Behavioural Brain Research 274 (2014) 326–333

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The impact of sex and menstrual cycle on the acoustic startle response Diana Armbruster a,∗ , Alexander Strobel a , Clemens Kirschbaum b , Burkhard Brocke a a b

Institute of Personality and Individual Differences, Technische Universitaet Dresden, Dresden 01062, Germany Institute of Biopsychology, Technische Universitaet Dresden, Dresden, Germany

h i g h l i g h t s • • • • •

We investigated impact of sex and menstrual cycle on acoustic startle response. The sample consisted of 107 male and 111 female healthy young adults. There was an influence of menstrual cycle but not of sex on ASR. ASR was elevated as expected during late luteal phase but also during ovulation. Higher ASR in mid-cycle is probably due to heightened auditory sensitivity.

a r t i c l e

i n f o

Article history: Received 17 April 2014 Received in revised form 1 August 2014 Accepted 4 August 2014 Available online 23 August 2014 Keywords: Acoustic startle response Fear Anxiety Sex differences Menstrual cycle Sex hormones

a b s t r a c t Sex differences in fear and anxiety have been widely reported although results are not entirely consistent depending on measures used. Also, a possible influence of the menstrual cycle is often not taken into account, and effect sizes are not always discussed. In a sample of healthy young adults (n = 111 women without hormonal contraceptives and n = 107 men) the acoustic startle response (ASR) and emotional ASR modulation were analysed. We found no significant effect of sex on ASR (p = .269) but a significant effect of menstrual cycle (p = .027, 2 = 0.105). Compared to men, women showed increased ASR during the late luteal phase probably reflecting elevated negative emotionality, and during ovulation which, however, might be due to increased auditory sensitivity and changes in general CNS arousal. Neither sex nor menstrual cycle affected startle modulation. Thus, at least in young adults, menstrual cycle but not sex per se appears to contribute significantly to ASR variance. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In numerous studies women have been found to report greater severity and number of fears as well as showing higher prevalence rates for most anxiety disorders compared to men [1]. These differences have been observed to be already present in children before the onset of puberty although they become more pronounced during adolescence and adulthood [2]. Such self-report based findings have received additional (though not entirely consistent) support in studies investigating sex differences at the peripheral physiological level: Depending on which measures are used women showed higher (e.g., heart rate at rest) or lower physiological reactivity (e.g., blood pressure, reactivity of the hypothalamic–pituitary–adrenal axis), while for instance heart rate during challenge resulted in no sex differences (for a review see [2]). Regarding anatomical,

∗ Corresponding author. Tel.: +49 351 463 36997; fax: +49 351 463 36993. E-mail address: [email protected] (D. Armbruster). http://dx.doi.org/10.1016/j.bbr.2014.08.013 0166-4328/© 2014 Elsevier B.V. All rights reserved.

chemical and functional sex differences at the level of the brain, Cahill [3] reviewed recent findings and concluded that the influence of sex in these domains is of vital importance and that in neuroscience sex should always be considered as a potential confounding factor. These differences have been attributed to distinct prenatal exposure to sex hormones resulting in long-term organisational changes of the central nervous system (CNS) while later occurring hormonal differences have been suggested to lead to transient effects [4]. However, as Hyde [5] rightly pointed out, aside from considering significant effects of sex one has to take into account effect sizes which have frequently been neglected in the past. In analyzing findings of 46 meta-analyses she concluded that 78% of the considered psychological sex differences ranging from cognitive abilities, personality traits, emotional reactivity and motor skills, were close to zero or small including, for instance, neuroticism scores [5]. When contrasting such opposing views it has to be emphasized that they are mostly based on different types of data: while Hyde built the gender similarities hypothesis on phenotypic psychological

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responses [5], Cahill mainly focussed on structural and functional brain features [3]. This discrepancy touches on the old problem that physiological measures have not always been found to correspond to accompanying self-reports (e.g. [6]). Furthermore, it has also been argued that sex differences in brain organisation and function might result nevertheless in similar outcomes at the level of psychological testing and that on the other hand sex differences in performance might nonetheless be associated with similar brain activation patterns [7,8]. Thus, disentangling sex differences at different levels along the way from brain activation patterns to reported emotional differences remains a challenge. In addition to the lack of effect sizes and the contradictory findings in different outcome measures, experimental studies comparing men and women often failed to take the influence of menstrual cycle phases into account although gonadal hormones have for instance been implicated in emotional regulation [9,10] as well as in cognitive function [11,12]. However, most problematic is the fact that sex as a possible influence is often not investigated at all. In sum, the actual degree of sex differences regarding distinct facets of anxiety and fear is still uncertain particularly regarding the underlying biological mechanisms. Here, we focussed on differences between men and women in a well established physiological measure of fear for which conflicting results concerning sex differences were reported: the acoustic startle response (ASR). The ASR comprises fast contractions of skeletal and facial muscles as well as closing of the eyes and acceleration of the heart rate after sudden high-intensity noise bursts [13]. In human studies the eye blink component of the startle reflex is usually assessed by electromyographic (EMG) recordings of the orbicularis oculi muscle response [14]. The underlying circuit of the ASR consists of the sensory receptors and the auditory nerve, the cochlear nucleus, the ventrolateral lemniscus, the nucleus reticularis pontis caudalis (PnC), and spinal motoneurons, which give rise to the ASR [13,15]. Since the stimuli commonly used to evoke the startle response are aversive and have been suggested to induce a state of fear or anxiety [16] the ASR itself might be regarded as an indicator of innate fear. ASR magnitude is reduced when the startling stimulus is immediately (30–500 ms) preceded by a weaker stimulus (prepulse inhibition; PPI; [13]). Furthermore, ASR magnitude can be modulated by cognitive processes such as attention [17,18] as well as emotional states triggered by the presentation of pleasant stimuli resulting in pleasure-attenuated startle (PAS) or unpleasant stimuli leading to fear-potentiated startle (FPS; e.g., [19–21]). Affective modulation of the startle response, particularly FPS, has been demonstrated to occur as early as 300 ms after picture onset although the overall startle magnitude in that time frame is still inhibited compared to later startle onset times [22–24]. Sex differences in affective startle modulation have been reported although findings are not entirely consistent. Stronger FPS has been found in women compared to men (e.g. [25–27]) while other studies found no sex differences in FPS or PAS [28,29]. Furthermore, it has been repeatedly demonstrated that compared to young men premenopausal women show smaller PPI [30–33] while there are no differences in PPI between postmenopausal women and men of similar age [32]. In addition, the effects of sex on ASR, which shows substantial interindividual variance even in healthy individuals [34], have been investigated although findings are also inconsistent with some studies reporting no effects (e.g. [32,35–37]) while others found significant differences between men and women: Kofler et al. [38] investigated ASR in several muscles as well as different ASR parameters (e.g., latency, startle probability, ASR area under the curve) and reported overall stronger ASR in women compared to men but no differences in startle probability and latency measured over the orbicularis oculi, although ASR area under the curve over this muscle was larger in females.

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Unfortunately, no effect sizes were reported. Recently, Quevedo et al. [39] reported higher response probability in ASR in females in a sample of healthy individuals with an effect size of 2 = 0.05. Similarly, women showed an overall greater startle amplitude (effect size: Cohen’s d = 0.80) as well as greater FPS than men in an emotional startle paradigm [27]. These conflicting findings in humans are mirrored in animal studies using different strains of rats. There were no sex differences in startle responses in Sprague-Dawley rats [40] while startle magnitudes were larger in male compared to female Wistar albino and hooded rats [41] and in Long-Evans rats [42]. However, it should be noted that the startle response in rodents is usually assessed by whole body ballistic ground reaction forces which has been found to be dependent on body weight which is usually higher in males [41]. One probable reason for the inconsistencies regarding sex differences in humans might be the lack of control for menstrual cycle phase or for the use of hormonal contraceptives. Those studies which did investigate the influence of menstrual cycle phase reported varying results depending on startle parameter used. In a sample of healthy women and patients with premenstrual dysphoric disorder (PMDD) Epperson et al. [43] reported heightened ASR in women with PMDD during the luteal compared to follicular phase, while there was no difference in healthy controls. However, in another study PMDD patients showed heightened ASR during both the follicular and the luteal phase compared to healthy women while there was no difference in ASR between the two cycle phases within the PMDD or the healthy control group [44]. Similarly, Jovanovic et al. [45] and Kumari et al. [46] found no differences in ASR magnitudes during follicular vs. luteal cycle phase in healthy samples although a significant effect of menstrual cycle phase was found on PPI [45]. Jovanovic et al. also investigated the influence of menstrual cycle phase in a longitudinal approach in 14 women. Again, no effect on ASR but a significant effect on PPI emerged [45]. In sum, in healthy women ASR seems not to be influenced by menstrual cycle phase but it did impact PPI in healthy participants as well as ASR in PMDD patients. However, some of these findings are based on comparatively small samples and not all studies included men as a comparison group. Thus, in the present study we aimed to investigate in a larger sample of healthy young adults (1) whether men and women differ in the startle response and its affective modulation, (2) the size of such a potential effect and (3) the possible influence of menstrual cycle phase on ASR as well as PAS and FPS. In addition, sex differences in stimulus ratings as well as questionnaire data on anxiety-related states and traits were analyzed.

2. Material and methods 2.1. Participants The sample originally consisted of 114 female and 109 male students of the Technische Universitaet Dresden. Of these five participants had to be excluded during data preprocessing due to excessive EMG artifacts or because of virtually no startle responses (non-responder), leaving 218 adults (111 female and 107 male) for the final sample (mean age 23.24 yrs., SD = 3.136, range 18–34 yrs.). Age did not differ significantly between the male and female sample (p = .698). All participants were of of German/Middle European ancestry. They were all non-smokers and none of the female participants used hormonal contraceptives. Prior to the experiment all participants underwent a telephone screening in order to assess physical and mental health. To be included in this study participants had to have normal or corrected to normal vision. Also, they could not have been diagnosed

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with hearing disorders in the past, could not have current hearing problems of any kind and no past or recent exposure to extremely loud noises. Furthermore, reported current psychological problems were an exclusion criterion as were past diagnoses of psychological disorders. No severe physical impairment or illness (e.g., cardiovascular diseases, diseases of the respiratory tract, liver diseases, diseases of the kidneys and the urinary passage, intestinal diseases and disorders, metabolic disorders, muscle diseases, blood diseases, severe infectious diseases, severe allergies, autoimmune diseases, thyroid diseases, head injuries) should be present. No participant used drugs and no participant reported to be under severe stress at the time of the experiment. As result of the pre-experimental screening a total of 16 potential subjects were not invited to participate because they did not meet all required criteria. Assessment of menstrual cycle phase was based on information given by the participant regarding (a) the first day of the last period, (b) the length of their menstrual cycle and (c) its regularity which were then used to calculate cycle phase. During a prior telephone screening female participants were asked if their menstrual cycle was approximately regular. Only those who answered in the affirmative were invited. Participants were informed that on the day of testing data regarding menstruation onset, cycle length and cycle irregularities would be obtained and they were thus asked to consult their menstrual calendar in order to ensure maximum accuracy. Only participants who reported no more than 3 days variation in cycle length were included in the final analysis concerning the impact of menstrual cycle phase. Eight participants who had reported more variation had to be excluded. Cycle length and the onset of the last menstruation were used to calculate the start of the next menstruation. Time of ovulation during the current cycle was estimated by subtracting 14 days from this date. Menstrual cycle phases were thus determined as follows: midcycle (ovulation) included days 13–15 prior to the next menstruation. Early luteal phase was defined as the 7 days following midcycle (days 16–22 for a 28 days cycle) while the late luteal phase comprised the 6 days immediately preceding the estimated onset of the next menstruation (days 23–28 for a 28 days cycle). Follicular phase consisted of the days prior to the estimated next midcycle (days 1–12 for 28 days cycle) with the first half designated as early follicular and the second half as late follicular phase. There were no significant differences in age between the cycle stage sub-samples (p = .300). Participants were informed about the aims of the study, gave written informed consent and were reimbursed for participation. The study design was approved by the Ethics Committee of the German Psychological Association. 2.2. Materials and design Acoustic startle probes were administered alone and during viewing of visual stimuli. To elicit a startle response, a single 50 ms burst of white noise (95 dB SPL with an instantaneous rise time) was presented binaurally over Eartone A3 Audiometric Insert Earphones (Aearo Company, Indianapolis, IN, USA). Pictorial stimuli used consisted of 48 affective pictures. 40 color pictures were selected from the International Affective Picture System (IAPS) [47] and eight angry or fearful “Ekman-faces” [48] were used. The pictures comprised four exemplars of 12 semantic contents (6 unpleasant, 3 neutral and 3 pleasant) which were grouped into four blocks. Each picture was presented for 6 s followed by a variable intertrial interval (ITI, 11–24 s). During picture viewing, an acoustic startle probe was administered at 0.5, 2.5, or 4.5 s after picture onset in 9 of the 12 trials per block. The timing was balanced across content categories. One picture in each category was presented without a startle probe. No more than two pictures of the same valence and no more than two pictures with the same startle onset time could occur consecutively. Otherwise, stimulus order

was pseudo-randomized. For half of the participants, the order of the four blocks was reversed. Finally, in each block, three startle probes were delivered during the ITIs to measure baseline startle responses. 2.3. Physiological data collection and reduction The eyeblink component of the startle response was measured by recording EMG activity over the orbicularis oculi muscle beneath the left eye. The raw EMG signal was amplified by a SynAmps amplifier (NeuroScan Inc., El Paso, TX, USA), sampled at 1000 Hz, filtered (30–200 Hz band pass), rectified and integrated. Startle magnitude was defined as EMG peak in a time window from 20 to 140 ms after probe presentation. 2.4. Affective rating Evaluative judgments of pleasure and arousal were measured using the Self-Assessment Manikin (SAM; [49]). The SAM valence scale shows a graphic figure with expressions ranging from happy to unhappy, and the SAM arousal scale displays a graphic figure with expressions ranging from calm to excited. Ratings of valence and arousal were made on nine-point-scales. 2.5. Self-report measures All participants completed the following questionnaires: The State–Trait–Anxiety–Inventory (STAI; [50]) which was used to evaluate current as well as longer lasting feelings of apprehension, tension, nervousness and worry. In addition, participants completed the NEO-Five Factory Inventory (NEO-FFI; [51]) from which the Neuroticism score was analyzed and the Tridimensional Personality Questionnaire (TPQ; [52]) from which the Harm Avoidance scale was used. Both scales assess negative emotionality characterized by anxiety, low mood, vulnerability, and hostility. 2.6. Procedure After a telephone interview checking basic inclusion criteria (e.g. age, health, medication, or menstrual cycle regularity) participants were scheduled for a laboratory session. Since circadian variation might impact acoustic startle magnitudes [53,54] the sessions started not sooner than 09:00 h and not later than 15:00 h. First, participants completed a demographic questionnaire and the state version of the STAI. Then, the startle paradigm was employed. Afterwards all pictures were presented a second time and were rated for valence and arousal using a computerized SAM version [49]. Following, participants filled out questionnaires in order to assess different personality traits (i.e., STAI trait version, NEO-FFI, TPQ). Finally, participants were debriefed, reimbursed, and thanked. 2.7. Statistical analysis Analyses were performed using SPSS for Windows (SPSS Inc., Chicago, IL, USA). Average startle magnitudes were calculated from the 48 startle variables, respectively, and were log-transformed because of their highly skewed distribution (KS-tests, p < .20). First, the average log-transformed startle magnitude which did not deviate significantly from univariate normality (KS-tests, p ≥ .598), was entered into an analysis of variance (ANOVA) with sex as betweensubjects factor. Second, an additional ANOVA with menstrual cycle (early follicular vs. late follicular vs. ovulation vs. early luteal vs. late luteal) as between-subjects factor was conducted. As a third step separate repeated measurement ANOVAs with sex and menstrual cycle, respectively, as between-subjects factors and condition (negative, neutral, positive, baseline) as within-subjects factor were

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Fig. 1. Mean log startle magnitudes and standard errors of means in the four conditions (baseline, negative, neutral, positive).

conducted in order to analyze whether affective startle modulation patterns differed between the sexes or across the menstrual cycle. The potential interplay between onset time (within-subject factor) and sex and menstrual cycle (as between-subject factors) was analyzed in a similar ANOVA. The impact of sex and menstrual cycle on picture ratings was analyzed with nonparametric tests since some of the valence and arousal ratings were not normally distributed (Kolmogorov–Smirnov-tests, p < .20). Finally the influence of sex and menstrual cycle on the questionnaire data was analyzed in a multivariate ANOVA.

3. Results 3.1. Startle modulation Startle magnitudes were significantly modulated by the presentation of emotional pictures (F2.7, 588.2 = 117.53, p < .001, 2 = 0.352; see Fig. 1). As contrast analyses revealed, positive pictures resulted in significantly decreased startle magnitudes compared to neutral pictures and baseline (PAS; p < .001, 2 = 0.538 and p < .001, 2 = 0.352, respectively). Negative pictures lead to increased startle responses compared to baseline (p = .002, 2 = 0.043), although there was no difference between negative and neutral pictures (p = .165). However, it should be noted that neutral pictures themselves resulted in increased startle responses compared to baseline (p < .001, 2 = 0.073). Startle magnitudes were significantly influenced by onset time (F1.5, 318.9 = 88.92, p < .001, 2 = 0.292). As expected, contrast analysis showed that the shortest onset time (0.5 s) resulted in smaller startle responses than an onset time of 2.5 s (p < .001). In turn, onset times of 2.5 s resulted in significant smaller startle magnitudes than at 4.5 s (p < .001). Moreover, there was an onset time by valence interaction effect (F3.4, 724.0 = 41.05, p < .001, 2 = 0.160) which was mostly due to the effects of positive pictures.

Fig. 2. Effect of sex and the menstrual cycle on the overall log acoustic startle magnitude (mean ± SEM).

3.2. Influence of sex and menstrual cycle on the startle response ANOVA revealed no effect of sex on the overall log. startle response (F1, 216 = 1.55, p = .269). However, within the female subsample there was a significant effect of menstrual cycle phase (F4, 98 = 2.87, p = .027, 2 = 0.105). Contrast analysis revealed that compared to men women showed significantly higher startle magnitudes during ovulation (p = .027) and during the late luteal phase (p = .039; see Table 1 and Fig. 2) while there was no difference between men and women during the early and late follicular as well as the early luteal phase (all p ≥ .361). Since the influence of sex and menstrual cycle might be confounded by emotional modulation of the startle response, their effect on baseline startle only was also analyzed. Similarly, there was no effect of sex per se on baseline startle (F1, 216 = 0.88, p = .347; see Table 1) but within the female subsample baseline startle responses were significantly modulated by menstrual cycle phase (F4, 98 = 2.58, p = .042, 2 = 0.095). Results of follow-up contrast analyses were comparable to those of overall startle response: women showed higher startle magnitudes during the late luteal phase (p = .042) and during ovulation, although the latter effect was only marginally significant (p = .052). There was no significant effect of sex or menstrual cycle on startle modulation by emotional pictures as indicated by the absence of a sex by condition (p = .178) and a menstrual cycle by condition (p = 0.316) interaction effect. However, while the interaction between cycle phase and onset time failed to reach significance (p = .087) there was a small interaction effect between sex and onset time (p = .038, 2 = 0.017; see Supplemental Table 1 and Fig. 1 for details). This effect was mostly due to the shortest onset time (0.5 s). Descriptively, women showed stronger startle responses compared

Table 1 Log startle magnitudes (SEM) for men and women. Women are additionally grouped according to menstrual cycle phase.

Men Women Women

Early follicular Late follicular Ovulation Early luteal Late luteal

N

Overall log startle magnitude (SEM)

Baseline log startle magnitude (SEM)

107 111 22 18 17 18 28

0.809 (0.072) 0.916 (0.068) 0.849 (0.122) 0.649 (0.155) 1.209 (0.153) 0.681 (0.162) 1.112 (0.133)

0.818 (0.069) 0.907 (0.068) 0.857 (0.119) 0.655 (0.154) 1.165 (0.157) 0.655 (0.166) 1.114 (0.142)

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to men during onset times of 0.5 s, although the contrast failed to reach significance (p = .117). 3.3. Influence of sex and menstrual cycle on affective picture ratings The medians of the ratings for picture categories were first compared using the nonparametric Wilcoxon-tests for paired samples. The median valence ratings on the SAM scale for unpleasant, neutral, and pleasant pictures were 3.06, 5.59 and 6.09, respectively, and the median arousal ratings were 3.94, 1.47 and 3.32, respectively. All two-way comparisons for valence and for arousal were highly significant (all p ≤ .001). Nonparametric Mann–Whitneytests revealed effects of sex on valence ratings of emotional but not neutral pictures (p = .206): Women rated unpleasant pictures as more negative (p = .007) and pleasant pictures as less positive than men (p = .005). There was also a sex difference regarding the arousal rating of unpleasant pictures with women reporting more arousal (p = .002; all other p ≥ .206; see Supplemental Table 2). These sex differences in valence ratings of emotional pictures and arousal ratings of unpleasant pictures, respectively, were further modulated by menstrual cycle phase (nonparametric Kruskal–Wallis tests p < .050; all other p ≥ .294). Detailed comparisons revealed that there were no SAM rating differences between men and women during the early follicular phase (all p ≥ .352) and during ovulation (all p ≥ .095). In addition, during early luteal phase only the arousal rating of unpleasant pictures differed between women and men (p = .005; all other p ≥ .105). However, during late follicular and late luteal phases there were significant sex differences in the valence ratings of unpleasant and pleasant as well as the arousal ratings of unpleasant pictures (all p ≤ .026; all other p ≥ .072; see Supplemental Table 3 and Figs. 2 and 3). 3.4. Influence of sex and menstrual cycle on self-report measures While there were no sex differences in STAI state (F1, 216 = 0.48, p = .490) or trait anxiey (F1, 216 = 0.71, p = .399) significant effects of sex emerged for NEO-FFI neuroticism (F1, 216 = 10.90, p = .001; 2 = 0.048) and TPQ harm avoidance (F1, 216 = 5.89, p = .016; 2 = 0.027) with women reporting higher scores. Regarding the impact of menstrual cycle phase there was an effect approaching significance on NEO-FFI neuroticism (F4, 98 = 2.33, p = .061; 2 = 0.087). Contrast analyses revealed that this was due to reduced scores during ovulation compared to late luteal phase (p = .025) while the comparison to early follicular phase was marginally significant (p = .086). Although there was no significant main effect of menstrual cycle on STAI state anxiety (p = 0.181), trait anxiety (p = .123) or TPQ harm avoidance (p = .570) in the female subsample, the lowest scores were again reported during ovulation with the largest differences to late luteal and early follicular phase. In order to investigate whether the sex differences in neuroticism and harm avoidance was modulated by the menstrual cycle contrast analyses were conducted. There were no differences in neuroticism and harm avoidance between men and women during ovulation and early luteal phase (all p ≥ .478) while women had higher scores in both traits during early and late follicular and late luteal phase (all p ≤ .031). 4. Discussion ASR magnitudes vary considerably even in healthy subjects. Startle eye blink magnitudes have been found to be 30–45 times larger in the most responsive participants compared to the least reactive individuals [34]. Our own data confirms this magnitude: the averaged ASR (integrated raw data) of the most responsive participant was 43.3 times larger than that of the least

responsive participant (after exclusion of non-responders). In a study with more than 200 healthy participants we examined the role of sex as a possible contributing factor to this substantial inter-individual variability. We found no difference between premenopausal women (without hormonal contraceptives) and men of comparable age in ASR. Consistently, there were also no sex differences in self-reported state and trait anxiety although women reported higher neuroticism and harm avoidance scores. Regarding ASR, inter-individual differences appear to be due to other factors than sex such as, for instance, environmental or genetic variation. For example, ASR heritability has been reported to be in the range of 59–61% [55] and 67% [56]. Nevertheless, previous studies reported conflicting results on the impact of sex on ASR: while some found no difference between men and women [32,35–37] others reported larger startle magnitudes in women at least in some ASR parameters [27,38,39]. Aside from differences in the startle paradigms used, these contradictory findings might be partly the result of varying effects of hormonal contraceptives or the menstrual cycle which were not controlled for in the majority of these studies. In our sample ASR and overall startle magnitudes differed significantly over the course of the menstrual cycle resulting in larger startle magnitudes during ovulation and the late luteal phase. These were the only times when startle magnitudes in women were significantly larger than in men while there were no sex differences during the early and late follicular and the early luteal phases. It should be noted, however, that the size of this effect was only small to moderate (2 = 0.105). Nevertheless, this finding points to the necessity to include information about cycle phase in data analysis if only to reduce the often substantial error variance. If information on menstrual cycle phase is not considered, emerging sex effects might be misinterpreted as a stable differences between men and women when in fact they might be based on fluctuating changes associated with the menstrual cycle. Contrary to our findings, no differences in ASR between follicular and luteal phase were reported in healthy women in several studies [43,45,46]. However, in some of these studies the investigated samples were comparatively small which would have made it harder to detect a rather minor effect. Intriguingly, women with PMDD were found at least in one study to show increased ASR during the luteal phase – despite a small sample size [43] suggesting a considerably larger effect of menstrual cycle phase in a clincal sample. Nevertheless, our finding of elevated ASR during is consistent with numerous reports on affective changes including higher levels of anxiety, depression, irritability or affective lability during the late luteal phase [57]. In general, hormonal changes over the course of the menstrual cycle effect nearly all systems of the body [11, reviews in: 58] including cognitive [12,59] and affective functions [10]. Given the extensive distribution of steroid receptors throughout different tissues and the fact that gonadal steroids modulate transcription of numerous genes, widespread and variable effects of steroids result, many of which are context- and developmental-stage dependent [60]. In several brain regions involved in mood regulation as well as arousal and reward processing, functional changes depending on steroid concentration have been documented including the prefrontal cortex, parietal and temporal cortex, hippocampus, amygdala, and striatum (overview: [60]). Furthermore, both baseline activity of the hypothalamus–pituitary–adrenal (HPA) axis and its response to stress are regulated by ovarian steroids [60]. Declining progesterone levels in connection with changes in the levels of its metabolite allopregnanolone, which interacts with GABA receptors, have been suggested as possible underlying cause of premenstrual dysphoria (overiew in [61]). Progesterone withdrawal (PWD) in female rats was found to result in an elevated ASR, providing an animal model supporting this notion [42]. However, declining progesterone levels cannot be the sole reason for

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heightened negative affect since in some women symptoms of emotional negativity start at ovulation and during the early luteal phase, i.e. before the progesterone decline occurs. Furthermore, progesterone administration during the late luteal phase does not lead to improvement in PMDD patients (review: [62]). Thus, alternatively, it has been suggested that mood changes during the late luteal phase might be the result of the preovulatory estradiol peak, or the postovulatory increase in progesterone, or both [62]. Furthermore, no differences in the production of gonadal steroids in women with vs. without PMS have been found suggesting that it might not be the fluctuation of steroid hormonal concentrations per se but rather an enhanced responsiveness to these changes that leads to late luteal symptoms [62]. Additional modulating factors include serotonin since serotonergic agents (e.g., SSRIs) are an effective treatment for premenstrual symptoms including negative affect. Furthermore, dopamine [63,64], endogenous opiod peptides [65] and the corticotrophin releasing factor (CRF; [66]) interact with changing steroids and thus might also influence physiological as well as affective changes across the menstrual cycle. In sum, late luteal phase has been found to be consistently associated with reduced well-being marked by mental as well as physiological symptoms in a substantial portion of premenopausal women [57,67]. Several biological mechanisms including changes in the concentration of gonadal hormones and their metabolites as well as their interactions with neurotransmitter and hormonal systems have been suggested as contributing factors in PMS etiology [62,65,68]. Our finding of larger startle responses during the late luteal phase is thus probably an indicator of a general shift towards heightened negative emotionality at the end of the menstrual cycle even in non-clinical females. Self-report data showed a general pattern of reduced anxiety and neuroticism during mid-cycle compared to other cycle phases. There were significant differences between late luteal phase – marked by higher anxiety scores – and ovulation, but there were for instance no significant differences between late luteal and early follicular phase. Thus, while higher anxiety scores during late luteal phase match larger startle magnitudes at that time period, this does not apply to other cycle phases. Overall, changes in startle magnitudes and in self-report data across the menstrual cycle were not synchronous. However, it has been repeatedly shown that physiological parameters are not always consistent with accompanying self-reports [69–71]. More importantly, different mechanisms underlying larger startle responses during ovulation and the late luteal phase, respectively, need to be taken into account. While the late luteal phase has been consistently characterized by higher anxiety, depression and irritability, the mid-cycle phase has been repeatedly associated with heightened well-being and self-esteem (review: [11]). The results of the self-report measures in this study are partly in line with these findings. Hence, our finding of larger startle magnitudes during ovulation stands at first glance in contrast to the questionnaire data as well as to general findings of heightened positive affect during mid-cycle [11]. Ovulation is marked by a surge in luteinizing hormone (LH), peaking follicle-stimulating hormone (FSH) and high estrogen [11]. Estrogen, in turn, has been associated with less negative emotionality contributing to the state of higher well-being during mid-cycle. However, estrogen has also positive effects on hearing. Women have not only been found to have a somewhat better hearing than men in general [11,72], but to show increased hearing sensitivity during ovulation [73,74], which might have contributed to the larger startle magnitudes in response to acoustic startle stimuli. Nevertheless, it should be noted that there are also some inconsistent findings regarding functional changes in the auditory system across the menstrual cycle (review: [74]). Additionally, positive effects of estrogen on general CNS arousal [75,76] might have also influenced startle magnitudes.

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Furthermore, effects of estrogen on muscle strength and on motor activity might be considered. The mid-cycle phase has also been associated with increased muscle strength [77,78], there are, however, several inconsistent findings reporting no influence of cycle phase on muscle strength [79–81] or muscle and tendon properties [82]. Furthermore, none of these studies investigated facial muscles, which limits their comparability to our results. Affective startle modulation was not significantly influenced by sex in our sample which is in line with some previous findings [28,29] but contrary to other findings [25–27]. There was also no effect of menstrual cycle on FPS or PAS although there were sex differences in the valence and arousal ratings of the affective pictures. Consistent with previous reports (review: [25]) unpleasant pictures were rated more negative and more arousing by women while men rated pleasant pictures more positive. This effect was further modulated by menstrual cycle phase and sex differences and was only present during the late follicular and late luteal phase. The rating pattern during the late luteal phase is consistent with other findings of increased negative emotionality [57] which is, however, not true for the late follicular phase. Nevertheless, our results suggest that the often reported sex differences in valence and arousal ratings (review: [25]) might not be a stable trait but shifting depending on cycle phase. Also, the discrepancy between subjective ratings being influenced by sex while startle modulation by the same set of stimuli did not differ between men and women, emphasizes again the often observed incongruity between physiological parameters and self-reports [69–71]. The three different onset times resulted in the expected differences in startle magnitudes with the shortest onset time (0.5 s) resulting in the smallest response while 4.5 s lead to the largest responses. This effect was not significantly influenced by menstrual cycle phase while sex showed only a small effect (2 = 0.017) which was mostly due to the shortest onset time (0.5 s). Taken together, the underlying biological mechanisms of changes in overall startle magnitudes across the menstrual cycle are probably impacted by changing ovarian steroid concentrations in interaction with further neurotransmitter and hormonal pathways. Startle magnitude has been independently found to be impacted by neuromodulators such as dopamine and norepinephrine (e.g., [83–85]), serotonin (e.g., [86–88]) or CRF (overview: [89]) which also interact with each other (e.g., [90]). In turn, estrogen as well as progesterone impact the release of norepinephrine, dopamine and serotonin via a wide variety of mechanisms including effects on pre- and postsynaptic receptors and neurotransmitter release (for a review see: [91]). Estrogen also impacts HPA-axis function by down-regulating CRF gene transcription [66]. Furthermore, this interacting network of hormonal and neurotransmitter pathways probably also contributed to the observed differences in affective picture rating. Interestingly, while there was no effect of sex on the affective startle modulation the same set of pictures was rated somewhat differently by men and women pointing to additional biological as well as probably cultural influence factors. There are several limitations to this study. Firstly, we investigated only physically and psychologically healthy individuals. Since anxiety-related disorders have been reported to be more frequent in women, particularly our female sub-sample might not be representative of the general population. Thus, our findings need to be replicated in population based samples. Secondly, we only included women who did not use hormonal contraceptives. Although usage varies widely across different cultures, at least in most western countries a substantial percentage of women between the ages of 16–49 years uses hormonal contraceptives [92]. Hormonal contraceptives have been found to influence several psychophysiological parameters of affective processing such as for instance the cortisol stress response [93]. Future studies on the startle reflex should thus also investigate the potential

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effects of hormonal contraceptives. Thirdly, our findings regarding the influence of menstrual cycle phase are based on cross-sectional data and should be replicated in samples using longitudinal data (such as in the study by [45]; but preferably testing over the course of several cycles). Fourthly, menstrual cycle phase was identified based solely on participants’ information and should in future studies assessed by the use of hormone levels. In light of the inconsistent findings regarding sex differences in ASR magnitudes, the implications of our results seem nevertheless clear: menstrual cycle phase should be included when investigating startle parameters. We strongly advocate for a consideration of such effects in order to reduce error variance. Furthermore, our results suggest that contrary to findings investigating self-reports of fear and anxiety, non-clinical women – when averaged across the menstrual cycle – and men do not show significant differences in overall startle responses. Only during the late luteal phase and during ovulation women showed higher ASR magnitudes. Our findings also underline the need for reporting and discussing effect sizes. In addition, potential reasons for inconsistencies in changes of physiological vs. self-rated anxiety parameters across the menstrual cycle should be addressed. Acknowledgements The authors would like to thank Ulrich Buhss for excellent work in processing and analyzing the EMG data. This work was supported by the Deutsche Forschungsgemeinschaft (KI 537/20-1, 20-3). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2014.08.013. References [1] Pigott TA. Gender differences in the epidemiology and treatment of anxiety disorders. J Clin Psychiatry 1999;60(Suppl 18):4–15. [2] McLean CP, Anderson ER. Brave men and timid women? A review of the gender differences in fear and anxiety. Clin Psychol Rev 2009;29:496–505. [3] Cahill L. Why sex matters for neuroscience. Nat Rev Neurosci 2006;7:477–84. [4] Hines M. Gender development and the human brain. Annu Rev Neurosci 2011;34:69–88. [5] Hyde JS. The gender similarities hypothesis. Am Psychol 2005;60:581–92. [6] Lacey JI, Lacey BC. Verification and extension of the principle of autonomic response-stereotypy. Am J Psychol 1958;71:50–73. [7] Bell EC, Willson MC, Wilman AH, Dave S, Silverstone PH. Males and females differ in brain activation during cognitive tasks. NeuroImage 2006;30:529–38. [8] Halari R, Sharma T, Hines M, Andrew C, Simmons A, Kumari V. Comparable fMRI activity with differential behavioural performance on mental rotation and overt verbal fluency tasks in healthy men and women. Exp Brain Res 2006;169:1–14. [9] Toufexis DJ, Myers KM, Davis M. The effect of gonadal hormones and gender on anxiety and emotional learning. Horm Behav 2006;50:539–49. [10] van Wingen GA, Ossewaarde L, Bäckström T, Hermans EJ, Fernandez G. Gonadal hormone regulation of the emotion circuitry in humans. Neuroscience 2011;191:38–45. [11] Farage MA, Osborn TW, MacLean AB. Cognitive, sensory, and emotional changes associated with the menstrual cycle: a review. Arch Gynecol Obstet 2008;278:299–307. [12] Schöning S, Engelien A, Kugel H, Schäfer S, Schiffbauer H, Zwitserlood P, et al. Functional anatomy of visuo-spatial working memory during mental rotation is influenced by sex, menstrual cycle, and sex steroid hormones. Neuropsychologia 2007;45:3203–14. [13] Koch M. The neurobiology of startle. Prog Neurobiol 1999;59:107–28. [14] Blumenthal TD, Cuthbert BN, Filion DL, Hackley S, Lipp OV, van Boxtel A. Committee report: guidelines for human startle eyeblink electromyographic studies. Psychophysiology 2005;42:1–15. [15] Davis M, Falls WA, Campeau S, Kim M. Fear-potentiated startle: a neural and pharmacological analysis. Behav Brain Res 1993;58:175–98. [16] Leaton RN, Cranney J. Potentiation of the acoustic startle response by a conditioned stimulus paired with acoustic startle stimulus in rats. J Exp Psychol Anim Behav Process 1990;16:279–87. [17] Alhadad SS, Lipp OV, Purkis HM. Modality-specific attentional startle modulation during continuous performance tasks: a brief time is sufficient. Psychophysiology 2008;45:1068–78.

[18] Filion DL, Dawson ME, Schell AM. The psychological significance of human startle eyeblink modification: a review. Biol Psychol 1998;47:1–43. [19] Lang PJ, Bradley MM, Cuthbert BN. Emotion, attention, and the startle reflex. Psychol Rev 1990;97:377–95. [20] Vrana SR, Spence EL, Lang PJ. The startle probe response: a new measure of emotion? J Abnorm Psychol 1988;97:487–91. [21] Bradley MM, Codispoti M, Cuthbert BN, Lang PJ. Emotion and motivation I: defensive and appetitive reactions in picture processing. Emotion 2001;1:276–98. [22] Codispoti M, Bradley MM, Lang PJ. Affective reactions to briefly presented pictures. Psychophysiology 2001;38:474–8. [23] Globisch J, Hamm AO, Esteves F, Ohman A. Fear appears fast: temporal course of startle reflex potentiation in animal fearful subjects. Psychophysiology 1999;36:66–75. [24] Stanley J, Knight RG. Emotional specificity of startle potentiation during the early stages of picture viewing. Psychophysiology 2004;41:935–40. [25] Bradley MM, Codispoti M, Sabatinelli D, Lang PJ. Emotion and motivation II: sex differences in picture processing. Emotion 2001;1:300–19. [26] Gard MG, Kring AM. Sex differences in the time course of emotion. Emotion 2007;7:429–37. [27] Bianchin M, Angrilli A. Gender differences in emotional responses: a psychophysiological study. Physiol Behav 2012;105:925–32. [28] Dichter GS, Tomarken AJ, Baucom BR. Startle modulation before, during and after exposure to emotional stimuli. Int J Psychophysiol 2002;43:191–6. [29] Hillman CH, Rosengren KS, Smith DP. Emotion and motivated behavior: postural adjustments to affective picture viewing. Biol Psychol 2004;66:51–62. [30] Swerdlow NR, Auerbach P, Monroe SM, Hartston H, Geyer MA, Braff DL. Men are more inhibited than women by weak prepulses. Biol Psychiatry 1993;34:253–60. [31] Kumari V, Gray JA, Gupta P, Luscher S, Sharma T. Sex differences in prepulse inhibition of the acoustic startle response. Pers Indiv Differ 2003;35:733–42. [32] Kumari V, Aasen I, Papadopoulos A, Bojang F, Poon L, Halari R, et al. A comparison of prepulse inhibition in pre- and postmenopausal women and age-matched men. Neuropsychopharmacology 2008;33:2610–8. [33] Aasen I, Kolli L, Kumari V. Sex effects in prepulse inhibition and facilitation of the acoustic startle response: implications for pharmacological and treatment studies. J Psychopharmacol 2005;19:39–45. [34] Blumenthal TD, Elden A, Flaten MA. A comparison of several methods used to quantify prepulse inhibition of eyeblink responding. Psychophysiology 2004;41:326–32. [35] Anokhin AP, Golosheykin S. Startle modulation by affective faces. Biol Psychol 2010;83:37–40. [36] Ludewig K, Ludewig S, Seitz A, Obrist M, Geyer MA, Vollenweider FX. The acoustic startle reflex and its modulation: effects of age and gender in humans. Biol Psychol 2003;63:311–23. [37] Hubbard CS, Ornitz E, Gaspar JX, Smith S, Amin J, Labus JS, et al. Modulation of nociceptive and acoustic startle responses to an unpredictable threat in men and women. Pain 2011;152:1632–40. [38] Kofler M, Muller J, Reggiani L, Valls-Sole J. Influence of gender on auditory startle responses. Brain Res 2001;921:206–10. [39] Quevedo K, Smith T, Donzella B, Schunk E, Gunnar M. The startle response: developmental effects and a paradigm for children and adults. Dev Psychobiol 2010;52:78–89. [40] Koch M. Sensorimotor gating changes across the estrous cycle in female rats. Physiol Behav 1998;64:625–8. [41] Blaszczyk J, Tajchert K. Sex and strain differences of acoustic startle reaction development in adolescent albino Wistar and hooded rats. Acta Neurobiol Exp (Wars) 1996;56:919–25. [42] Gulinello M, Orman R, Smith SS. Sex differences in anxiety, sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal. Eur J Neurosci 2003;17:641–8. [43] Epperson CN, Pittman B, Czarkowski KA, Stiklus S, Krystal JH, Grillon C. Lutealphase accentuation of acoustic startle response in women with premenstrual dysphoric disorder. Neuropsychopharmacology 2007;32:2190–8. [44] Kask K, Gulinello M, Bäckström T, Geyer MA, Sundström-Poromaa I. Patients with premenstrual dysphoric disorder have increased startle response across both cycle phases and lower levels of prepulse inhibition during the late luteal phase of the menstrual cycle. Neuropsychopharmacology 2008;33:2283–90. [45] Jovanovic T, Szilagyi S, Chakravorty S, Fiallos AM, Lewison BJ, Parwani A, et al. Menstrual cycle phase effects on prepulse inhibition of acoustic startle. Psychophysiology 2004;41:401–6. [46] Kumari V, Konstantinou J, Papadopoulos A, Aasen I, Poon L, Halari R, et al. Evidence for a role of progesterone in menstrual cycle-related variability in prepulse inhibition in healthy young women. Neuropsychopharmacology 2010;35:929–37. [47] Lang PJ, Bradley MM, Cuthbert BN. International Affective Picture System (IAPS): instruction manual and affective ratings. Gainesville, FL: Center for Research in Psychophysiology. University of Florida; 1999 [Technical Report A-4]. [48] Ekman P, Friesen WV. Pictures of facial affect. Palo Alto: Consulting Psychologists Press; 1976. [49] Lang PJ. Behavioral treatment and bio-behavioral assessment: computer applications. In: Sidowski JB, Johnson JH, Williams TA, editors. Technology in mental health care delivery systems. Norwood: Ablex; 1980. p. 119–37. [50] Spielberger CD, Gorsuch RL, Lushene HE. STAI manual for the State-Trait Anxiety Inventory. Palo Alto: Consulting Psychologist Press; 1970.

D. Armbruster et al. / Behavioural Brain Research 274 (2014) 326–333 [51] Costa PT, McCrae RR. Revised NEO Personality Inventory (NEO PI-R) and NEO Five-Factor Inventory (NEO-FFI). Odessa: Psychological Assessment Resources; 1992. [52] Cloninger CR, Svrakic DM, Przybeck TR. A psychobiological model of temperament and character. Arch Gen Psychiatry 1993;50:975–90. [53] Miller MW, Gronfier C. Diurnal variation of the startle reflex in relation to HPAaxis activity in humans. Psychophysiology 2006;43:297–301. [54] Frankland PW, Ralph MR. Circadian modulation in the rat acoustic startle circuit. Behav Neurosci 1995;109:43–8. [55] Anokhin AP, Golosheykin S, Heath AC. Genetic and environmental influences on emotion-modulated startle reflex: a twin study. Psychophysiology 2007;44:106–12. [56] Hasenkamp W, Epstein MP, Green A, Wilcox L, Boshoven W, Lewison B, et al. Heritability of acoustic startle magnitude, prepulse inhibition, and startle latency in schizophrenia and control families. Psychiatry Res 2010;178: 236–43. [57] Halbreich U, Borenstein J, Pearlstein T, Kahn LS. The prevalence, impairment, impact, and burden of premenstrual dysphoric disorder (PMS/PMDD). Psychoneuroendocrinology 2003;28(Suppl 3):1–23. [58] Farage MA, Neill S, MacLean AB. Physiological changes associated with the menstrual cycle: a review. Obstet Gynecol Surv 2009;64:58–72. [59] Hausmann M, Slabbekoorn D, Van Goozen SH, Cohen-Kettenis PT, Güntürkün O. Sex hormones affect spatial abilities during the menstrual cycle. Behav Neurosci 2000;114:1245–50. [60] Rubinow DR, Schmidt PJ, Pathophysiology I:. The role of ovarian steroids. In: O’Brien PMS, Rapkin AJ, Schmidt PJ, editors. The premenstrual syndromes: PMS and PMDD. London: Informa; 2007. p. 83–98. [61] Cunningham J, Yonkers KA, O’Brien S, Eriksson E. Update on research and treatment of premenstrual dysphoric disorder. Harv Rev Psychiatry 2009;17:120–37. [62] Yonkers KA, O’Brien PM, Eriksson E. Premenstrual syndrome. Lancet 2008;371:1200–10. [63] Küppers E, Ivanova T, Karolczak M, Beyer C. Estrogen: a multifunctional messenger to nigrostriatal dopaminergic neurons. J Neurocytol 2000;29:375–85. [64] Dluzen D, Horstink M. Estrogen as neuroprotectant of nigrostriatal dopaminergic system: laboratory and clinical studies. Endocrine 2003;21:67–75. [65] Rapkin AJ, Kuo J. Neurotransmitter physiology: the basics for understanding premenstrual syndrome. In: O’Brien PMS, Rapkin AJ, Schmidt PJ, editors. The premenstrual syndromes: PMS and PMDD. London: Informa; 2007. p. 69– 81. [66] De Nicola AF, Saravia FE, Beauquis J, Pietranera L, Ferrini MG. Estrogens and neuroendocrine hypothalamic–pituitary–adrenal axis function. Front Horm Res 2006;35:157–68. [67] Wittchen HU, Becker E, Lieb R, Krause P. Prevalence, incidence and stability of premenstrual dysphoric disorder in the community. Psychol Med 2002;32:119–32. [68] Halbreich U. The etiology, biology, and evolving pathology of premenstrual syndromes. Psychoneuroendocrinology 2003;28(Suppl 3):55–99. [69] Gross JJ, Levenson RW. Emotional suppression – physiology, self-report, and expressive behavior. J Pers Soc Psychol 1993;64:970–86. [70] McNeil DW, Ries BJ, Turk CL. Behavioral assessment: self-report, physiology, and overt behavior. In: Heimberg RG, Liebowitz MR, Hope DA, Schneier FR, editors. Social phobia: diagnosis, assessment, and treatment. New York: Guilford Press; 1995. p. 202–31. [71] Pennebaker JW. The psychology of physical symptoms. New York: SpringerVerlag; 1982.

333

[72] Hultcrantz M, Simonoska R, Stenberg AE. Estrogen and hearing: a summary of recent investigations. Acta Otolaryngol 2006;126:10–4. [73] Al-Mana D, Ceranic B, Djahanbakhch O, Luxon LM. Alteration in auditory function during the ovarian cycle. Hear Res 2010;268:114–22. [74] Al-Mana D, Ceranic B, Djahanbakhch O, Luxon LM. Hormones and the auditory system: a review of physiology and pathophysiology. Neuroscience 2008;153:881–900. [75] Schober J, Weil Z, Pfaff D. How generalized CNS arousal strengthens sexual arousal (and vice versa). Horm Behav 2011;59:689–95. [76] Devidze N, Lee AW, Zhou J, Pfaff DW. CNS arousal mechanisms bearing on sex and other biologically regulated behaviors. Physiol Behav 2006;88:283–93. [77] Sarwar R, Niclos BB, Rutherford OM. Changes in muscle strength, relaxation rate and fatiguability during the human menstrual cycle. J Physiol 1996;493(Pt 1):267–72. [78] Bambaeichi E, Reilly T, Cable NT, Giacomoni M. The isolated and combined effects of menstrual cycle phase and time-of-day on muscle strength of eumenorrheic females. Chronobiol Int 2004;21:645–60. [79] de Jonge XAKJ, Boot CRL, Thom JM, Ruell PA, Thompson MW. The influence of menstrual cycle phase on skeletal muscle contractile characteristics in humans. J Physiol – London 2001;530:161–6. [80] Lebrun CM, Mckenzie DC, Prior JC, Taunton JE. Effects of menstrual-cycle phase on athletic performance. Med Sci Sport Exer 1995;27:437–44. [81] Gür H. Concentric and eccentric isokinetic measurements in knee muscles during the menstrual cycle: a special reference to reciprocal moment ratios. Arch Phys Med Rehab 1997;78:501–5. [82] Kubo K, Miyamoto M, Tanaka S, Maki A, Tsunoda N, Kanehisa H. Muscle and tendon properties during menstrual cycle. Int J Sports Med 2009;30:139–43. [83] Lähdesmäki J, Sallinen J, MacDonald E, Scheinin M. Alpha2A-adrenoceptors are important modulators of the effects of d-amphetamine on startle reactivity and brain monoamines. Neuropsychopharmacology 2004;29:1282–93. [84] Davis M. Neural systems involved in fear-potentiated startle. Ann N Y Acad Sci 1989;563:165–83. [85] Kehne JH, Davis M. Central noradrenergic involvement in yohimbine excitation of acoustic startle: effects of DSP4 and 6-OHDA. Brain Res 1985;330:31–41. [86] Liechti ME, Geyer MA, Hell D, Vollenweider FX. Effects of MDMA (ecstasy) on prepulse inhibition and habituation of startle in humans after pretreatment with citalopram, haloperidol, or ketanserin. Neuropsychopharmacology 2001;24:240–52. [87] McQueen DA, Overstreet DH, Ardayfio PA, Commissaris RL. Acoustic startle, conditioned startle potentiation and the effects of 8-OH-DPAT and buspirone in rats selectively bred for differences in 8-OH-DPAT-induced hypothermia. Behav Pharmacol 2001;12:509–16. [88] Mansbach RS, Geyer MA. Blockade of potentiated startle responding in rats by 5-hydroxytryptamine1A receptor ligands. Eur J Pharmacol 1988;156:375–83. [89] Risbrough VB, Stein MB. Role of corticotropin releasing factor in anxiety disorders: a translational research perspective. Horm Behav 2006;50:550–61. [90] Conti LH. Interactions between corticotropin-releasing factor and the serotonin 1A receptor system on acoustic startle amplitude and prepulse inhibition of the startle response in two rat strains. Neuropharmacology 2012;62:256–63. [91] Zheng P. Neuroactive steroid regulation of neurotransmitter release in the CNS: action, mechanism and possible significance. Prog Neurobiol 2009;89:134–52. [92] United Nations Department of Economic and Social Affairs PD. World Contraceptive Use 2010 2011. [93] Kirschbaum C, Kudielka BM, Gaab J, Schommer NC, Hellhammer DH. Impact of gender, menstrual cycle phase, and oral contraceptives on the activity of the hypothalamus–pituitary–adrenal axis. Psychosom Med 1999;61:154–62.