iScience Article

Human ovulatory phase-increasing odors cause

positive emotions and stress-suppressive effects in
males
Graphical abstract Authors
Nozomi Ohgi, Mika Shirasu,
Yusuke Ogura, ..., Rieko Kawamura,
Hirosato Takikawa, Kazushige Touhara

Correspondence
ktouhara@g.ecc.u-tokyo.ac.jp

In brief
Human physiology; Natural sciences;
Chemistry; Biological sciences

Highlights
• Axillary odor is more pleasant, fragrant, and less vinegary
during the ovulatory phase

• The three compounds that increase during the ovulatory

phase were identified

• Ovulatory phase compounds reduce unpleasantness of

axillary odor and enhance its preference

• Ovulatory phase compounds reduce axillary odor stress and

enhance female face impressions

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Article
Human ovulatory phase-increasing
odors cause positive emotions
and stress-suppressive effects in males
Nozomi Ohgi,1,3 Mika Shirasu,1,3 Yusuke Ogura,1 Yukei Hirasawa,1 Masako Okamoto,1 Rieko Kawamura,1
Hirosato Takikawa,1 and Kazushige Touhara1,2,4,*
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
2International Research Center for Neurointelligence (WPI-IRCN), Institutes for Advanced Study, The University of Tokyo, Tokyo, Japan
3These authors contributed equally
4Lead contact

*Correspondence: ktouhara@g.ecc.u-tokyo.ac.jp
https://doi.org/10.1016/j.isci.2025.113087

SUMMARY

In many animals, olfactory cues are key for recognizing their sex, kinship, and reproductive status. In humans,
it has been proposed that males find female body odors attractive during the ovulatory phase, coinciding with
peak fertility. However, the molecular and physiological foundations of this attractiveness remain unclear. In
this study, we combined sensory evaluation with gas chromatography-mass spectrometry for chemical anal­
ysis and identified volatile compounds that fluctuate across menstrual cycle phases. We identified three
ovulatory phase-increasing compounds that mitigate the basal unpleasant axillary odor, resulting in the
ovulatory-phase body odor being perceived as the most pleasant by males. Additionally, these compounds
alleviated hostility and stress induced by the basal axillary odor, leading to relaxation in males and an
enhanced positive impression of female facial images. Our results suggest that several ovulatory phase-
increasing compounds may contribute to the positive effects of ovulatory female odor on males, as proposed
in previous research.

INTRODUCTION dent on female hormones.9 It has been suggested that women’s

behavior and physical attractiveness may serve as identifiable
In many species, female body odors reflect their reproductive cues to fertility,10 and that men’s preferences are likely to in­
status, and odors around the time of ovulation attract males, re­ crease for physical cues associated with high estrogen
sulting in behavioral and/or physiological changes. For instance, levels.11,12 However, in a recent study by Zetzsche et al., no dif­
male hamsters are attracted to estrous vaginal discharge, facili­ ference in pleasantness was observed in axillary samples
tating their copulatory behavior.1 In some non-human primates, collected overnight for 12 h at donor’s home during the men­
ovulatory odors from females induce inspection or exploratory strual cycle.13 In the same study, chemical analysis using ther­
behavior in males.2,3 Common marmosets exhibit an increase mal desorption (TD) tubes showed no correlation between axil­
in male testosterone levels in response to the ovulatory scent lary odor composition and fertility states. Therefore, while
emitted from females.4 Olfactory cues play a crucial role in suc­ female axillary odors may convey fertility-related information,
cessful mating in many animals, prompting the intriguing ques­ some inconsistencies still exist.
tion of whether humans utilize olfaction for male-female In this study, we explore various methods of axillary odor
interaction. collection and, using a more careful and rigorous approach,
In humans, several studies have suggested that female body aim to investigate whether body odor communicates information
odor signals periods of high fertility. For instance, vaginal secre­ about the menstrual cycle. First, we extended previous sensory
tions collected during the preovulatory and ovulatory phases are evaluation studies to investigate not only pleasantness of odors
rated as more pleasant than those from the menstrual, early but also perceived odor quality across the menstrual cycle. Sec­
luteal, or late luteal phases.5 Additionally, axillary odors during ond, we aimed to identify chemical profile of axillary odor at each
the fertile phase are perceived by men as more pleasant, sexy, phase of the menstrual cycle using gas chromatography-mass
or attractive compared to those during the menstrual and luteal spectrometry (GC/MS). We also aim to examine whether ovula­
phases.6,7 Another study indicated that high-fertility scents tory phase odors induce positive emotions and psychological ef­
collected from the armpit were chosen as more attractive than fects in males. The overarching goal is to address the question of
low-fertility scents.8 The fact that hormonal contraception results whether olfactory communication exists to facilitate relation­
in the loss of these effects suggests that attractiveness is depen­ ships between males and females in humans.

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Figure 1. Sensory evaluation of female axil­
lary odor during the menstrual cycle
Pleasantness (A) and intensity (B) ratings of 84
axillary odor samples collected from 21 females
during four phases (M: menstrual, F: follicular, O:
ovulatory, L: luteal) by male raters (n = 21).
Pleasantness was rated on a scale from -4
(extremely unpleasant) to +4 (extremely pleasant),
and intensity was rated on a scale from 0 (not
perceived) to 6 (extremely intense). Error bars
show standard errors. The effect of menstrual
phase on rating values was examined using a
linear mixed model (LMM), with rating values as
the dependent variable and menstrual cycle phase
as the explanatory variable. Statistical signifi­
cance of the differences between the O phase and
each of the other phases was evaluated by testing
the significance of the beta values (see STAR
Methods for details). *p < 0.05 and **p < 0.01.
See also Figure S1 and Table S1.

RESULTS were excluded because they may not accurately assess odor
quality (STAR Methods). The selection rate of 15 descriptors,
Axillary odor during the ovulatory phase is more based on quantitative descriptive analysis (QDA, see STAR
pleasant than that during the menstrual phase Methods), chosen by male raters (n = 16) to describe the axillary
To assess the intensity and pleasantness of axillary odor across samples is presented in Table 1. Among the 15 descriptors, with
different phases of the menstrual cycle, axillary odors were a focus on those with higher frequency of selection, ‘‘vinegary
collected on gauze from 21 female donors at each of the four odor’’ was checked 24–28 times in M, F, and L-phases, and 17
stages of their menstrual cycle (i.e., M[enstrual], F[ollicular], O times in the O-phase, with a lower number of selections in the
[vulatory], L[uteal]-phases; a total of 84 odor samples) O-phase. In contrast, ‘‘fragrant odor’’ was examined 37–40 times
(Figure S1A). Each phase of the menstrual cycle was determined in the M-, F-, and L-phases, and 53 times in the O-phase, with a
by the luteinizing hormone (LH) surge and body temperature, and higher number of selections in the O-phase. All these results sug­
the amounts of estradiol and progesterone in saliva were evalu­ gest that axillary odors during the O-phase are relatively
ated for some donors (Figure S1B). Given that previous reports pleasant.
suggest differences in body odor under stress from normal
body odor,14,15 we used the state-trait anxiety inventory (STAI) Chemical composition analysis of axillary odor identified
score to assess the anxiety state of the donors at the time of increasing or decreasing compounds for each phase
sample collection. No significant difference in anxiety state, as To explore the chemical profiles of female axillary odors during
indicated by the STAI score, was observed among the four the M-, F-, O-, and L-phases, we analyzed volatiles emitted
phases (see STAR Methods). Out of the 84 odor samples, each from the axilla during the menstrual cycle in 21 female donors,
male rater (n = 21) provided odor ratings (intensity and pleasant­ the same donors as in the previous section, using GC/MS.
ness) on samples from 10 to 11 donors (40–44 samples in total). Initially, we compared four different adsorbents, including sterile
Linear mixed model (LMM) analyses revealed that axillary odor gauze, polydimethylsiloxane (PDMS) membranes, Monotrap,
during the O-phase was rated significantly more pleasant than and Twister, for a female (Table S2 and Method for detailed sam­
those during M and L-phases (M vs. O: t(846) = 3.1, p = 0.006, ple preparation and analytical methods for each adsorbent). We
L vs. O: t(846) = 2.4, p = 0.048, Figure 1 and Table S1). There found that the PDMS membrane adsorbed the largest number of
was also a tendency for pleasantness to be higher compared compounds (Figure S2) and that its close fit to the armpit allowed
to the F-phase (F vs. O: t(846) = 2.2, p = 0.077, Figure 1 and for the efficient adsorption of low-volatile compounds such as
Table S1). When excluding raters with lower olfactory ability long-chain and unsaturated fatty acids secreted from the axillary
(Methods), the O-phase showed significantly higher pleasant­ surface (see Tables S3 and S4). Therefore, we used the PDMS
ness compared to the M, F, and L-phases (M vs. O: t(636) = membrane to sample axillary odors.
2.8, p = 0.017, F vs. O: t(636) = 2.5, p = 0.043, L vs. O: t(636) = Subsequently, 84 axillary odor samples (four-phase samples
2.4, p = 0.045, Table S1). These findings suggest that the from 21 female donors) collected using the PDMS membrane
O-phase tends to have a pleasant odor, with this tendency being were subjected to analysis using GC/MS combined with a ther­
more pronounced among raters with higher olfactory abilities. mal desorption system (Figure S3). Among numerous peaks
No statistically significant differences in intensity were observed identified following GC/MS analyses, we focused on compounds
among the phases. that were structurally identified using an authentic sample or that
Next, the axillary odor quality at each of the four phases was existed in more than five donors, at least in one phase. The re­
investigated using the check-all-that-apply (CATA) sensory sulting 98 peaks were further analyzed (average occurrence
profiling method. Raters with low odor identification quality rate in all phases of all compounds = 71%, see STAR

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Table 1. Sensory evaluation of the odor quality of female axillary odor during the menstrual cycle
Male raters, n = 16
Number of selections Selection rate (%) Cochran’s
Descriptor Q test Effect
Descriptor selection rate (%) M F O L M F O L (p value) size (η2Q)
Pleasant Fragrant 25% 37 37 53 40 22% 22% 32% 24% 0.044 0.016
Woody/Pencil-like 11% 16 23 15 17 23% 32% 21% 24% 0.41 0.0056
Sweet 6% 11 8 12 9 28% 20% 30% 23% 0.73 0.0026
Milky 5% 11 7 11 8 30% 19% 30% 22% 0.65 0.0032
Citrus 4% 5 6 13 6 17% 20% 43% 20% 0.14 0.011
Grassy/Green 4% 6 4 10 6 23% 15% 38% 23% 0.31 0.0070
Earthy 3% 4 7 4 2 24% 41% 24% 12% 0.22 0.0086
Yogurt 2% 4 0 8 3 27% 0% 53% 20% 0.027 0.018
Unpleasant Sweaty 29% 51 55 45 46 26% 28% 23% 23% 0.40 0.0058
Vinegary 14% 26 24 17 28 27% 25% 18% 29% 0.17 0.010
Sebaceous 13% 21 23 21 25 23% 26% 23% 28% 0.82 0.0019
Damp clothing 8% 15 13 12 14 28% 24% 22% 26% 0.90 0.0019
Dusty 6% 7 10 11 11 18% 26% 28% 28% 0.72 0.0027
Musty 5% 13 10 3 7 39% 30% 9% 21% 0.048 0.016
Stinky 5% 9 8 7 8 28% 25% 22% 25% 0.96 0.00061
Evaluation of the sensory profile of female axillary odors at each phase of the menstrual cycle using the check-all-that-apply (CATA) method (male
raters, n = 16). Descriptor selection rate indicates ‘‘number of descriptor selections/number of raters (16 males) × number of axillary odor samples
evaluated by each male rater (40 or 44 samples).’’ Number of selections indicates the ‘‘number of descriptor selections for each phase samples.’’ Se­
lection rate indicates ‘‘number of selections/number of total selection from M to L phase.’’ Cochran’s Q-test was used to evaluate the differences in
axillary odors between the four different phases.

Methods). To mitigate the effect of inter-individual data vari­ p values were 1,3-butanediol (ID41) (Z = 2.6, p = 0.005, r = 0.56),
ability, the peak area values were standardized using a robust octadecane (ID#45) (Z = 2.7, p = 0.0032, r = 0.60), and
standardization method. To discern changes in the secretion of 1-pentadecanol (ID#72) (Z = 2.6, p = 0.0043, r = 0.58)
the 98 compounds during the menstrual cycle, Ward’s method (Figures 2 and 3; Tables S3 and S4). In the L-phase, four com­
cluster analysis was performed using the mean of the robust Z pounds significantly increased, and the identified compounds
scores for each time period. The 98 compounds were arranged were hydrogen sulfide (ID#1) (Z = 2.1, p = 0.02, r = 0.45),
so that compounds with similar changes between the four 2-pyrrolidone (ID#58) (Z = 2.3, p = 0.01, r = 0.51), and hexahy­
phases were located in close proximity (Figure 2). This analysis dro-2H-azepin-2-one (ID#68) (Z = 1.9, p = 0.029, r = 0.41)
reveals several groups of compounds that tend to decrease dur­ (Tables S3 and S4).
ing the M-phase (top cluster) or increase during the O- and
F-phases (top or middle cluster, respectively). Men perceive the odor of ovulatory phase-increasing
To identify increasing or decreasing compounds at each compounds as more pleasant and comforting
phase, we searched for compounds that significantly increased In conjunction with the sensory evaluation (refer to Figure 1) and
above the median of all phases for each phase. A one-sample GC/MS analysis (refer to Figures 2 and 3), (E)-geranylacetone,
Wilcoxon signed-rank test (one-tailed) was performed to deter­ tetradecanoic acid, and (Z)-9-hexadecenoic acid emerged as
mine whether the amount of the compound in each phase was potential compounds contributing to the reduction of the un­
greater than 0 (median of the robust Z score). Out of the 98 com­ pleasantness of axillary odor during the O-phase. To probe
pounds, seven significantly increased during the O-phase how the increase in these three compounds during the
(Figures 2 and 3; Tables S3 and S4). Three compounds were O-phase influences the quality of axillary odor for men, we exam­
identified as (E)-geranylacetone (ID#49) (Z = 2.1, p = 0.016, r = ined changes in odor quality when the odor-O (odor-O: three
0.47), tetradecanoic acid (ID#86) (Z = 1.7, p = 0.043, r = 0.38), O-phase-increasing compounds) was introduced to a basal axil­
and (Z)-9-hexadecenoic acid (ID#94) (Z = 2.1, p = 0.019, r = lary odor, closely mimicking axillary odor16 (odor-A: a basal axil­
0.45) (Figure S4). In the M-phase, four compounds significantly lary odor), consistently present in the female axilla throughout
increased, and the identified compounds were octanal (ID#15) the menstrual cycle with little or no difference (refer to Star
(Z = 1.9, p = 0.031, r = 0.41) and 1-tetradecanol (ID#65) (Z = Methods and Table S6). Sensory evaluations for four odor stimuli
1.7, p = 0.045, r = 0.37) (Tables S3 and S4). The remaining two (Ctrl, no odor; odor-O, three O-phase-increasing compounds;
compounds with small p value (like ID#2) were unknown (not odor-A, basal axillary odor; odor-A+O, a mixture of odor-A and
identified) but representative m/z was obtained (Table S10). In odor-O; Table S6) were conducted on 72 male raters. The eval­
the F-phase, 26 compounds significantly increased, and of the uation terms included the 15 descriptors in Table 1, along with
15 compounds that could be identified, the three with the lowest intensity, pleasantness, preference, feminine/masculine, relax,

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and arousal/excitement. Figure 4 illustrates the results for 13 tering other participants, sat calmly in a chair, and were initially
terms where significant differences were observed in the sensory asked to perform a baseline multiple mood scale (MMS,
evaluation (all results are available in Table S7). Odor-O was Table S8) (a type of psychological test) assessment and saliva
weakly perceptible in intensity (t(64) = 5.4, q < 0.0005, r = collection (base condition). In session 1–3, the raters then wore
0.56), and the overall odor quality was deemed pleasant with a headset with a microphone, in which the experimenter
fragrant (t(64) = 3.9, q < 0.005, r = 0.44), citrus (t(64) = 3.1, applied an odor to the microphone portion without informing
q < 0.05, r = 0.36), and relaxing (t(60) = 3.6, q < 0.005, r = the raters, and rated female face images (impression rating)
0.42). The addition of odor-O to odor-A led to significantly higher and completed MMS, followed by saliva collection immediately
ratings for pleasantness (t(71) = 10.1, q < 0.0005, r = 0.77) and after removing the headset. The 19 male rater had counter-
preference (t(71) = 9.5, q < 0.0005, r = 0.75), a more feminine rat­ balanced exposure to Ctrl or odor-O in session 1 and 2. Of
ing for feminine/masculine (t(69) = 4.9, q < 0.0005, r = 0.51), and the 19 male raters, 9 were exposed to odor-A, and 10 were
lower ratings for intensity (t(71) = 4.0, q < 0.0005, r = 0.43) (odor-A exposed to odor-A+O in session 3.
+O in Figure 4A). Fragrant (t(71) = 2.9, q < 0.05, r = 0.32), citrus (t The analysis of MMS in sessions 1 and 2 (Figure 5A) revealed
(71) = 2.6, q < 0.05, r = 0.29), woody (t(71) = 3.7, q < 0.005, r = that ‘‘hostility’’ was significantly lower in the odor-O condition
0.40), sweet (t(71) = 2.3, q < 0.05, r = 0.27), and relaxing compared to the Ctrl condition (no odor) (q = 3.7, p = 0.05,
(t(65) = 3.6, q < 0.005, r = 0.41) received significantly higher rat­ d = 0.66) (Figure 5B, top panel, second from the left; Table
ings, whereas sweaty (t(71) = 5.0, q < 0.0005, r = 0.51), damp S14). The impression rating tasks during sessions showed a sig­
clothing (t(71) = 2.9, q < 0.05, r = 0.33), sebaceous (t(71) = 2.3, nificant increase in ‘‘boredom’’ (q = 4.2, p = 0.025, d = 0.90) and a
q < 0.05, r = 0.26), and stinky (t(71) = 4.8, q < 0.0005, r = 0.50) decrease in ‘‘concentration’’ (q = 4.5, p = 0.016, d = 0.84) in the
received significantly lower ratings in odor-A+O (Table S12). Ctrl condition compared to the base condition (before session 1),
This observation aligns with the finding that male raters associ­ with no significant difference observed between the odor-O and
ated fragrant and citrus scents when rating the ovulatory female base conditions (Figure 5B, top panel; Table S14). These findings
axillary odor (Table 1). suggest that odor-O has a positive psychological effect that
We posited a hypothesis that the three O-phase-increasing might decrease the feeling of ‘‘hostility.’’
compounds (odor-O) are pivotal female axillary odors evoking In session 3, a comparison of the scales for each odor condi­
pleasant and positive emotional changes in males. Thus, we tion and the corresponding control condition demonstrated
tested the effects of adding each of the odor-O compounds a significant decrease in ‘‘relaxation’’ in the odor-A condition
to odor-A for 37 male raters in terms of pleasantness. A single (t(6) = 2.5, p = 0.047, d = 0.69). However, this decrease was miti­
odor-O compound significantly reduced the unpleasantness of gated by adding odor-O into odor-A (odor-A+O condition)
odor-A, although the combined effect of the odor-O mix was (Figure 5B, middle and lower panel, forth column from the right;
more pronounced than that of a single compound (Figure 4B, Table S14). These results suggest that odor-O has a positive
A + O vs. A + G: t(36) = 2.1, p = 0.016, d = 0.48; A + O vs. A + psychological effect that increases the feeling of relaxation
T: t(36) = 1.8, p = 0.023, d = 0.45; and A + O vs. A + H: t(36) = when added to the basal axillary odor.
2.1, p = 0.012, d = 0.48, odor-G, T, and H are (E)-geranylace­ To delve into the relaxation effect of odor-O, we focused on
tone, tetradecanoic acid, and (Z)-9-hexadecenoic acid, α-amylase in saliva, known to be elevated in malodor stress.17
respectively; Tables S6, S12, and Figure S5). To evaluate We observed a significant negative correlation between
whether the same unpleasantness-suppression effect could α-amylase and the ‘‘relaxation’’ score in MMS at all time points
be observed in compounds that increase during phases other (p = 0.0005) (base, session 1 to 3, n = 19 × 4, Figure 5C), indi­
than the O-phase, a similar experiment was conducted using cating that the amount of α-amylase in saliva increases as the
the three compounds that increase during the F-phase ‘‘relaxation’’ score decreases. Comparing α-amylase levels in
(odor-F). Odor-F also demonstrated the effect of reducing the the base, Ctrl (no odor), and odor-O conditions revealed no sig­
unpleasantness of the basal axillary odor; however, the effect nificant differences (Figure 5D, left). Comparing the Ctrl(A) (Ctrl
was not as significant as that of odor-O (Figure 4C, A + O vs. data from raters who smelled odor-A in session 3, n = 7) and
A + F: q = 6.2, p < 0.005, d = 0.70). odor-A conditions showed a significant increase in α-amylase
under odor-A (t(6) = 2.9, p = 0.027, d = 0.94, Figure 5D, middle,
Psychological and physiological changes induced by the Table S14), whereas this increase was suppressed by adding
O-phase-increasing compounds on men odor-O (odor-A+O condition) (Ctrl data from raters who smelled
To investigate the psychological and physiological effects of A + O in session 3, n = 10). These results demonstrate that
the O-phase-increasing odor, we conducted an experiment odor-O has the effect of suppressing the increase in salivary
with the timeline depicted in Figure 5A. In essence, male raters amylase caused by a baseline axillary odor, correlating with
(n = 19) entered a private experimental room to avoid encoun­ the level of relaxation.

Figure 2. The secretion levels of the 98 compounds in each phase of the menstrual cycle
Cluster analysis with the Ward method was performed using the mean of the robust Z scores for each time period, and the 98 compounds were arranged such
that compounds showing similar changes between the four phases were located in close proximity to each other. The box extends from the 25th to the 75th
percentiles, and individual scores are plotted as points superimposed on the graph. The pink arrows indicate the three compounds that increased during the
ovulatory phase, and the yellow arrows indicate the three compounds that increased during the follicular phase used in the following experiments.
See also Tables S2, S3, S4, and S5; Figures S2 and S3.

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Figure 3. Variability in volatile compounds that significantly increased during ovulatory or follicular phases
Line graphs show the changes in the amounts of the three O-phase-increasing compounds (#49, #86, and #94) and the three identified compounds (#41, #45, and
#72) during the menstrual cycle. The chemical structure of each compound is shown in the graph. The y axis shows the robust Z score of the peak area of each
compound obtained using GC/MS analysis. The box extends from the 25th to the 75th percentiles; individual scores are plotted as points superimposed on the
graph, and the gray lines indicate the connection of each donor’s score. Statistical analyses were performed using a one-sample Wilcoxon signed-rank test (one-
tailed) to determine whether the amount of each compound was greater than 0 (median of robust Z score). The significance level was set at p < 0.05. *p < 0.05, and
**p < 0.01.
See also Figure S4.

Effects of the O-phase-increasing compounds on male the average ratings for these categories (Figure 5E, red line) and
evaluation of female faces employed it as the ‘‘attractiveness’’ in the subsequent analysis.
In the same experimental timeline (Figure 5A), we examined the Face images were divided into two groups based on attractive­
impact of the O-phase-increasing odor on the impressions male ness under Ctrl conditions: a low-rated group (4 images) and a
raters had for female faces. Images of eight female faces were high-rated group (4 images). It was observed that the attractive­
prepared (50% smiling, Figure 5E), and raters were instructed ness of faces in the low-rated group decreased with odor-A and
to rate their impression of each face in four categories (cate­ significantly increased with odor-A+O compared to the no-odor
gories: beautiful, elegant and intellectual, want to spend time condition (F (1, 8) = 9.2, p = 0.016, η2 = 0.004, Figure 5F, left,
with, want to keep gazing at). Because all four categories of 8 im­ Table S14). When the differences between odor-A and no-odor
ages exhibited similar rating trends under Ctrl conditions (odor-A - Ctrl(A) in Figure 5F) and odor-A+O and no-odor (A +
(Figure 5E, purple, blue, green, and orange lines), we calculated O - Ctrl(A + O) in Figure 5F) were calculated and compared, it

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Figure 4. Three ovulatory phase-increasing compounds positively affected the unpleasant quality of basal axillary odor
(A) Results of sensory evaluation for four odor stimuli (Ctrl: no odor; odor-O: O-phase-increasing three compounds; odor-A: a basal axillary odor; odor-A+O:
mixture of odor-A, and odor-O, Table S6). The y axis indicates the rating score (male, n = 61–72, mean ± SEM). Paired t test with FDR for all descriptors. *q < 0.05,
**q < 0.01, ***q < 0.005, and ****q < 0.001, #q < 0.0005. Intensity (0 = not perceived to +6 = extremely intense), pleasantness (− 3 = very unpleasant to +3 = very
pleasant), preference (− 3 = extremely dislike to +3 = extremely like), feminine/masculine (− 3 = very masculine to +3 = very feminine), relax (0 = not perceived
to +6 = extremely perceived), arousal/excitement (0 = not perceived to +6 = extremely perceived), and the top 15 descriptors in Table 1 (0 = not perceived to +6 =
extremely perceived). See also Tables S6 and S7.
(B) Evaluation of the intensity and pleasantness of each odor-O component y axis indicates the rating score (male, n = 37, mean ± SEM). (odor-G,-T, and-H: (E)-
geranylacetone, tetradecanoic acid, and (Z)-9-hexadecenoic acid, respectively; odor-A+G, A + T, A + H: mixtures of odor-A and odor-G, T, and H, respectively).
The odor-A results are shown for reference. Dunnett’s multiple comparison test was used for comparisons. *p < 0.05 and **p < 0.01. See also Figure S5.
(C) Evaluation of the intensity and pleasantness of each stimulus. y axis indicates the rating score (male, n = 37, mean ± SEM). (odor-F: mixture of three increasing
F-phase-compounds; odor A + F: mixture of odor-A and odor-F). The odor-F results are shown for reference. Tukey’s multiple-comparison test was used.
***p < 0.005 and ****p < 0.001.

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Figure 5. Physiological and psychological effects of the ovulatory compounds on males

(A) Experimental timeline. Yellow arrow: MMS; blue arrow: salivary collection; black arrow: impression-rating test using a computer while presented with each
odor from the headset microphone. See also Table S8.
(legend continued on next page)

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was evident that the attractiveness of low-rated images was palmitoleic acid, it is nearly odorless. It is derived from palmitic
significantly higher under odor-A+O than under odor-A (F (1, acid and is presumed to be a precursor to (E)-2-nonenal, a repre­
16) = 15, p = 0.0013, η2 = 0.1, Figure 5F, left). In the faces of sentative odor of ‘‘aging odor,’’ which is generated through its
the high-rated group, there were no significant differences be­ breakdown by skin bacteria.34,35 Although the three compounds
tween the odor stimuli condition and the no-odor state aforementioned increased during the ovulatory phase, it is
(Figure 5F, right). These results suggest that when odor-O was important to note that this study was designed as an exploratory
added to the basal axillary odor, a positive behavioral effect investigation. To minimize type II error, multiple testing correc­
was observed in terms of impression ratings. tion was not applied, which unfortunately increased susceptibil­
ity to type I error.
DISCUSSION Changes in axillary volatile compounds within the menstrual
cycle may be attributed to fluctuations in hormone concentra­
This study investigated the role of olfactory cues in human inter­ tions, including estradiol and progesterone, within the cycle. In
sexual communication, with a focus on the ovulatory phase of particular, estradiol receptors are expressed in many tissues
the menstrual cycle, and identified ovulatory phase-increasing and are believed to be involved in systemic metabolism and
odorants that elicited positive emotional, physiological, and sweat gland secretory activity. Studies have revealed that amino
behavioral effects, facilitating male-female interaction. acids and their derivatives in blood tend to increase during the
Key points to consider when analyzing body odors are compre­ menstrual phase and decrease during the luteal phase, whereas
hensiveness and reproducibility in a well-controlled condition. phospholipids and other substances in blood increase during the
Conventional research on axillary odor often relies on a single follicular phase and decrease during the luteal phase.36 In this
collection or analytical method, resulting in the detection of a study, we demonstrate that long-chain fatty acids, including tet­
limited number of compounds.18–21 In this study, we compared radecanoic acid and (Z)-9-hexadecenoic acid, increase from the
four collection adsorbents and GC/MS pretreatment methods. follicular phase to around ovulation and decrease during the
Based on the diversity of collected compounds and consistency luteal phase. This possibly reflects the degradation of blood-
with previous findings,22 we adopted a method combining derived lipids on the skin surface, which increase during the
PDMS and a thermal desorption system with GC/MS analysis. follicular phase. Moreover, several compounds also increased
This method proved to be the most efficient in detecting the during the follicular phase, and some of these tended to further
largest number of compounds among those tested and could increase during ovulation, suggesting the strong effects of estra­
reproducibly detect compounds from an average of more than diol on the variability of amounts of each compound during the
70% of the donors. However, since competitive effects are known menstrual cycle.
to occur with adsorbents, the composition of the compounds ad­ This study revealed that the odor of the three O-phase-
sorbed on PDMS cannot be said to be identical to that of the orig­ increasing compounds induces a sense of comfort and relaxa­
inal axillary odor. Through careful optimization of the analytical tion in men. Furthermore, when the odor of the basal axillary
method allowed for the identification of compounds during the odor was combined with the three O-phase increasing com­
menstrual cycle that affect male-female interaction. pounds, it not only enhanced the attractiveness of low-attrac­
The current knowledge on the identified three O-phase- tive-level female faces but also suppressed the increase in sali­
increasing compounds is as follows: (1) (E)-geranylacetone: it vary amylase, an indicator of stress-related bad odor, in
is produced by oxidation of squalene and emits a floral or green comparison to the odor of the basal axillary scent alone. It has
odor quality and is among the prominently recognized sub­ been reported that pleasant essential oil (like rose) enhance
stances in humans.23–27 (2) Tetradecanoic acid: it has an facial attractiveness,37 but it is noteworthy that such effects
oxidized sebum odor quality and is detected in skin, saliva,28 may also be present in components of body odor that fluctuate
breast milk, and amniotic fluid. Its primary production pathway with the menstrual cycle. If other compounds that increase dur­
is believed to be triglyceride hydrolysis by skin bacteria lipase,29 ing ovulation that could not be identified in this study can be clar­
or it may be derived from blood owing to its ubiquitous presence ified, the function of ovulatory axillary odor will be elucidated in
in cells and plasma.30,31 Human amniotic fluid and breast milk more detail. Previous reports have suggested that when others
contain this odorant to which newborns exhibit a suckling smell body odor collected in high-stress situations, they experi­
response.32,33 (3) (Z)-9-hexadecenoic acid: commonly termed ence anxiety from ambiguous facial expressions,38 and anxiety

(B) The y axis indicates the score for each mood on the MSS for each condition by male raters. The first row shows base (basal condition) vs. Ctrl (no odor) vs.
odor-O (three ovulatory-phase increasing compounds) (n = 19), the second row shows Ctrl(A) vs. odor-A (basal axillary odor) (n = 9), and the third row shows Ctrl
(A + O) vs. odor-A+O (mixture of A and odor-O) (n = 10). *p < 0.05, **p < 0.01, and ***p < 0.005.
(C) Salivary α-amylase and ‘‘relaxation’’ scores in MMS at all time points (baseline and sessions 1–3, n = 76) are negatively correlated by simple linear regression
analysis. This indicates that the amount of α-amylase in the saliva increased with lower relaxation in this experiment. Dotted lines are 95% confidence intervals of
the best fit lines.
(D) The y axis shows the amount of salivary α-amylase under each condition. α-amylase significantly increased under odor-A stimulation (n = 7) but not under
odor-A+O (n = 10). *p < 0.05.
(E) Ratings for images of 8 females by male raters (n = 19) under Ctrl conditions for four impression categories (purple, blue, green, and orange lines). The average
values of these categories (red line) are indicated (mean ± SE).
(F) Average rating of a low-rated group (8 images) and a high-rated group (8 images) under each condition and the differences between odor and Ctrl conditions
(mean ± SE). *p < 0.05 and **p < 0.01.

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and nervousness increase,39 whereas when others smell body Material availability
odor collected in a happy state, a facial expression and percep­ This study did not generate new unique reagents.
tual processing style indicative of happiness were shown to be
induced.40 Additionally, women who smelled their partner’s Data and code availability
body odor experienced less stress.41,42 Body odors from new­ • GC/MS raw data supporting the current study will not be deposited in a
borns has been reported to activate reward-related cerebral public repository because we are still analyzing the data for projects not
areas in their mothers43 and mother’s odor did not cause typical reported in this study; however, these data will be available from the
brain responses to fearful stimuli in their child.44 These findings corresponding author upon reasonable request.

are implying that while body odor conveys negative emotions,

• We used publicly available code for the analyses.
• Subject profile and time schedule (Figures 1 and 5), statistical data
it also acts in a positive direction and inhibit negative emotions. (Figures 1, 4, and 5), and the information of authenticated and unknown
In addition to body odor, several reports indicate various phys­ compounds (Tables S3 and S4) are compiled in the Excel format tables.
ical changes in females during the menstrual cycle. The voice
during the ovulation period is perceived as the most attractive
ACKNOWLEDGMENTS
to men,45 and it has been demonstrated that the frequency of
the voice also increases during the fertile phase.46,47 Photo­ We thank all Touhara members for help in conducting experiment. This work
graphs of a female face during the ovulation phase are perceived was supported by the ERATO Touhara Chemosensory Signal Project to K.T.
as more desirable than other times.48 The redness of the face has (JPMJER1202), the JST Mirai program to K.T. (JPMJMI17DC and
been reported to decrease after the first day of menstruation and JPMJMI19D1), and JSPS KAKENHI grants (18K14651 and 22K06418 to M.
increases toward ovulation, although it is not detectable by the S.; 21K13546 to Y.H.; 18K02477, 18H04998, and 21H05808 to M.O.; and
JP18H05267 and JP23H05410 to K.T.), and Grant for Women Scientists in
naked eye.49 It appears that physical features that enhance ap­
Challenging Research by Japan Society for Bioscience, Biotechnology, and
peal to the opposite sex are heightened during the ovulation
Agrochemistry (JSBBA) to M.S.
phase. Together with our findings, although females with highly
attractive faces appear to be less affected by olfactory informa­
tion, other females may use the ovulatory phase-increasing odor AUTHOR CONTRIBUTIONS
and other physical features synergistically to attract the interest
N.O., M.S., and K.T. designed research; N.O., M.S., Y.O., Y.H., M.O., and R.K.
of the opposite sex during the reproductive period.
performed research; M.S., N.O., H.T., and K.T. analyzed data; M.S., N.O., and
In other mammals, ovulatory body odors function as a releaser K.T. wrote the paper.
pheromone, causing apparent behavioral changes such as
attraction and providing information regarding the reproductive
stage of female individuals. However, a uniform intrinsic attrac­ DECLARATION OF INTERESTS
tive behavior is not observed between males and females in
The authors declare that they have filed a patent application related to the con­
highly rationalized human society. Our study highlights an tent of this manuscript (PCT/JP2024/024888).
intriguing hypothesis that the role of ovulatory odors might
have shifted in the course of evolution in humans from the clas­
sical behavior-releasing pheromone to a communicative tool STAR★METHODS
that positively affects emotions in males and induces relaxation
Detailed methods are provided in the online version of this paper and include
and a positive impression toward females, thereby facilitating
the following:
male-female interaction. Such a role could function as a discreet
but effective cross-sex olfactory signal in a highly developed hu­ • KEY RESOURCES TABLE
man society governed by intellect rather than instinct. • EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
○ Ethics
○ Female axillary odor donors
Limitations of the study ○ Odor raters

It is known that the composition of axillary odor is partially influ­ • METHOD DETAILS
enced by genetic factors,50 and that the perception of body odor ○ Menstrual cycle assessment
○ Stress related emotional state assessment in axillary odor sampling
is also affected by genetic background51,52 and cultural
○ Collection of axillary odor for sensory evaluation
context.53,54 Therefore, by conducting sensory evaluations and ○ Procedure for sensory evaluation of female axillary odor
chemical analysis of axillary odor across diverse ethnic groups ○ Odor questionnaire for axillary odor
beyond the Japanese population, while accounting for the ef­ ○ Generation of descriptive terms

fects of genetic polymorphisms associated with odor percep­ ○ GCMS analysis

tion, it should be possible to perform a detailed investigation ○ Collection of axillary odors to compare the four phases of menstru­

into the factors that influence the evaluation of pleasantness or ation

○ Separation of E- and Z-isomers of geranylacetone
unpleasantness of menstrual cycle-related body odor. ○ Odor evaluation using O-phase-increasing odor

• QUANTIFICATION AND STATISTICAL ANALYSIS

RESOURCE AVAILABILITY ○ Statistical analysis of sensory evaluation of female axillary odor
○ Statistical analyses of 98 axillary odor compounds in each phase of

Lead contact the menstrual cycle

Further information and requests for resources should be directed to and will be ○ Statistical analyses of sensory evaluations and physiological and

fulfilled by the lead contact, Kazushige Touhara (ktouhara@g.ecc.u-tokyo.ac.jp). psychological experiments

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Received: June 4, 2024 Contributor to Axillary and Foot Odor Associated with an Acidic Note.
Revised: September 6, 2024 Chem. Biodivers. 12, 248–258. https://doi.org/10.1002/cbdv.201400272.
Accepted: July 8, 2025
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STAR★METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Chemicals, peptides, and recombinant proteins
Geranylacetone Tokyo Chemical Industry Co., Ltd. Cat#G0236
Tetradecanoic acid Tokyo Chemical Industry Co., Ltd. Cat#M0476
(Z)-9-hexadecenoic acid Tokyo Chemical Industry Co., Ltd. Cat#H0072
Acetic acid FUJIFILM Wako Pure Chemical Co. Cat#017-00251
Propanoic acid Tokyo Chemical Industry Co., Ltd. Cat#P0500
Butanoic acid Tokyo Chemical Industry Co., Ltd. Cat#B0754
2-methylpropanoic acid Tokyo Chemical Industry Co., Ltd. Cat#I0103
3-methylbutanoic acid Tokyo Chemical Industry Co., Ltd. Cat#M0182
Octanoic acid Kanto Chemical Co., Inc. Cat#07132-33
Decanoic acid Tokyo Chemical Industry Co., Ltd. Cat#D0017
Software and algorithms
IBM SPSS version 27 IBM Co. https://www.ibm.com/support/pages/
downloading-ibm-spss-statistics-27
GraphPad Prism Ver9.5.1 GraphPad Software https://www.graphpad.com/updates
GCMS solution software Shimadzu https://www.shimadzu.com/an/products/
gas-chromatograph-mass-spectrometry/
gc-ms-software/gcmssolution/index.html
NIST 17 National Institute of Standards https://chemdata.nist.gov/dokuwiki/
and Technology doku.php?id=chemdata:nist17
Wiley Registry of Mass Wiley https://sciencesolutions.wiley.com/
Spectral Data, 9th edition solutions/technique/gc-ms/wiley-
registry-of-mass-spectral-data/
Other
Open Essence Kit FUJIFILM Wako Pure https://labchem-wako.fujifilm.com/
Chemical Corporation us/category/00368.html
PDMS membranes Goodfellow Cambridge Limited Cat#751-624-16
Gas Chromatography Mass Shimadzu GCMS-TQ8030
Spectrometer -TQ8030
A thermal desorption system OPTIC4 GL Science OPTIC-4
Salivary Estradiol ELISA Kit Salimetrics https://salimetrics.com/assay-kit/
salivary-estradiol-elisa-kit/
Salivary Progesterone ELISA Kit Salimetrics https://salimetrics.com/assay-kit/
salivary-progesterone-elisa-kit/
Salivary α-Amylase Kinetic Enzyme Assay Kit Salimetrics https://salimetrics.com/assay-kit/
salivary-alpha-amylase-enzymatic-assay/
One Step Ovulation Urine Test Strip Doctor’s Choice https://blerdology.co/hairannkennsayaku/
he-20-ovltnc.html

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Ethics
The study received approval from the Ethics Committee of the University of Tokyo and adhered to the principles of the Declaration of
Helsinki. Informed written consent was obtained from all participants before conducting the experiments.

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Female axillary odor donors

A total of 29 females participated as odor donors, providing axillary odors during each phase of their menstrual cycles. Inclusion
criteria required regular menstrual cycles (averaging between 25 and 30 days), overall good health without medication use (including
oral hormonal contraceptives), absence of pregnancy, no gynecological diseases, chronic conditions, inflammations of the axillary
surface, or smoking habits. Axillary odors were collected during the menstrual cycle (menstrual, follicular, ovulatory, and luteal
phases). Eight females were excluded based on the following criteria: (i) an unstable menstrual cycle during the study, (ii) poor phys­
ical condition, (iii) scheduling conflicts, and (iv) a negative ovulation test result. Therefore, 21 females (age: mean ± SD = 23.7 ± 2.9
years) who provided samples from all four phases were included for further analysis. The donor profiles are shown in Table S9. Do­
nors were instructed to abstain from sleeping with or having sexual intercourse with another person, consuming garlic, spicy or herb-
infused foods, and alcohol from the day before each collection day until the end of the collection period. Additionally, they were asked
to refrain from using antiperspirants, perfumes, scented deodorants, and scented soap powder on each collection day until the end
of the collection period. All female donors were required to shave their axillary hair before the odor collection day. In this way, we try
our best to prevent sampling of exogenous compounds, but it is fair to say that it is difficult to exclude 100%. Therefore, we carefully
considered a possibility of exogenous origin after we identified compounds.

Odor raters
Sensory evaluation of female axillary odor
Twenty-one raters (males: n = 21, age: mean ± SD = 24 ± 2.9 years) were recruited for evaluating axillary odor samples. Raters were
heterosexual, single, free of respiratory, brain and nervous system, mental, and chronic diseases, not taking anabolic steroids, and
nonsmokers. Raters were informed that they would be evaluating various odors, including female-related odors. In this study, the
influence of sex and gender was not evaluated.
Odor evaluation using O-phase-increasing odor
Seventy-two single men (age: mean ± SD = 29 ± 5.4 years, the score of Open Essence Kit (OPE score): 10 ± 1.2) were recruited as
odor raters. All raters were heterosexual, did not suffer from respiratory, brain and nervous system, mental, and chronic diseases, did
not take anabolic steroids, and were nonsmokers. The ability to identify odors was measured using the Open Essence Kit, and all
raters scored 8 or higher, indicating no problem in identifying odor quality.
In this study, the influence of sex and gender was not evaluated.

METHOD DETAILS

Menstrual cycle assessment

The identification of menstrual phases relied on multiple methods, including LH surge assessment using LH test kits (One Step Ovula­
tion Urine Test Strip, Doctor’s Choice, USA), basal body temperature (BBT) measurements (n = 21), donor’s self-reported onset and
end dates of menstrual bleeding, and salivary estradiol and progesterone measurements (n = 12, for donors participating in the later
period).
Donors provided information on the onset of their last menstrual period, the length of the menstrual phase, and the usual cycle
length before axillary odor collection. This was used to estimate the length of their cycle and the onset of their next menstrual period.
During the collection period, donors were also asked to report the first and last dates of their menstrual bleeding during the collection
period via email and axillary odor was collected during their menstrual phase. After menstruation, LH test kits were used twice daily
(morning and evening) to detect the LH surge. When positive results was prompted, donors were instructed to notify the laboratory
and axillary odor was collected within one day. BBT was measured using a thermometer (A basal thermometer M-172L, OMRON,
Japan) immediately upon waking and recorded in a BBT management application (WOMANCOM, https://kisotaion.web-db.ws/
system/servlet/womancom.user, Japan) during the collection period. Additionally, saliva samples (1 mL) were collected from 12 do­
nors at the time of axillary odor collection, and the levels of female hormones (estradiol and progesterone) were measured using
enzyme-linked immunosorbent assay kits (SALIVARY 17β-ESTRADIOL ENZYME IMMUNOASSAY KIT, SALIVARY PROGESTER­
ONE ENZYME IMMUNOASSAY KIT, Salimetrics, USA). As saliva sampling for hormone analysis began with the latter half of the
21 participants, hormone data are presented for 12 donors. In addition to the previously mentioned periods, axillary odor was
collected during the period after menstruation ended and before LH tests turned positive, as well as during the period after a positive
LH test turned negative and before the onset of menstruation. Samples collected during these periods were classified as follicular,
ovulation or luteal phase samples, based on the results of BBT.

Stress related emotional state assessment in axillary odor sampling

We used STAI scores to determine the anxiety of the donors at the time of sample collection. Stress-related emotional state was as­
sessed using the anxiety state scale of STAI-State (STAI-S), which consists of 20 questions. Anxiety is a negative emotional state, char­
acterized by the reaction of an organism to stressors.55 STAI-S has been reported to be a sensitive tool to evaluate the increase of anx­
iety in response to various stressors.56 STAI-S scores ranged from 20 (low anxiety) to 80 (high anxiety) and a score of 42 or higher

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indicates the presence of anxiety symptoms. The mean STAI-S scores for each phase were as follows: M-phase = 32.6 ± 5.48 points,
F-phase = 33.7 ± 9.16 points, O-phase = 34.9 ± 10.3 points, and L-phase = 34.9 ± 7.72 points. No significant difference in anxiety state
was observed among the four phases (p = 0.423, Friedman’s test).

Collection of axillary odor for sensory evaluation

Before odor collection, the surface of each axilla underwent cleaning with a moist hot towel and 70% ethanol using sterile gauze to
minimize potential confounding factors, especially from cosmetic products. Four sterile gauzes (5 cm × 5 cm) (Oosaki Medical Corp.,
Japan) were placed on both axillae, secured with baby skin surgical tape (3M Japan, Japan) for 4 h, with the order of gauze changing
every hour. After removal, eight gauzes were immediately frozen at − 80◦ C in a 50-mL glass vial (NICHIDENRIKA-GLASS CO., LTD.,
Japan) until sensory evaluation. Donors changed into clothes (UNIQLO, Japan) washed with fragrance-free detergent and remained
at rest in a room with a temperature of 26◦ C and constant humidity during odor collection.

Procedure for sensory evaluation of female axillary odor

Sensory evaluations occurred twice daily at 10:00 a.m. and 2:00 p.m. Gauze samples were thawed 2 h before each session. Two of
eight pieces from each donor were made into a set, secured with a double clip, and placed in a glass vial. Each rater evaluated axillary
odor samples from four menstrual phases from 10 or 11 different female donors (40 or 44 axillary odor samples) and one control sam­
ple (a new sterile gauze). Each rater was seated in a chair in front of a desk in a well-ventilated, temperature-controlled experimental
room. The raters were asked to fill out the odor questionnaire immediately after sniffing each sample. The odor questionnaire con­
sisted of 22 questions (described in the next section) related to the sensory characteristics of the odor samples. The order of gauze
samples was counterbalanced, and the evaluations took 60–90 min, with short breaks allowed every 12 samples.
All raters were required to refrain from eating odor-producing foods and drinking alcoholic beverages for 24 h before the testing
because these activities may affect olfactory performance. They also refrained from using perfume, scented body cream, hand lotion,
lipstick, and scented deodorant on the day of the evaluation.

Odor questionnaire for axillary odor

The questionnaire consists of two-halves. The first half assesses odor intensity (0 = not perceived to +6 = extremely intense) and
pleasantness (− 4 = extremely unpleasant to +4 = extremely pleasant). In the second half, raters use a CATA format57 to rate the
odor quality of axillary samples using 20 descriptors (Sweaty, Vinegary, Fragrant, Sebaceous, Woody/Pencil-like, Damp clothing,
Musty, Sweet, Earthy, Stinky, Grassy/Green, Citrus, Yogurt, Milky, Spicy, Sulfuric, Grapefruit peel-like, Dusty, Fishy, Meaty).
Descriptor selection was based on the QDA method (details in the ‘‘generation of descriptive terms’’ section). Descriptors with a se­
lection rate below 2% were excluded from subsequent analyses (Table 1).
For the CATA analysis, data from raters scoring 8 or higher on the Open Essence Kit (OPE) (FUJIFILM Wako Pure Chemical Cor­
poration, Japan) were used to rigorously evaluate odor quality (single men: n = 16, age: mean ± SD = 23 ± 2.2). The Open Essence Kit
measured odor identification ability, an enhanced version of OSIT-J,58 widely used in Japan, involves 12 odors familiar to the Jap­
anese population.59 Raters chose 1 of 6 alternatives for each odor: 4 odor names, including a correct name, ‘‘not detected,’’ and
‘‘unknown.’’ Scores were calculated based on the total number of correct answers for the 12 odors. Scores range from 0 to 12,
with scores of 8 or higher indicating satisfactory odor identification ability for all age groups.60

Generation of descriptive terms

We selected 20 descriptors using the following procedure.

(1) First, the sensory terms were generated via a pilot test comprising an open-ended questionnaire that was administered to 15
raters (male: n = 8, female: n = 7). They evaluated 17 samples of axillary odor during the menstrual cycle (menstrual phase: n =
4, follicular phase: n = 4, ovulatory phase: n = 6, luteal phase: n = 3). The raters sniffed the axillary odor samples in Teflon
squeeze bottles (Big Boy, Japan) and immediately described the quality of the odor after sniffing each sample. The list of de­
scriptors consisted of 103 terms. Of the 103 terms, those used only once were omitted.
(2) The next procedure was conducted by five experts (male: n = 2, female: n = 3), involved in sensory evaluation and analytical
chemistry, to summarize the terms of the descriptor list. When several terms suggested a synonym, the most common word
was selected. After eliminating redundancies and controversial terms, the descriptor list had a total of 45 terms.
(3) To ensure that the 45 terms were sufficient to evaluate axillary odors, we conducted a pilot evaluation. Five raters assessed 21
odor samples (menstrual phase: n = 4, follicular phase: n = 4, ovulatory phase: n = 5, luteal phase: n = 8) and rated the 45 terms
on a seven-point scale (0 = not perceived to +6 = extremely perceived). We retained characteristic descriptors that were likely
to be important to discriminate between samples, as well as descriptors that were commonly perceived in most samples and
were considered important in assessing axillary odors. Furthermore, we re-confirmed the definitions of terms that were
believed to have multiple definitions and could cause individual perception-related differences. Terms with high-frequency
correlations were consolidated. Thus, the final selection resulted in 20 descriptors.

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GCMS analysis
Four different sampling techniques and associated GC/MS analysis
To determine the appropriate sampling method for the analysis of volatile organic compounds (VOCs) in axillary odors, we compared
four different sampling methods. These included (1) sterile 100% cotton gauze (Oosaki Medical Corp., Japan), which is widely used
for body odor collection18,19; (2) PDMS membranes (Cat. No.: 751-624-16, Goodfellow Cambridge Limited) that are suitable for
capturing body odor VOCs due to their high adsorption capacity and flexibility61,62; (3) Monotrap (GL Sciences, Japan); and (4)
Twister (GERSTEL Inc., USA) adsorbents, which are widely used for odor analysis and are easy to handle.63
To compare each method, axillary odors were obtained from a female (laboratory member who met the inclusion/exclusion criteria
in the method of ‘‘female axillary odor donors,’’ age = 27) axilla using all four methods on the same day. The PDMS membranes were
first placed on the axillae surface, covered with PET film cut from Flek-Sampler (OMI ODOR-AIR SERVICE Corporation, Japan), and
secured to the skin using waterproof films (New Tac Kasei, Japan) for 2 h (refer to ‘‘collection of axillary odors to compare the four
phases of menstruation’’). Subsequently, sterile gauze (5 cm × 5 cm) was placed on the surface of the axillae and held in place for 2 h
using baby skin surgical tape. Monotrap or Twister was placed on the surface of the axillae and covered with a plate (TissueTec Cry­
omold No. 2, Sakura Finetech Inc Japan.) and secured with waterproof film for 2 h. Following removal, PDMS membranes, Monotrap,
and Twister samples were transferred to a 2-mL glass vial (Thermo Fisher Scientific Inc., USA) and immediately frozen at − 30◦ C. The
gauze samples were immediately frozen at − 80◦ C in a 50-mL glass vial (NICHIDENRIKA-GLASS CO., LTD., Japan). While the sam­
ples were attached to the axilla, the donor was instructed to change into clothes (UNIQLO, Japan) that were washed with fragrance-
free detergent.
We analyzed the axillary odor samples obtained from the four sampling methods using the appropriate equipment and conditions
for each sample. The sterile gauze sample was analyzed by GCMS-QP2010 Ultra (Shimadzu, Japan) (SH-Stabilwax column of
30 m × 0.53 mm i.d. with a film thickness of 1 μm) combined with the large volume static headspace system (7650HS-CTS, Entech,
USA). The gauze samples were placed in 500-mL inert glass vials (EN39-75500W, Entech, USA) and equilibrated in the oven for
20 min (80◦ C) before extracting the headspace into the capillary traps. The headspace (200 cc) was directed to the capillary traps,
followed by backflushing directly to the GC analytical column. The column temperature was programmed to rise from 40◦ C (5 min
hold) to 230◦ C (66 min hold) at 10◦ C/min. The interface temperature was maintained at 230◦ C, and the ion source temperature at
230◦ C. Mass spectra were obtained in full-scan mode (range: m/z 29–400) at 70ev electron impact.
PDMS membrane, Twister, and Monotrap samples were analyzed using GCMS-TQ8030 (Shimadzu, Japan) (Stabilwax column of
60 m × 0.32 mm i.d. with 0.5-μm film thickness) combined with a thermal desorption system OPTIC4 (GL Science, Japan). The con­
ditions of thermal desorption controlled by OPTIC4 are as follows. The vaporization chamber increased from 35◦ C to 230◦ C at 5◦ C/s,
septum purge flow 5mL/min, sample sweep time 30 s, column flow 2mL/min, transfer time 2 min, split flow 10mL/min, cryotrap low
temperature − 150◦ C (130 s hold), cryotrap high temperature 250◦ C, cryotrap heat ramp rate 60◦ C/s, and the GCMS column temper­
ature was programmed to rise from 50◦ C (2.5 min hold) followed by 10◦ C/min to 150◦ C, then 5◦ C/min to 230◦ C (61.5 min hold). The
interface temperature was maintained at 230◦ C, and the temperature of the ion source was 230◦ C. Mass spectra were obtained in
full-scan mode (range: m/z 29–400) at 70ev electron impact. Volatile compounds were detected using the automatic peak detector
(GCMS solution software; Shimadzu, Japan), and individual peaks were identified using the NIST 17 (National Institute of Standards
and Technology, USA) and the Wiley Registry of Mass Spectral Data, 9th edition (Wiley, USA). Visual inspection and fragment match­
ing were used to confirm peaks with >70% similarity in the library search. Moreover, about half of the compounds were confirmed by
the MS and retention time of purchased authenticated compounds with >95% similarity. A peak retention index (RI) was subse­
quently calculated to compare the analysis results for each condition. If a peak detected in the axillary odor sample was also detected
in the control sample (experimental room), only the peak detected with a peak area value more than twice that of the control was
adopted as the body odor compounds and the total number of such peaks were calculated (Figure S2). The threshold of more
than twice was set to avoid picking up noise while ensuring that no potential candidates were excluded.

Collection of axillary odors to compare the four phases of menstruation

PDMS membranes were utilized to collect axillary odor for GC/MS. Small pieces of PDMS membrane (15 mm × 5 mm × 0.45 mm)
were cut from a sheet and conditioned in an oven at 260◦ C for 1 h with nitrogen flowing. These conditioned pieces were immediately
placed in Low Adsorption (LA) Qsert vials (Cat no: 29663-U, MERCK, USA) and stored at − 30◦ C before axillary odor collection. Before
applying the PDMS membrane to the axilla, the surface of both axillae was wiped with a moist hot towel and cleaned with 70%
ethanol using sterile gauze. Conditioned PDMS membranes were then placed on the surface of both axillae, covered with polyethy­
leneterephthalate (PET) film cut from Flek-Sampler (OMI ODOR-AIR SERVICE Corporation, Japan), and secured to the skin with
waterproof films (New Tac Kasei, Japan). The PDMS membranes were left on the skin for 2 h, following which they were removed
using clean tweezers, inserted into an OPTIC DMI sample insert (60-μL micro vial) (GL Science, Japan), and placed in 2-mL glass
vials (Thermo Fisher Scientific Inc., USA). Blank PDMS membranes, placed on a PET film on a desk in the experimental room,
were taken and analyzed as controls. Each glass vial was injected with 1 μL of 100 ppm toluene-D8 solution diluted with methanol
(Cat no: 233382, Sigma-Aldrich, US) as an internal standard. After sampling, the PDMS membranes were immediately stored at
− 80◦ C. Odor collection with the PDMS membrane was performed before axillary odor collection with gauze in the ‘‘collection of
axillary odor for sensory evaluation’’ section. During odor collection, donors were instructed to change into clothes (UNIQLO, Japan)
washed with fragrance-free detergent and were kept at rest in a room with a temperature of 26◦ C and constant humidity (48 ± 5%).

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We collected 84 axillary odor samples using PDMS membranes from November 2017 to March 2018, and all GC/MS data were
obtained from January 2018 to April 2018 using the aforementioned methods. Volatile compounds were detected using the auto­
matic peak detector (GCMS solution software; Shimadzu, Kyoto, Japan), and individual peaks were verified using NIST 17 (National
Institute of Standards and Technology, USA) and the Wiley Registry of Mass Spectral Data, 9th edition (Wiley, USA). Compounds with
fatty acid characteristics, even if they did not match the database exactly, were labeled as ‘‘long-chain fatty acid’’ (Table S4) ‘‘Un­
known compound’’ refers to a compound with an RI or mass spectrum that matched between samples but could not be identified.
Subsequently, the peaks were aligned, and the area of each peak was calculated using the GCMS solution software (Shimadzu,
Japan) by automatic integration. The compounds information is shown in Tables S10 and S11. For statistical analysis, 98 compounds
that could be identified by authentic compounds or had a minimum occurrence rate of 24% (equivalent to 5 donors) or more in the
phase in which they were detected in the largest number of people (average occurrence rate in all phases of all compounds = 71%)
were used. Of the 98 compounds, of which 52 compounds were identified by authentic compounds, 28 compounds were detected in
the control sample.
Because the samples were carefully stored and analyzed in a short period of time, there was little difference in the peak areas of the
internal standard toluene-D8 between axillary samples, so absolute peak areas were used in subsequent statistical analyses. When
compounds were not detected by GCMS solution (LOD&LOQ, a signal-to-noise ratio of 3 was considered), we set 0 for peak area.

Separation of E- and Z-isomers of geranylacetone

A mixture of geometrical isomers of geranylacetone (Tokyo Chemical Industry Co., Japan) was purified using column chromatog­
raphy. Silica gel was impregnated with 8% AgNO3,64, (hexane/ethyl acetate = 9:1) to obtain its E- and Z-isomers. The ratio of E-
and Z-isomers of the fractionated samples was determined at each timepoint using GC/MS, and finally, the (E)-geranylacetone
was purified to 99.6%. The geometry of each isomer was confirmed by observing diagnostic nuclear Overhauser effect (NOE) cor­
relations. The purified (E)-geranylacetone (purity >99.6%) was used in this study.

Odor evaluation using O-phase-increasing odor

Odor solution
For experiments involving the O-phase-increasing three compounds, we prepared an odor mixture solution containing the same
compounds with their respective ratios as those detected from odor-O. All samples were soluble in Ethanol (EtOH), and it was
thus used as the solvent. The final concentration of the solution was adjusted so that the 60 μL of odor-O solution used in each exper­
iment contained three times the median of the O-phase-increasing compounds detected by the 2-h collection of axillary odors using
the PDMS membrane. The concentration of the model axillary odor solution was set at approximately 1.8 times the intensity of the
axillary odor when gauze was collected for 4 h in Figure 1 (equivalent to a total of 7.2 h). Therefore, the concentration of the ovulation-
inducing component solution was set at 3 times the concentration of the 2-h PDMS collection (6 times the total concentration), which
was estimated to be slightly lower due to individual differences. For quantification, diluted authentic compounds were applied to the
same size of PDMS membranes used for the axillary, analyzed in the same manner, and quantified from a calibration curve.
A model mixture of axillary odors,12 the mixture of seven fatty acids (Table S6), modified to match our results, was used as the basal
axillary odor (odor-A). Due to practical constraints during reagent preparation, the concentrations used in this study are slightly
different from those reported in the original publication. GC/MS analysis did not reveal phase specificity in the compounds used
as odor-A, except propanoic acid, which is increased in the F-phase (Table S3). The odor intensity (median = 2.89, SE = 0.147,
Figure 4A) of the basal axillary odor was set at approximately 1.8 times the odor intensity (mean = 1.61, SE = 0.107, Figure 1B) of
the female axillary gauze collected for 4 h.
Furthermore, we prepared a mixture of odor-A and odor-O as odor-A+O, and a solution containing one compound of each of the
three O-phase-increasing compounds in odor-A as odor-A+G, odor-A+T, and odor-A+H. As the control in the odor phase, we pre­
pared an odor mixture solution containing the compounds detected in the axillary odor of a follicular female (odor-F), and the final
concentration was adjusted three times the median of the F-phase-increasing compounds detected by the 2-h collection of axillary
odor using the PDMS membrane. Additionally, a mixture of odor-A and odor-F was prepared as odor-A+F. The required amount of
each odorant solution was pipetted onto the top of a mouillette (5.5 × 8.5 × 2.5 cm, Hiromichi, Japan) attached in a loop on a 32-mm
double clip (ASKUL Corporation, Japan) and allowed to wait for 10 min until the solvent EtOH had completely evaporated.
Sensory evaluation
Odor ratings of (Ctrl, no odor; odor-O, three O-phase-increasing compounds; odor-A, basal axillary odor; odor-A+O, mixture of odor-
A & odor-O, Table S6) were obtained for intensity (0 = not perceived to +6 = extremely intense), pleasantness (− 3 = very unpleasant
to +3 = very pleasant), preference (− 3 = extremely dislike to +3 = extremely like), feminine/masculine (− 3 = very masculine to +3 =
very feminine), relax (0 = not perceived to +6 = extremely perceived), arousal/excitement (0 = not perceived to +6 = extremely
perceived), and the top 15 descriptors in Table 1 (0 = not perceived to +6 = extremely perceived) were performed.
Thirty-seven single men of 72 single men participated in the evaluation (age: mean ± SD = 30 ± 5.5 years, OPE score: 9.8 ± 1.2).
Odor ratings of (odor-A, basal axillary odor; odor-A+O, mixture of odor-A & odor-O; odor-A+G, mixture of odor-A & (E)-geranylace­
tone; odor-A+H, mixture of odor-A & (Z)-9-hexandecenoic acid; odor-A+T, mixture of odor-A & tetradecanoic acid; odor-A+F,
mixture of odor-A & odor-F, Table S6) were obtained for intensity (0 = not perceived to +6 = extremely intense) and pleasantness
(− 3 = very unpleasant to +3 = very pleasant). All other descriptors are presented in Figure S5.

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Physiological and psychological experiments

To explore the psychological and physiological effects of the O-phase-increasing odor, we conducted an experiment with the time­
line shown in Figure 5A for single men (n = 19, age: mean ± SD = 27 ± 4.5 years, OPE score: 9.2 ± 1.7). The experiments started at
10:30 a.m. or 1:00 p.m. The order of odor stimulation was counterbalanced, and we employed a double-blind experimental design. In
Base session, each rater entered the experimental room (private room), sat quietly in a chair, and received experimental instructions
displayed on monitors in front of the raters and explained verbally by a female experimenter. First, raters completed the baseline
MMS, a type of questionnaire (details provided in the ‘‘MMS’’ section). They were then instructed regarding the saliva collection pro­
cedure, and baseline saliva was collected (details are provided in the ‘‘Saliva assay’’ section). Subsequently, the rater was instructed
to wear a headset with an odor (Session1: Ctrl or odor-O, Session2: odor-O or Ctrl, Session3: odor-A or odor-A+O, The odors pre­
sented in each session are noted in Table S13) on the microphone65 for 22–25 min and was subjected to three sessions of impression
ratings of female facial images using a computer (details are provided in the ‘‘impression rating of female facial images’’ section) and
MMS. Saliva was collected immediately after removing the headset. Of the 19 single men, 9 received odor-A and 10 received odor-A
+O in Session 3. The raters were unaware that the microphone of the headset was scented. We explained to raters upon informed
consent that a debriefing will be performed upon request, but nobody requested.
MMS
The MMS, a self-reporting questionnaire developed for Japanese individuals to measure multiple mood-emotional states at a given
time, was used to assess the emotions of the participants.66 The MMS has proven to be a sensitive tool for assessing emotions in
response to various stimuli. MMS can measure general emotional states, unlike STAI-S, which focuses on anxiety states, and the
positive and negative affect schedule (PANAS), which measures more abstract emotions and moods such as positive or negative
effects.67 There are five subcategories within the eight major emotional categories (Depression/Anxiety, Hostility, Boredom, Liveli­
ness, Relaxation/Tranquility, Friendliness/Amicability, Concentration/Focus, Startle/Surprise) in MMS, each of which is scored
from 0 (not perceived) to 3 (clearly perceived). Thus, the score for each major category ranges from 0 to 15 points (details are shown
in Table S8).
Saliva assay
Saliva was collected without orofacial movement using polypropylene tubes (Cryovial 2 mL, Salimetrics LLC, USA).68 The collected
samples were frozen at − 80◦ C until analysis. Salivary α-amylase activity (U/min) was measured using the Salivary α-Amylase Kinetic
Enzyme Assay Kit (Salimetrics LLC, USA) at the Yanaihara Institute Inc. in Japan. It is worth noting that one participant could not
collect saliva, and two participants faced challenges collecting saliva during the experiment on several occasions.
Impression rating of female facial images
We obtained one smiling and one neutral image each of eight young female faces from Getty Images (https://www.gettyimages.co.
jp/), AIST facial expression database 2017 (https://unit.aist.go.jp/harc/kao_CECRT/kaohyoujoDB_HP.html), iStock (https://www.
istockphoto.com/jp), Shutterstock (https://www.shutterstock.com/ja), and PIXTA (https://pixta.jp/). We then used Future Baby
Maker | FaceFilm (Jiangguo Yan, App Store) to create a morphing image in 9 steps with 0% for neutral images and 100% for smiling
images, and 50% smiling face images of eight females were used in this experiment (Figure 5D). During the odor stimulation sessions
1–3, raters rated their impression of each face using four categories (beautiful, elegant & intellectual, want to spend time with, want to
keep gazing at; ratings: 0 = not perceived, 5 = extremely strong). Each image was presented on the monitor for 2 s, followed by a 10-s
presentation of the page on which the ratings for the four categories were administered. If a response was not received within 10 s,
the next image was forcefully displayed. Presentation (Neurobehavioral Systems, USA) was used for data collection.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis of sensory evaluation of female axillary odor

The effect of menstrual cycle status on the perceived odor intensity and pleasantness was analyzed using linear mixed models
(LMMs). In each model, the response variable was the rating scores, and the fixed factor was the menstrual cycle phase at the
time of odor collection (four categories: M, F, O, and L-phase), which was used as dummy variables. The models specified random
intercepts for odor raters and odor donors. All analyses were conducted in R, version 3.5.2, using the lmerTest package (lmerTest
package; https://cran.r-project.org/web/packages/lmerTest/index.html) (Figure 1 and Table S1). All statistical tests were two-tailed,
and the significance level was set at p < 0.05. For the CATA questions, the number of selections and descriptor selection rate (%)
were determined for each descriptor. Cochran’s Q-test was performed for 15 descriptors (descriptor selection rate >2%) to evaluate
significant differences in axillary odors from four different phases. IBM SPSS version 27 (IBM Co., USA) and GraphPad Prism Ver9.5.1
(GraphPad Software, USA) were used. A priori power analysis using G*Power 3.1 indicated that 23 raters were enough in the con­
dition of effect size f = 0.25, α level = 0.05, Power (1-β err prob) = 0.8 in ANOVA (Repeated measures, within factors).

Statistical analyses of 98 axillary odor compounds in each phase of the menstrual cycle
A priori power analysis using G*Power 3.1 indicated that 22 donors were enough in the condition of effect size d = 0.57, α level = 0.05,
Power (1-β err prob) = 0.8 in Wilcoxon signed-rank test (one sample case). We converted the peak area of 98 compounds into a robust
Z score for standardization. Robust z-scores were calculated as follows: robust z = (x – median)/normalized interquartile range
(NIQR), where ‘‘x’’ is the respective peak area value, and median and NIQR were those of the respective female donors. The cluster

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analysis used in Figure 2 is ward method with squared euclidean distance. IBM SPSS version 27 (IBM Co., USA) was used for these
analyses. The p-value was calculated using a one-sample Wilcoxon signed-rank test (one-tailed) to determine whether the amount of
the compound in each phase was greater than 0 (median of the robust Z score), and the significance level was set at p < 0.05
(Tables S3 and S4). To test whether it is possible to discriminate between the ovulatory phase and other phases using the 98 com­
pounds that were used in this analysis, conditional logistic regression on the corresponding data for 21 donors was performed using
the COXREG command of SPSS ver. 27 (Conditional logistic regression using COXREG (ibm.com)). First, the problem of multicolli­
nearity was encountered, so the Spearman’s coefficient for the 98 compounds was calculated. Based on > 0.7, 13 variables (#5, 6,
14, 19, 24, 56, 83, 89, 90, 91, 92, 93, 94) were removed. A logistic regression analysis was conducted using forward selection (likeli­
hood ratio) for the remaining 85 variables (Table S5).

Statistical analyses of sensory evaluations and physiological and psychological experiments

For the analyses shown in Figure 4, a priori power analysis using G*Power 3.1 indicated that 67 raters were sufficient for t test (Dif­
ference between two dependent means (matched pairs)) in Figure 4A with an effect size dz = 0.35, α level = 0.05, Power (1-β err
prob) = 0.8, while 34 raters were sufficient for Figures 4B and 4C under the same conditions with an effect size dz = 0.5. Paired t
test with false discovery rate (FDR) was used for the 21 descriptors in Figure 4A (control vs. odor-O, odor-A vs. odor-A+O). Dunnett’s
multiple comparison test among the four types of odors is shown in Figure 4B (odor-A+O vs. A + G, A + H, A + T) and Tukey’s multiple
comparison test is shown in Figure 4C (odor-A vs. A + O, A + F, and odorA + O vs. odorA + F). All statistical tests were two-tailed, and
the significance level was set at q < 0.05 or p < 0.05. IBM SPSS version 27 (IBM Co., USA) and GraphPad Prism Ver9.5.1 (GraphPad
Software, Boston, USA) were used for statistical analyses. The Statistical data is shown in Table S12.
For the analyses shown in Figure 5, a priori power analysis using G*Power 3.1 indicated that 24 raters were enough in the condition
of effect size f = 0.25, α level = 0.05, Power (1-β err prob) = 0.8 in ANOVA (Repeated measures, within-between interaction). One-way
ANOVA (within-participants) was performed using the Greenhouse-Geisser method with Tukey’s multiple comparison test as a post
hoc test (base vs. Ctrl vs. odor-O) or paired t test (Ctrl(A) vs. odor-A, Ctrl(A + O) vs. odor-A+O) (Figures 5B and 5D). We also performed
two-way ANOVA for within-subject design, or if some values are missing, those data were analyzed by fitting a mixed effect model
(REML) (Ctrl(A) vs. odor-A, Ctrl(A + O) vs. odor-A+O), and two-way ANOVA or REML (Ctrl(A) vs. Ctrl(A + O), odor-A – Ctrl (A) vs. odor-A
+O – Ctrl(A + O)) adjusted by the Greenhouse-Geisser method (Figure 5F). Simple linear regression analysis was performed for sali­
vary α-amylase and relaxation score of MMS (Figure 5C). All statistical tests were two-tailed, and the significance level was set at
p < 0.05. IBM SPSS version 27 (IBM Co., USA). GraphPad Prism Ver9.5.1 (GraphPad Software, Boston, USA) were used for statistical
analysis. The Statistical data is shown in Table S14.

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