), and fertile women in the luteal phase reported to occur in women throughout the menstrual (n=16), aged 23.0±3.9 (range 19–31 y), with a BMI cycle: studies have shown higher levels of BDNF during 19.6±1.6 (range 16.8–22.0). Each group was assessed the luteal phase than in the follicular phase (Cubeddu both in the follicular phase (days 0–14) and in the luteal et al. 2011). Hormone replacement therapy (HRT) is phase (days 15–28) of the menstrual cycle. Menstrual able to increase BDNF levels from menopausal levels phase was determined based on self-reporting.
to approximately those observed in the follicular phase Prior to enrollment, participating subjects gave (Begliuomini et al. 2007). Furthermore, estradiol (E2) their written informed consent and they were asked to is induced by BDNF in neural cells of the rat brain, answer a questionnaire regarding age, weight, height, including those in the hypothalamus and hippocampus and menstrual cycles. Subjects with oral infective dis- (Scharfman & MacLusky 2008). E2 plays an important eases, current illness, under medication, or with aller- role in the modification of intra-tissue (Scharfman & gies, or a family history, or overweight (BMI ≥25.0) MacLusky 2008) and plasma levels of BDNF (Begliuo- were excluded from this study. The mean menstrual mini et al. 2007). Interestingly, the BDNF profile of pre- cycles of all subjects lasted 27.9±4.8 days. menstrual syndrome is characterized by a low plasma level in the luteal phase, despite increases in E Saliva collection (Cubeddu et al. 2011). Measurement of plasma BDNF For 5 min prior to sample collection, subjects did not levels during the menstrual cycle may have important consume any food or drink, or brush their teeth. All implications in women's health and reproduction.
samples were collected between 1 and 4 pm, within a The sensitivity and precision of the saliva E2 assay 10-min period, to minimize any possible effect of diur- are comparable to those of assays of serum E2 for assess- nal variation. All saliva samples were collected using ing changes in hormone levels (Lu et al. 1999). The the Salivette (Sarstedt Co. Ltd., Nümbrecht, Germany) measurement of E2 and progesterone concentrations in absorbent method according to the manufacturer's saliva has become increasingly common over the past instructions. Briefly, participants were instructed place several years (Dame et al. 1989). The measurement of a cotton roll on the floor of the mouth for 5 min, or saliva E2 and progesterone levels during the post-awak- until the cotton was fully saturated with saliva, and then ening period could provide information regarding the spit the cotton into the Salivette tube, which was sub- hypothalamus-pituitary-ovary axis response to awak- sequently stored on ice. The tubes were centrifuged at Neuroendocrinology Letters Vol. 35 No. 3 2014 • Article available online: http://node.nel.edu
Chisa Matsuki, et al. 626 × g for 15 min at 4 °C and were then stored at –80 °C Tab. 1. Differences between follicular phase and luteal phase.
until required for use. Upon thawing, the samples Luteal phase
were centrifuged once more to ensure complete debris removal, using the same conditions.
BDNF assay Human BDNF was detected by sandwich ELISA according to the manufacturer's instructions (CYT306; Millipore Corp, Bedford, MA, USA). All assays were performed in F-bottom 96-well plates (Nunc, Wies-baden, Germany). Tertiary antibodies were conjugated to horseradish peroxidase and color was developed with tetramethylbenzidine and measured at 450/570 nm. The BDNF content was quantified against a standard curve; the detection limit of <4 pg/mL was calibrated with known amounts of human recombinant BDNF. All samples were tested twice, and the mean was calculated. Cross-reactivity to related NTs (NGF, NT-3, and NT-4) was less than 3%. Intra- and inter-assay coefficients of variation were 3.7% and 8.5%, respectively. Concentra- tions were expressed as pg/mL (Goto et al. 2012).
E2 and progesterone assays Progesterone (pg/ml) The saliva ELISA kit for measuring free 17β-estradiol and progesterone were used according to the manufac-turer's instructions (IBL, Hamburg, Germany) (Rijn et al. 2011). The lowest detectable levels of E Luteal phase
gesterone that could be distinguished from the zero standard were 0.4 pg/mL for E2 and 3.8 pg/mL for pro- gesterone, at the 95% confidence limit. The intra- and inter-assay coefficients of variation were 2.6% and 2.1% for E2, and 4.7% and 5.3% for progesterone, respectively.
Parameters and statistical analysis r = 0.543p = 0.030 ELISA data are reported as a mean ± SD. Statistical analysis was carried out using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). We used the Mann– Whitney test to evaluate differences between follicular Progesterone (pg/ml) and luteal phase subjects for each parameter. A correla-tion index (Spearman's index) was computed in order to Fig. 1. Scatterplot showing the relationship between saliva E2 levels
investigate the relationship among saliva BDNF levels and saliva progesterone levels of subjects in the follicular phase and those in the luteal phase.
2 as well as progesterone levels. The p-values of 0.05 or less were accepted as significant.
Figure 1. Saliva BDNF variations strongly correlated with saliva E2 changes in the follicular phase and in Differences among parameters between the luteal phase (Figure 2). Although saliva BDNF and follicular phase and luteal phase subjects progesterone levels correlated in individuals in the fol- Data from subjects in the follicular phase or luteal licular phase, no correlations were noted in individuals phase, as determined by ELISA, are compared in in the luteal phase, as shown in Figure 3. Table  1. Although these levels tended to be higher in individuals in the luteal phase than those of subjects in Variation of saliva BDNF, E2, and the follicular phase, the differences were not significant.
progesterone during menstrual cycleBy classifying subjects by every 4 days according to the Correlation index phase of their menstrual cycle, we investigated the vari- There were positive correlations between saliva E2 and ations in saliva BDNF and sex hormone levels during progesterone levels for subjects in the follicular phase the whole of the menstrual cycle (Figure 4). BDNF and for individuals in the luteal phase, represented in levels were found to show two peaks: one lower peak Copyright 2014 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu
BDNF and E2 in saliva r = 0.721p Progesterone (pg/ml) Luteal phase
Luteal phase
Progesterone (pg/ml) Fig. 2. Scatterplot showing the relationship between saliva BDNF
Fig. 3. Scatterplot showing the relationship between saliva BDNF
levels and saliva E2 levels in subjects in the follicular phase and levels and saliva progesterone levels of subjects in the follicular those in the luteal phase.
phase and those in the luteal phase.
10.007±8.154). Moreover, the levels of progesterone peaked at the 13th–20th day (13th–16th day: 98.591±52.624, 17th–20th day: F (pg/ml)
Saliva E2 and progesterone con- centrations tended to be higher in subjects in the luteal phase than in those in the follicular phase, but these differences, as determined using an ELISA system, were not statistically significant. In addi-tion, there were significant correla- Fig. 4. Changes in saliva BDNF, E2, and progesterone levels during the menstrual cycle in
tions between E2 and progesterone young healthy women. Average levels for every 4 days of the cycle are presented.
levels in the saliva of both groups of subjects. Lu et al. reported that at the 13th–16th day (14.916±7.702) and one tall peak the sensitivity and precision of saliva E2 and proges- at the 21st–24th day (22.309±29.990) during a normal terone assays were comparable with assays of blood menstrual cycle. Similar to BDNF, E2 levels also showed E2 and progesterone for assessing changes in hormone two peaks (13th–16th day: 8.858±4.863; 21st–24th day: levels (Lu et al. 1999). It is well-known that blood and Neuroendocrinology Letters Vol. 35 No. 3 2014 • Article available online: http://node.nel.edu
Chisa Matsuki, et al. saliva concentrations of E2 and progesterone are related the quality of salivary gland function (Laine & Leimola- (Celec et al. 2009; Dame et al. 1989). Since sex hormone Virtanen 1996). It is thought that E2 plays crucial roles levels, including those of E2 and progesterone, are high- in the salivary gland. It is possible that saliva BDNF may est in blood during the luteal phase (Cubeddu et al. be induced and/or released from acini cells, mediated by 2011; Okifji & Turk 2006), our results can be attributed E2–ER interaction, and is not only derived from blood. to the increase of E2 and progesterone concentrations In addition, the saliva BDNF levels of subjects in in blood. If the number of subjects had been sufficient, the follicular phase tended to be lower than those in the difference between levels of individuals in the fol- the luteal phase; thus, it was reasonable that a decrease licular and luteal phase may have been significant.
in saliva BDNF was induced by a decrease in blood We found that there was a strong linear correlation E2 levels. Thus, measurement of saliva BDNF may be between BDNF and E2 levels in saliva. Experimen- useful to determine the effects of HRT as a marker of tal studies on animal models showed that estrogen increased blood BDNF.
treatment of ovariectomized rats improved learn- The relationship between progesterone and BDNF ing acquisition and memory performance (O'Neal levels in subjects in the follicular phase was significant, et al. 1996). Indeed, E2 increased the dendritic spine but this correlation was non-significant in the luteal number though a BDNF-dependent mechanism (Segal phase. In recent years, it has been shown that proges- & Murphy 2001). In humans, it has been reported that terone attenuated the E2-mediated increase in the levels there is a positive relationship between plasma BDNF of BDNF and the BDNF receptor, TrkB, in cultured hip- and E2 levels during the menstrual cycle, based on a pocampal slices (Aguirre & Baudry 2009). Progesterone study of five healthy women, with a correlation index probably suppresses BDNF expression (Bimonte-Nel- ranging from 0.6144 to 0.8542 (Begliuomini et al. son et al. 2004). However, the interplay between BDNF 2007). Interestingly, BDNF levels were increased by and progesterone remains controversial (Cubeddu et HRT (including E2) in subjects showing a low plasma al. 2011). Our results also suggest that BDNF levels are BDNF level, such as amenorrhoeic women and post- strongly correlated with E2 levels, rather than with pro- menopausal women (Begliuomini et al. 2007). HRT gesterone levels in saliva, which supports the previous also improved cognitive functions in postmenopausal report by Begliuomini et al. (2007). women (Genazzani et al. 2005; Prelevic et al. 2005). Several studies have shown that there are associa- These previous studies and our results established that tions between a decrease in blood BDNF and neurolog- estrogens and BDNF were reciprocally and positively ical diseases, such as depression, Alzheimer's disease, involved in various situations. and Parkinson's disease (Connor et al. 1997; Parain et al. Moreover, when we investigated the fluctuations 1999; Tsukinoki & Saruta 2012). In addition, individu- in the levels of BDNF and sex hormones during the als with premenstrual syndrome and mood disorder menstrual cycle, the fluctuation pattern of BDNF was in the postnatal phase have been reported to demon- similar to that of E2. Sex hormone levels in blood differ strate lower blood BDNF levels than healthy women in terms of their peaks during the menstrual cycle: E2 is (Cubeddu et al. 2011; Lommatzsch et al. 2006). More- elevated in the pre-ovulatory and middle luteal phase, over, it has been shown that a long-term deficiency of whereas progesterone is increased during the middle BDNF, such as that observed during menopause, may luteal phase (Begliuomini et al. 2007). Interestingly, in pose a higher risk for the development of mental dis- the present study, reflecting the changes in blood levels, orders (Begliuomini et al. 2007). BDNF seems to be saliva E2 levels showed two peaks (13th–16th and 21st– strongly involved in neurological diseases related to the 24th day), although saliva progesterone levels differed reproductive cycle of women. It would be interesting to from the blood levels, because it was not examined con- determine the saliva BDNF level profiles in various dis- secutively throughout the whole menstrual cycle in all eases known to be associated with a low blood BDNF subjects. Thus, our findings indicated the probability that changes in saliva BDNF levels are associated with those of E2 during the menstrual cycle. This suggests that measurement of saliva BDNF may be useful in monitoring reproductive health in women.
We found that saliva BDNF and E2 levels were closely On the other hand, the source of saliva BDNF has related in healthy young women. In particular, for first never been investigated in detail, although saliva clearly time, this relationship was investigated throughout the contains BDNF protein, as determined by western menstrual cycle, including the follicular phase and the blotting and ELISA analysis. Saruta et al. reported that luteal phase. Since blood BDNF levels may be modi- BDNF mRNA and protein are expressed in the acini fied during the menstrual cycle, depending on E2 levels, cells of the human salivary gland (Saruta et al. 2012). In measurement of the saliva BDNF–E2 balance may be addition, human salivary gland tissue expresses estro- useful for monitoring reproductive function. Examin- gen receptor (ER) mRNA, as determined by RT-PCR ing saliva BDNF levels may also provide useful infor- (Leimola-Virtanen et al. 2000), but does not express E2. mation for assessing women's mental health, although Additionally, HRT can improve both the quantity and further studies are needed to support this proposal. Copyright 2014 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu
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Neuroendocrinology Letters Vol. 35 No. 3 2014 • Article available online: http://node.nel.edu

Source: http://www.graduate.kdu.ac.jp/dessertation/pdf/h2509/%E7%94%B2%E7%AC%AC444%E5%8F%B7_Associations%20Between%20Brain-Derived%20Neurotrophic%20Factor%20and%20Estradiol%20in%20Women%E2%80%99s%20Saliva.pdf