Dental pulp pain in young and postmenopausal women: a pilot study

Abstract Introduction This pilot study was aimed to compare pulpal pain provoked by electrical and thermal (cold) stimuli in healthy young women during various phases of menstrual cycle and postmenopausal women. Material and methods The study included 20 regularly menstruating healthy women and 20 postmenopausal women. Electrical (electrical pulp tester) and cold (refrigerant spray) stimuli were performed on mandibular central incisors, twice in regularly menstruating (menstrual and luteal phases) and once in postmenopausal women. Results were expressed as pain threshold values for electrical pulp stimulation (0-80 units) and pain intensity scores (visual numeric scale, from 0 to 10) for cold stimulation. Results In young women, higher pain electrical threshold (p=0.484) and pain sensitivity score (p=0.015) were observed in luteal in comparison to menstrual phase. In postmenopausal women, electrical pain threshold was significantly higher while pain intensity score was significantly lower than in young women, regardless of the menstrual phase and painful stimuli. Conclusion Lower responsiveness to dental pulp pain was obtained in young women in luteal phase and postmenopausal women.


INTRODUCTION
A large number of clinical and experimental studies on sex differences in various painful conditions have shown their higher prevalence in women [1,2]. Migraine headache, tension-type headache, fibromyalgia, rheumatoid arthritis, osteoarthritis, irritable bowel syndrome, and temporomandibular disorders (TMD) are more common in women than in man [1,[3][4][5][6][7][8]. Furthermore, women experience higher pain intensity, pain frequency and longer duration of pain [1,9]. Women are also more likely to consult physicians because of various pain conditions [1]. Sex differences in pain response can be ascribed to social, neurophysiological, genetic, and immunological influences, as well as to the effects of gonadal hormones. A large number of findings suggest gonadal hormones may affect pain perception in women [1,10]. In the lifetime hormonal levels modulate significant changes in clinical pain conditions in women. Namely, TMD and migraine headache usually appear after puberty and peak during the reproductive period, in the 20-45-age range [10]. Pain intensity in TMD, migraine headache, and other painful clinical conditions increases towards the end of the menstrual cycle and during the first days of menstruation [11,12]. These findings suggest that decrease in estrogen and progesterone levels during perimenstrual phase cause onset and increase of pain. Hormonal milieu during early luteal phase (decrease in estrogen concentration with rise in progesterone concentration) has also been related to increased migraine pain intensity. Similarly, TMD pain level can increase during ovulation, the phase characterized by the rapid change in estrogen level [11]. Conversely, pain fluctuation during menstrual cycle has not been observed in patients with fibromyalgia [12].
Changes in pain conditions could be expected after the reproductive period due to both ageing process and other medical causes. Studies have shown that in postmenopausal period the prevalence of TMD and migrenous headaches decreases [10], but prevalence of other pain syndromes such as fibromyalgia and osteoarthritis increases [10,13]. These findings suggest that changes in pain conditions in postmenopausal period depend on the pain modality and characteristics [14]. Unlike clinical pain conditions, it is not yet clear if pain sensitivity on various acute stimuli in healthy women (experimental pain) depends on hormonal status. Namely, several authors have indicated increased pain sensitivity on various stimuli in healthy women during various menstrual phases [15,16], while others have shown an absence in pain response variability during menstrual cycle [17,18]. Hormones affect numerous sites for pain sensitivity modulation: primary afferent fibres, spinal cord, brainstem, and cerebrum [19]. As gonadal hormone receptors have been identified throughout the nervous system [20] it is possible that these hormones affect numerous sites to modulate pain. It has been demonstrated that gonadal hormones interact with nociceptive processes at multiple levels of the peripheral and central nervous system [12,21]. However, the exact role of gonadal hormones in nociceptive modulation is rather complex and yet not fully understood [22].
Fluctuation in ovarian hormones during menstrual cycle and in various life periods may affect physiological and pathological responses of dental pulp. Namely, estrogen receptors have been detected in the pulp tissue in women in reproductive age, as well as in menopausal women, regardless of age [23]. Also, it has been proven that lower estrogen level during menstrual phase in regularly menstruating and menopausal women is related to lower pulp blood flow [24]. However, according to Tófoli et al. [25], who investigated anaesthetic efficacy and pain induced by dental anaesthesia in regards to menstrual phase, no association between menstrual cycle and investigated parameters of dental anaesthesia has been found, both in healthy regularly menstruating women and women using contraceptives. The influence of hormonal changes during menstrual cycle and postmenopausal period on the pulp pain sensitivity has not yet been investigated. Better insight into these interactions might be of great importance to improve the treatment efficacy.
The aim of this pilot study was to compare the pulp pain response provoked by electrical and thermal (cold) stimuli in young healthy women during various phases of menstrual cycle and postmenopausal women.

MATERIAL AND METHODS
Forty healthy and pain-free women were selected from patients attending yearly routine check-ups, or subjects accompanying the patients referred to the Specialist Clinic of Faculty of Medicine, University of East Sarajevo, Republic of Srpska, Bosnia and Herzegovina. The subjects were divided into the two groups: the group of regularly menstruating women (menstrual cycle defined as varying between 26 and 28 days), aged 20 to 40 years (mean age 32.8±5.9) (nS=20), and the group of postmenopausal women (at least 2 years after menopause), aged 50 to 65 years (mean age 59.6±4.4) (n=20). The inclusion criteria were: the presence of vital mandibular central incisor free of caries, restorations, crowns or veneers and tooth wear, without pulp and periodontal disease. Further criteria were no known medical condition, pregnancy, intake of oral contraceptives, any drugs on a regular basis or any medications that could alter pain perception for at least 24 hours proceeding each test session. Furthermore, there was no recent history of orthodontic treatment, periodontal treatment or trauma. Only one tooth per patient was included.
Each regularly menstruating woman was tested twice, once during the menstrual phase (1-5 days of menstrual cycle) and once during the luteal phase (days 17-23). Postmenopausal women were tested once. Dental pulp sensitivity was tested by electrical and thermal (cold) stimuli. Before test the tooth surface was isolated and dried by cotton rolls. The electric test was done using digital pulp tester (Analytical Technologies, Redmond, WA) with reads from 0-80 units. As a conducting medium, toothpaste was applied lightly to the electrode. The probe was placed on the incisal third of the buccal surface. Participants were asked to raise their hand on first detection of painful sensation. The lowest current intensity that provoked pulp response was considered as pain threshold.
Cold pulpal testing was done by a refrigerant spray (Endo-Frost -50C; Coltene/Whaledent, Altstatten, Switzerland), applied to the incisal third of the buccal surface of the tooth using a cotton pellet for 15 seconds, or until participant indicated a response. Participant was asked to rate her pain on a 0 to 10 numeric rating scale (NRS), with 0 representing no pain and 10 indicating the worst pain the subject has ever experienced. The test was repeated once if there was no response. A 0 value was marked if no response was obtained. A recovery period of at least 2 minutes was allowed after each pulp test.
Data were analyzed using SPSS 19.0 (SPSS, IBM Corp., Armonk, NY). The means and SDs for parametric data, and frequencies for nonparametric data were calculated. To compare pain sensitivity induced by electrical stimulus between the various phases of menstrual cycle and between the two groups of participants Student's t-test for unpaired sample and independent t-test were used, respectively. To compare difference in prevalence and intensity of pain induced by cold stimulus between two phases of menstrual cycle McNemar and Wilcoxon signed-rank tests were used, while intergroup analysis was performed by Chi-square and Mann-Whitney tests. The level of significance was assessed at p<0.05. Table 1 shows the frequency of pulp response to the applied stimuli. The reaction of the pulp to electrical stimulus was observed in all subjects and in all periods of measurement. On the other hand, the number of teeth that were responsive to cold stimulation was lower in postmenopausal women (65%) compared to women in menstrual (100%; p = 0.008) and luteal phase (85%) of the cycle. Mean values and standard deviations (SD) of stimulus threshold and intensity of pain in studied groups are shown in Graphs 1 and 2. The sensitivity analysis in the pulp with respect to the cycle phase showed greater pulp sensitivity to electrical (lower threshold level) and cold stimuli (higher pain intensity) in the menstrual compared to the luteal phase, however, the difference was statistically significant only for cold stimulation (p = 0.015). The pulp in postmenopausal women showed significantly higher threshold level to electric stimulus and significantly lower pain intensity to cold stimulus compared to women with regular menstrual cycle, regardless of the cycle phase (Graphs 1 and 2).

DISCUSSION
In order to clarify the possible influence of gonadal hormonal fluctuations on pain sensitivity of the pulp, the current research examined the effects of electric and cold stimuli in women of reproductive age, within two phases of menstrual cycle (menstrual and luteal), and postmenopausal women. The results showed that luteal phase in women with regular menstrual cycle characterized by high levels of gonadal hormones, and menopause -a period of chronic reduction in the level of gonadal hormones, are periods of lower pulp sensitivity.
In our study, pulpal pain was induced by electric and cold stimuli in healthy women without the presence of other painful conditions (experimental pain model). Evaluation of pain sensitivity in healthy subjects is important for investigation of various aspects of mechanisms that underlie pain. Sensory manifestations and sensory-motor interaction of previously clearly defined pain can be determined on experimental pain model. Moreover, experimental pain model is valuable for pharmacological and clinical studies in order to quantify the sensitivity of the nociceptive system in pain patients, as well as to predict clinical pain and clinical pain outcomes [26]. Research on sensitivity of healthy women to various stimuli during menstrual cycle have not given clear evidence of experimental pain hormonal dependence [12,27]. Some authors have found that pain perception varies during the cycle [15,16], while others have not confirmed a link between pain and hormonal fluctuations during the cycle [17,18]. Discrepancies in results can be attributed to methodological differences, such as type of observed experimental pain, stimulated tissue, a measuring outcomes (threshold or intensity) and functional division of the menstrual cycle with no measurement of gonadal hormone concentrations [12,27]. An interesting fact is that the brain cortex activity induced by experimental painful stimuli varies between menstrual phases with high levels of estrogen and phase characterized by low hormone levels, and these changes are not always accompanied by altered response to pain [28]. Unlike the experimental pain, it has been shown that pain in certain clinical pain conditions varies during menstrual cycle [10]. In these patients, many other factors can influence the perception of pain during menstrual cycle. Also, patients with pain conditions may have altered sensitivity to hormonal changes during the cycle.
The current study investigated the pulp reaction to electrical and cold stimuli. Electric pulp test and cold test using cold spray are the most frequently used tests for investigation of pulp sensitivity [29]. Various types of stimuli have been applied in experimental studies that investigate the relationship between hormonal status and pain and its characteristics. It has been observed that various stimuli differently activate nociceptive afferent fibres in the tissue. Electric stimuli trigger all classes of afferent neurons, while thermal stimuli only A-δ and C fibres [30]. Also, certain types of stimuli can cause stress, while others activate endogenous regulatory mechanisms of pain. Therefore, hormonal status can have different effects on different stimuli. In fact, it has been shown that menstrual cycle has opposite effect on pain caused by electrical stimulus from pain caused by other painful stimuli (pressure, cold, warm, ischemic pain) [31]. In our study, higher pulp sensitivity to electrical as well as to cold stimulation was observed in the menstrual phase. Although various groups of nerve fibres contribute in the occurrence of various types of pain, in dentin-pulp complex both stimuli cause  activation of peripherally placed A-δ fibres that are mediators of sharp, localized pain. Centrally positioned C fibres that are responsible for the appearance of dull, long-term pain were not activated during testing, unless greater intensity of electricity was applied or there was an irritation that caused tissue damage. It is important to note that in addition to the type of stimulation, pain sensitivity varies on the type of tested tissue and the depth of tissue to which the stimulus is applied [15].
In women of reproductive age increased pain sensitivity, especially to cold stimulation, was recorded during menstrual phase, phase characterized by low levels of estrogen and progesterone, while lower sensitivity was present during luteal phase when estrogen and progesterone levels were high. Gonadal hormones, estrogen and progesterone, can express pronociceptivne as well as antinociceptive effects depending on the overall hormonal profile [12]. It is believed that at the end of luteal and in early follicular phase, when the levels of both gonadal hormones are low, sympathetic tone, inflammatory response, the synthesis of prostaglandins and affective symptoms reach their highest level, while descending inhibitory systems are at the lowest level [12]. This explains why perimenstrual phase is period of increased sensitivity to various painful conditions. On the other hand, in the middle luteal phase, gonadal hormones reduce pain sensitivity through increased GABA-tic tone and increased cortisol secretion [12]. These mechanisms could explain differences in pulp sensitivity during investigated periods of menstrual cycle observed in the current study.
Compared with reproductive age women, in menopausal women sensitivity of pulp was lower, regardless of the applied stimulus and the phase of cycle. Although the influence of gonadal hormones in the reproductive age on clinical pain conditions is somewhat clarified, data on their impact in menopause are different [27]. A study that compared the incidence of various painful conditions in menopausal women with premenopausal period, has found that menopausal changes depend on the type and characteristics of pain [14]. Headaches and cervical / lumbar pain decreased or even disappeared, while developed or intensified joint pain affected more than half of women. Pain intensity and painful area also change with menopause. In most of the cases, high intensity pain that would affect larger area remained unchanged or decreased, while low intensity pain and pain that affects smaller less area increased [14]. The impact of ageing process on the pulp tissue and its sensitivity should not be ignored. Reduction of painful pulp reactions in elderly is attributed to reduction in number of nerve fibres, creation of secondary and tertiary dentin and tubule occlusion [32]. However, significant impact of age on dental pulp response caused by electrical stimulation was not found [33,34]. Reaction to electrical test was obtained in all subjects, in women of reproductive age as well as menopausal women. On the other hand, it has been shown that the sensitivity of pulp to cold stimuli decreases with age [35]. Unlike electrical test, which is dependent on the ions movement, cold stimulus causes pain response by dentinal fluid movement [36], which can partly ex-plain reduction of pulp sensitivity to cold in menopausal women. In the current study response to cold stimulation was not obtained in 35% of menopausal women even after two repeated measurements. An interesting finding is that the application of cold spray caused a painful reaction in all subjects in the menstrual phase, but not in 15% of subjects in the luteal phase, what could be misinterpreted in clinical conditions as a loss of tooth vitality. Studies have shown that the response of dental pulp to cold stimulation is the most reliable in this particular age group (21-50 years) [29]. Findings of decreased pain sensitivity in the luteal phase of menstrual cycle characterized by high levels of sex hormones, as well as reduced sensitivity during menopause, a period of chronic reduction in the levels of gonadal hormones, suggest that if there is an association between gonadal hormone levels and pain sensitivity, it is not simple.
Potential variations were not examined in this study. Namely, the division of menstrual cycle in phases was performed on the basis of medical history, without measurement of gonadal hormone concentration. Measuring gonadal hormones levels is important for precise determination of menstrual cycle phase, as well as in determining the correlation between hormonal levels and painful response. However, only a few studies have investigated the influence of gonadal hormone concentration on experimental pain until now. Secondly, sensitivity of the pulp is measured only in the two phases of menstrual cycle. However, to determine the relationship between hormonal status and pain, a research should include more cycle phases.