Different Brain Activation Patterns to Pain and Pain-related Unpleasantness during the Menstrual Cycle

Eighteen normal nonpregnant menstruant volunteers (aged 23.11 ±1.91 yr) were recruited from local university students and were paid for their participation. The Medical Ethics Committee of Yonsei University Wonju College of Medicine (Wonju, Kwangwon-Do, South Korea) approved this study. All of the volunteers gave written, informed consent, which acknowledged that (1) they would experience experimental thermal pain, (2) no tissue damage would result from this stimulation, (3) all of the methods and procedures were clearly explained, and (4) they were free to withdraw from the experiment at any time. All the participants were right-handed, and those with amenorrhea, irregular or recently missed menstrual periods, a previous history of any neurologic or psychiatric disorders, hormonal medication within the past 6 months, use of central nervous system–active medication or illicit drugs, or regular consumption of nicotine or alcohol were excluded. The participants were asked to refrain from smoking and alcohol and caffeine consumption for 24 h before the study. The participants were examined twice, once in the follicular phase and once in the luteal phase. Half of the participants were tested first during the follicular phase, and the other half were tested first during the luteal phase. They were also asked about their last menstrual period and the average cycle duration. The monitoring of the luteal phase was conformed by records of the next menstrual bleeding and progesterone level (progesterone: 0.15–1.4 ng/ml [follicular], 1.6–21.0 ng/ml [luteal], 5.2–23.0 ng/ml [midluteal]). The follicular or the luteal day of each participant was determined by the last menstrual period and the next menstrual bleeding as well as by the serum estradiol and progesterone level (table 1). The distal phalanges and distal two thirds of the middle phalanges of the middle finger of the left hand were immersed into an 850-ml water bath made of expandable polystyrene (at 48° or 35°C). A research assistant helped to exchange the 48°C water box for the 35°C water box.

Figure 1shows the stimulation paradigm. The participants were informed 15 s in advance that a 48°C (pain) or 35°C (control) stimulation was to begin (anticipation period [prestimulation, precontrol, respectively]). A 30 s-finger immersion stimulation at 48° or 35°C (pain stimulation, control stimulation) was followed by a 30 s-poststimulation, postcontrol period, respectively. This 168-s block was repeated five times for each participant. The participants wore insert earphones connected to a microphone through which they received instructions during each period. Switching among each period was verbally cued. To reduce the effect of circadian fluctuations on the levels of other hormones (e.g. , corticosteroids), the first and second experimental session were conducted at the same time of day and conducted in an identical manner with a 2-week (nine participants) or 10-day (nine participants) interval between the two scanning sessions. To minimize the level of anxiety and to familiarize the participants with the finger immersion in a hot bath, the participants underwent a 30 s-finger immersion stimulation at 48°C of the right middle finger before beginning the session. During scanning of the first three blocks of a total of five blocks, the temperature of water was decreased from 48°C to 47.5°C. We designed an interscan resting interval of 5 min between the third and fourth block. During this 5-min interval, the research assistant increased the temperature of the water from 47.5° to 48°C. On the visual analog scale, 0 indicated no sensation or no unpleasantness, and 100 indicated the most intense pain or unpleasant feeling imaginable. At an interscan interval of 5 min between the third and fourth block and the end of the stimuli series, participants were asked to rate pain intensity and unpleasantness. The pain and unpleasantness ratings were averaged for each participant. Then, a group average was acquired.

A venous blood sample was drawn before each fMRI investigation to determine the estradiol, progesterone, and testosterone levels in the right forearm. The estradiol (E²), progesterone (P⁴), and testosterone levels were measured using a γ-counter (COBRA 5010 II; Packard, Downers Grove, IL) with kit Coat-A-COUNT Estradiol (DPC, Los Angeles, CA), Coat-A-COUNT Progesterone (DPC), and Coat-A-COUNT Testosterone (DPC). The intraassay and interassay coefficients of variation were 4.0 and 4.2% for estradiol, and 3.50 and 4.30% for progesterone, respectively. The coefficients of variation of testosterone were 5.90%.

The hormonal levels of estradiol, progesterone, and testosterone and the pain and unpleasantness ratings during the follicular and luteal phases were compared using a paired t  test. An overall P  value of less than 0.05 was considered significant.

Before the scan, the participants were instructed to keep their eyes closed, to stay awake, and to refrain as much as possible from moving throughout the imaging session. After being placed in a comfortable position, the head was immobilized with padded ear muffs and a foam headrest, and a plastic bar was placed across the bridge of the nose. The images were acquired using a KASIT 3-T MRI scanner (ISOL Technology, Gwangju, Kyonggi-Do, Korea) with a quadrature head coil. After a T1-weighted scout image, high-resolution anatomic images were acquired using a magnetization-prepared rapid gradient echo pulse sequence with time to echo (TE) =16 ms, repetition time (TR) = 2,800 ms, flip angle = 60°, and matrix size = 192 × 256 mm. The T2*-weighted functional data were acquired using an echo planar imaging pulse sequence of TE = 30 ms, TR = 3,000 ms, flip angle = 80°, and matrix size = 64 × 64 mm. Twenty-eight slices of the echo planar images were obtained with a slice thickness of 5 mm. An experimental run consisted of a total of five blocks, and a series of tasks were performed in each block: pain (anticipation of pain stimulation − pain stimulation [48°C]− poststimulation) − control (anticipation of control stimulation − control stimulation [35°C]− poststimulation). One hundred seventy-two volumes for the first three blocks and 116 volumes for the remaining two blocks were acquired in each experimental run. The initial five images for each scan were discarded, and all images scanned during cueing were also discarded.

The image data were analyzed using SPM99 (Wellcome Department of Cognitive Neurology, London, United Kingdom). The images for each subject were motion-corrected and realigned using the first scan as a reference. The T1 anatomical images were coregistered with the mean of the functional scans (echo planar images), which were then normalized to the SPM T1 template in the atlas space of Talairach and Tournoux. Finally, the images were smoothed by applying a gaussian filter with an 8-mm full width at half-maximum. Difference images in each participant were generated by subtracting the blood oxygen level–dependent signal data obtained during the precontrol (15 s), control stimulation (35°C, 30 s), and postcontrol (30 s) from the blood oxygen level–dependent signal data obtained during prestimulation (15 s), pain stimulation (48°C, 30 s), and poststimulation (30 s), respectively. We took the prestimulation versus  precontrol, pain versus  control, and poststimulation versus  postcontrol t contrasts as the index of anticipation of pain, pain response, and poststimulation response in the follicular and luteal phase. The resulting contrast images were then used in random effect analyses at the group level. The group data for 18 participants in the follicular and luteal phase were analyzed. The brain areas activated in the follicular compared with the luteal group (n = 18, respectively) or in the luteal compared with the follicular group (n = 18, respectively) were compared using voxel-by-voxel paired t  test. The brain activations in paired t  test were reported if the P  value was less than 0.005 or 0.001 (the follicular group in the poststimulation period) or 0.007 (the luteal group in the poststimulation period) uncorrected for multiple comparisons, and if the cluster size was more than 10 voxels. To acquire the simple regression between brain activation patterns and sex hormone levels (or visual analog scale scores of pain intensity and unpleasantness), the individual pain-control contrast for all participants was extracted, and sex hormone levels or visual analog scale scores of pain intensity and unpleasantness were entered into the regression model as a covariate. The resulting images were displayed on SPM99 graphic window at P < 0.001, with more than 10 voxels. The volume of interest with a 1-mm radius sphere at peak coordinate was selected, and the parameter estimates were acquired. The Pearson’s correlation coefficients between these parameter estimates and sex hormone levels (or visual analog scale scores of pain intensity and unpleasantness) were calculated with correlation analysis using SPSS 12.0E (SPSS Inc., Chicago, IL).

The estradiol and progesterone levels were significantly higher in the luteal phase than in the follicular phase (table 2). No significant differences between the follicular and luteal phases were observed for the testosterone levels (table 2). The pain ratings and pain-related unpleasantness ratings were significantly higher in the luteal phase than in the follicular phase (table 2).

In the anticipation period, the activated brain regions during the follicular phase included the inferior frontal gyrus, middle frontal gyrus, medial frontal gyrus, and frontal lobe (table 3and fig. 2A), whereas during the luteal phase, they included the cerebellum, precentral gyrus, uncus, superior temporal gyrus, amygdala, middle temporal gyrus, and parahippocampal gyrus (table 3and fig. 2B). In the thermal pain stimulation period, the areas of brain activation during the follicular phase included the precentral gyrus, postcentral gyrus, cerebellum, occipital lobe, superior frontal gyrus, putamen, and parahippocampal gyrus (table 4and fig. 2C), whereas during the luteal phase, they included the thalamus (right medial dorsal nucleus) and superior temporal gyrus (table 4and fig. 2D). In the poststimulation period, the activated brain regions during the follicular phase were the superior frontal gyrus, postcentral gyrus, precentral gyrus, cerebellum, inferior frontal gyrus, and middle frontal gyrus (table 5and fig. 2E). The brain regions activated during the poststimulation period of the luteal phase included the superior temporal gyrus, medial frontal gyrus, cerebellum, inferior frontal gyrus, precuneus, and parahippocampal gyrus (table 5and fig. 2F).

The activation of the left precentral gyrus was negatively correlated (Pearson correlation coefficient [r ]=−0.626, P < 0.001) with pain-related unpleasantness ratings in the pain stimulation period. The activation of the left precentral gyrus was positively correlated (r = 0.761, P < 0.001) with serum testosterone levels in the pain stimulation period of the follicular phase. There was a positive correlation (r = 0.540, P = 0.001) between activation of the right thalamus and pain-related unpleasantness ratings in the pain stimulation period. There was a negative correlation (r =−0.524, P = 0.001) between activation of the right thalamus and serum testosterone levels in the pain stimulation period.

In anticipation of pain, dorsolateral prefrontal cortices and right medial frontal gyrus were activated during the follicular phase, whereas the left uncus (medially curved anterior end of the parahippocampal gyrus), left superior temporal gyrus, left amygdala, right middle temporal gyrus, and right parahippocampal gyrus (part of the hippocampal complex just inferior to the hippocampus proper) were activated during the luteal phase. It may be possible that activation of the dorsolateral prefrontal cortices during the anticipation period of the follicular phase decreased pain perception during the pain stimulation period of the follicular phase. This is supported by a report that placebo analgesia which engages opioid or nonopioid mechanisms increases the brain activity of the prefrontal cortex during anticipation of pain.8During the anticipatory period of the follicular phase, women are likely able to earlier anticipate and adapt or cope with impending noxious events than during the luteal phase. Activation of the medial frontal gyrus and dorsolateral prefrontal cortices during the anticipation period of the follicular phase may decrease pain-related unpleasantness during the pain stimulation period of the follicular phase. There are reports that the positive emotion (presentation of happy facial expressions) activated the medial frontal cortex,9whereas unpleasant emotion activated the bilateral occipitotemporal cortex and cerebellum and the left parahippocampal gyrus, hippocampus, and amygdala.10In our study, the superior temporal gyrus (temporal pole) was activated during the anticipation period of the luteal phase. Previous studies found that increasing intensity of sad and angry facial expression was associated with enhanced activity in the temporal pole and superior temporal gyrus.11,12During the anticipation period of the luteal phase, the right middle temporal gyrus was activated. Recent study has shown that negative (relative to positive) autobiographical memories differentially increased neural activity in the right middle temporal gyrus only.13This study and our study suggest that the temporal pole and the right middle temporal gyrus may be implicated in the processing of pain-related unpleasantness. In the current study, activation of the left uncus and right parahippocampal gyrus and left amygdala during the anticipation period of the luteal phase suggests that participants during the luteal phase may feel more pain-related unpleasantness than during the anticipation period of the follicular phase. The amygdala is believed to be one of the most important brain regions for processing emotions involving fear.14Lorazepam (anxiolytic drug) administration attenuated the blood oxygen level–dependent fMRI signal in the bilateral amygdala in a dose-dependent manner during an emotion face assessment task.15This result suggests that the amygdala is activated by anxiety. Interactions between the amygdala and the hippocampal complex during emotional events have been reported.16Recent research showed that viewing fearful or disgusting slides increased the level of pain perception and unpleasantness, indicating that both the sensory–discriminative and affective–motivational dimensions of pain are amplified by an unpleasant emotion.17The anticipatory period of pain during the luteal phase could activate the brain areas that may increase pain-related unpleasantness. Inadvertent adaptation to noxious stimuli can cause anticipatory anxiety or exacerbate the emotional distress of a painful event.18In our experiment, women in the luteal phase were likely to have lower adaption to painful stimuli than in the follicular phase, although cerebellum and precentral gyrus involving motor preparation and defense mechanism were activated.

During the pain stimulation period of the follicular phase, the primary and secondary somatosensory cortex (S1 and S2) were activated. It is known that both primary somatosensory cortex (S1) and secondary somatosensory cortex (S2) are commonly activated in heat pain studies and that the nociceptive input into these regions at least partially underlies perception of the sensory features of pain.19During pain stimulation of the follicular phase, activation of prefrontal cortices may decrease pain perception compared with the luteal phase. The putamen and cerebellum and precentral gyrus involving motor preparation and defense mechanism related to antinociceptive behavior20were activated during the pain stimulation of the follicular phase. The activation of these areas may facilitate the participant’s effective coping with the pain stimulus. In our study, pain-related unpleasantness ratings were significantly lower in the follicular phase than in the luteal phase. The activation of the left precentral gyrus was negatively correlated with pain-related unpleasantness ratings (r =−0.626, P < 0.001) in the pain stimulation period and positively correlated with serum testosterone levels (r = 0.761, P < 0.001) in the pain stimulation period of the follicular phase. The activation of the left precentral gyrus increased when unpleasantness ratings decreased and serum testosterone levels increased. These results are supported by a report that endogenous testosterone or administration of testosterone increases analgesia, anxiolysis, and cognitive performance of male rats.21These mechanisms may explain the reason for the degree of pain and unpleasantness ratings experienced during the follicular phase being less than that in the luteal phase. However, during the pain stimulation period of the follicular phase, the activation of parahippocampal gyrus may be associated with the pain-related unpleasantness, even though the rating of unpleasantness was less than that in the luteal phase.

The right thalamus and superior temporal gyrus (temporal pole) were activated during the pain stimulation period of the luteal phase. In addition to the pain stimuli, attentional processes and vigilance have also been shown to increase thalamic activity.22In our study, because the thalamus was activated contralaterally to the noxious stimuli, these responses may merely reflect a sensory component that would be supposed to predominate contralaterally to the pain stimuli. However, the activation of the right thalamus was positively correlated with pain-related unpleasantness ratings (r = 0.540, P = 0.001) and negatively correlated with serum testosterone levels (r =−0.524, P = 0.001) in the pain stimulation period. The activation of the right thalamus increased when unpleasantness ratings increased and serum testosterone levels decreased. Therefore, these thalamic responses to the pain stimuli can be considered as a part of both discriminative and affective networks involved in pain processing.

During the poststimulation period of the follicular phase, the postcentral and precentral gyrus and prefrontal cortices were activated. Because the primary somatosensory cortex (S1, postcentral gyrus) was activated, residual pain could continue until the poststimulation period, although the degree of pain intensity (pain rating) was less than that experienced in the pain stimulation period. The activation of the precentral gyrus and prefrontal cortices may suggest that the motor preparation and defense mechanism related to pain modulation could continue until the poststimulation period.

During the poststimulation period of the luteal phase, the parahippocampal gyrus and temporal pole were activated. The activation of these areas may be related to an increase of pain-related unpleasantness. Also, the activation of the medial frontal gyrus in the poststimulation period of the luteal phase may be related to an increase of pain-related unpleasantness. In addition to positive emotions, negative emotions have been known to engage the medial prefrontal cortex.23The activation of the precuneus in the poststimulation period of the luteal phase may be associated with an increase in pain intensity and pain-related unpleasantness. Previous study reported less activation in the precuneus with hypnosis during painful heat stimuli.24These may suggest that the pain and pain-related unpleasantness could continue until the poststimulation period, although the degree of pain intensity and pain-related unpleasantness were less than that experienced in the pain stimulation period.

The temporal pole was activated during the anticipation, pain stimulation, and poststimulation periods of the luteal phase. The activation of the temporal pole during the entire luteal phase may be closely associated with an increase of the pain and pain-related unpleasantness. The above-mentioned results suggest that changes in the sex hormones and their related central nervous system alterations in women may decrease the feelings of pain and pain-related unpleasantness during the follicular phase and increase the feelings of pain and pain-related unpleasantness during the luteal phase.

The limitations and merits of this study are as follows. Because of the irregular menstrual cycle of some volunteers and schedules at the fMRI institute, each follicular day and luteal day for all participants could not be controlled exactly. Therefore, the same follicular day and luteal day of the 18 participants could not be compared. However, instead of comparing one point of follicular and luteal days, we could compare the follicular phase with the luteal phase throughout the menstrual cycle for 18 participants by examining each participant twice. Instead of 75-s hand immersion stimulus at 46.5°–47.5°C25and 30 s-hand immersion stimulus at 50°–52°C26used in previous reports,25,2630 s-finger immersion stimulus at 48°C (a much smaller area than the hand and temperature lower than 50°–52°C, resulting in a weaker pain intensity than previous studies25,26) was selected because it was thought to be an appropriate thermal stimulus for detecting different responses to pain and pain-related unpleasantness between the follicular and the luteal phase. Therefore, pain-specific areas of the brain were not fully activated. However, if the temperature was increased more than 48°C and hand immersion was used, pain-specific brain regions could be fully activated, whereas we could not detect different responses to pain and pain-related unpleasantness between the follicular and the luteal phase. Because previous study has shown that brain activations continue for 20 s or so after thermal pain is removed,8we selected the 30-s poststimulation period. In clinical practice, this situation resembles postprocedural pain continuing after medical procedures.

In conclusion, the brain areas that may decrease pain perception were activated during the follicular phase, whereas the brain areas that may increase pain perception were activated during the luteal phase. The results of this study suggest that pain and unpleasantness may be decreased during the follicular phase and increased during the luteal phase. During the follicular phase, women may have a higher ability to anticipate and adapt or cope with pain stimuli than during the luteal phase. Therefore, during surgical and medical procedures, requirements for anesthetic and analgesic or anxiolytic drugs may be reduced during the follicular phase and increased during the luteal phase. Although the precise, functional relation between the sex hormones and fMRI signal changes during the follicular and luteal phases requires further clarification, this study emphasizes the importance of considering the effects of sex hormones in nonpregnant menstruant women when designing clinical or neuroimaging studies or when treating patients for pain and pain-related unpleasantness.

The authors thank all the staff of the fMRI Laboratory, Brain Science Research Center, Korea Advanced Institute of Science and Technology (Daejeon, South Korea) for technical assistance. The authors thank Myung Soon Kim, M.D. (Professor, Radiology, Yonsei University Wonju College of Medicine, Wonju, Kwangwon-Do, South Korea), and Jae Jin Kim, M.D. (Associate Professor, Department of Psychiatry, Yonsei University College of Medicine), for their participation in helpful discussions. The authors also thank Margaret Jean Storey, R.N., Ph.D. (Director, Yonsei University Wonju Community Center for People with Disabilities), and Jin Ok Park, B.A. (Director, Unis English School, Wonju, Kwangwon-Do, South Korea), for correcting the English usage in this article.