Olfactory/Pheromonal Input, Human Female Sexual Preferences and Behavior

Abstract
Chemical signals communicate affective and motivational states by eliciting hormonal effects that may translate to unconscious behavioral affects in non-human animals. In human females, androstenol elicits hormonal effects. Androsterone is a species-specific metabolite of dehydroepiandrosterone (DHEA) and a putative human pheromone. Mammalian conditioning paradigms suggest that androstenol may condition hormonal effects that are unconsciously associated with the potential behavioral affects of androsterone. In Experiment 1 of the present study, we evaluated the interaction of ovulatory phase human female subjects with a male confederate who wore either a standardized androstenol / androsterone mixture diluted in propylene glycol, or who wore propylene glycol alone during a cooperative task. We found that when the confederate wore the olfactory/pheromonal mixture, female subjects were more likely to display “hair flipping” and rated the confederate higher in being “funny” and “more comfortable to be around”. In Experiment 2 the study was repeated, with similar effects, using sandalwood essential oil as a masking odor to keep the confederate blind to his condition. We found that when the confederate wore the olfactory/pheromonal mixture, female subjects were more likely to make eye contact with the confederate and laugh during the interaction. They also rated themselves as being more attracted to the confederate. These results suggest that combining the known hormonal effects of a putative human pheromone (e.g., androstenol) with the possible behavioral affects of androsterone may help to extend non-human animal models of chemical communication to humans.
Rejected Submission
A role for putative pheromones in human behavior has not yet been clearly demonstrated. The traditional definition of pheromones originated with Karlson and Luscher in 1959, who wrote: “Pheromones are defined as substances which are secreted to the outside by an individual and received by a second individual of the same species, in which they release a specific reaction, for example, a definite behaviour, or a developmental process (p. 55).” Pheromones in most mammals are understood to be detected in the vomeronasal organ (VNO), processed in an accessory olfactory bulb, and act via gonadotrophin releasing hormone (GnRH) neurons. GnRH neurons prompt the secretion of GnRH, the hormone responsible for altering luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels that trigger puberty, produce gametes, and regulate the estrus cycle (Boehm, Zou, and Buck, 2005). The pheromone induced change in LH levels subsequently alters the levels and ratios of androgens and estrogens, which may be responsible for the modifications in neurotransmission made during the development of sexual preferences in mammals (Kohl, 2006).
Controversy over whether humans are capable of detecting pheromones has centered on whether or not humans have a functioning vomeronasal organ (VNO). Knecht and Lunstrom (2003) found a VNO epithelium outlining a VNO duct in the nasal septum in human subjects. They did not find a corresponding accessory olfactory bulb or nerves leaving the VNO. However, a vestigial VNO does not void the concept of human pheromones. A second group of receptors were recently discovered in the mouse olfactory epithelium that recognized volatile amines and one type of mouse pheromone. The genes encoding these pheromone-detecting trace amine-associated receptors have also been found in humans, suggesting that pheromone processing may be possible directly through the main olfactory processing pathway, eliminating a need for a functioning human VNO (Liberles and Buck, 2006).
The difference between whether an individual of the same species is deemed to be responding to an odor (i.e., olfactory input) or to pheromones (i.e., pheromonal input) may lie only in generalized conscious detection thresholds. Besides, detection thresholds are not individually established in humans either for odors or for putative human pheromones. This makes it difficult to determine whether any individual’s conscious processing either of olfactory input, or of pheromonal input is a factor. Thus, here we use the term olfactory/pheromonal input in an attempt to eliminate controversial definitions.
According to Kohl (2006), sexual preferences are genetically predisposed and primarily influenced by olfactory/pheromonal input from the social environment. In his mammalian model, species-specific responses to visual, auditory, and other sensory input are secondarily conditioned by olfactory/pheromonal input from the social environment. These species-specific responses condition changes in physiology and mood. Humans may consciously associate the conditioned changes with odors as is common with the development of visual preferences for different foods, which are more obviously based on the food’s chemical appeal (Gottfried, et al., 2006). Similarly then, during the development of sexual preferences, conditioned changes are unconsciously associated with behavioral affects of olfactory/pheromonal input on hormones. In other mammals, the development of food and mate preferences is a function of olfaction and pheromones, and other mammals are unlikely to make any conscious associations with their physiology and mood during either the development of their food preferences or the development of their mate preferences. Thus, there are animal models for olfactory/pheromonal conditioning of food preferences and mate preferences.
A direct effect of olfactory/pheromonal input on genes in hormone-secreting nerve cells occurs with concurrent exposure to non-olfactory sensory input (e.g., visual, auditory, or tactile input) from the social environment during the development of mammalian sexual preferences and their likely expression in sexual behavior. The affects of olfactory/pheromonal input on sexual behavior appear to be unconsciously associated with the direct effect of olfactory/pheromonal input on hormones during sexual development (Kohl, 2006). In contrast, the direct effect, if any, of non-olfactory/pheromonal input on hormones during the development of mammalian sexual behavior has not been detailed. Unconscious associations with the behavioral affects of olfactory/pheromonal input on hormones can be expected to rely on reinforcement of the most likely physiological response; a downstream effect of nerve cells that secrete gonadotropin releasing hormone (Kohl, 2006; Kohl, Atzmueller, Fink, and Grammer, 2001).
Gonadotropin releasing hormone (GnRH)-directed change in levels of luteinizing hormone (LH) is a well-known downstream effect of olfactory/pheromonal input from opposite sex conspecifics. This LH response to males is found, for example, in female mice (Coquelin and Bronson, 1980); in female rats (Rajendren, Dudley, and Moss, 1993); in female ferrets (Wersinger and Baum, 1997); and in human females (Preti, Wysocki, Barnhart, Sondheimer, and Leyden, 2003). However, the LH response to olfactory/pheromonal stimuli may not be species specific.
Species specificity is required in the traditional definition of pheromones (Karlson and Luscher, 1959). This may help to explain why no human pheromone has been isolated via bioassay (Wysocki and Preti, 2004). Researchers may never be able to isolate a human pheromone (singular) that fits a definition initially used to describe the effect of insect pheromones (plural). We did not attempt to isolate a human pheromone but instead we used a likely olfactory/pheromonal mixture: a combination of putative human pheromones.
The GnRH-directed LH response of human females exposed to olfactory/pheromonal input is as readily apparent as it is in other mammals. Androstenol (5a-androst-16-en-3a-ol), a boar pheromone that alters behavior in female pigs, is present in the axillary secretions of human males (Austin and Ellis, 2003; Dufort, Soucy, Lacoste, and Luu-The, 2001). It elicits an LH response from human females (Shinohara, et al., 2000). This LH response is linked to changes in mood (Preti et al., 2003). Shinohara et al. (2000) proposed that androstenol is the axillary constituent that is responsible for the change in LH. Androstenol is known for its pleasant musky odor, but not for any properties that enable cross species comparisons of exposure-altered human female sexual behavior (Cowley and Brooksbank, 1991). Thus, as may be obvious, the link between olfactory/pheromonal input and either the mood or the behavior of any mammalian male or female is more complex than the input’s effect on GnRH-directed LH. The LH response is merely the first measurable response in humans that links olfactory/pheromonal input to a sequence of neuroendocrine events that link sensory input from the social environment to endocrinology and behavior. Subsequently, LH and follicle stimulating hormone (FSH) control the release of steroid hormones, such as estrogens and testosterone that influence sexual behavior via their effects on neurogenesis (Kohl, 2006). In mice, LH has recently been linked to estrogen-associated hippocampal and olfactory neurogenesis in adult females, a finding that speculatively extends from mice to humans the data on mate preferences for dominant male olfactory/pheromonal input (Mak, Enwere, Gregg, Pakarainen, Poutanen, Huhtaniemi, and Weiss, 2007). Furthermore, estradiol (E2) is an estrogen that may act directly as a neurotransmitter, enabling more rapid behavioral changes (e.g., after approximately 15 minutes) with mammalian female exposure to male olfactory/pheromonal stimuli (Taziaux, Keller, Bakker, and Balthazart, 2007). Similarly, in mice and thus perhaps in humans, a fragment of the GnRH peptide may act directly as a neurotransmitter (Moss and Dudley, 1990) which would enable an even more rapid behavioral response (e.g., in milliseconds) to olfactory/pheromonal input. From a molecular biochemical perspective, it now appears that olfactory/pheromonal input can elicit responses with times that parallel responses that are attributed to visual input.
Studies of individual constituents present in olfactory/pheromonal stimuli that might influence levels of hormones or the behavior of women have been performed and linked to physiological and mood changes, but not to any change in women’s behavior (Jacob and McClintock, 2000; Lundstrom, Goncalves, Esteves, and Olsson, 2003; Lundstrom and Olsson, 2005; Spencer, McClintock, Sellergren, Bullivant, Jacob, and Mennella, 2004). Reports of affects on nervousness, tension, and other negative feeling states, even when they are observed with concordant changes in autonomic physiology (Grosser, Monti-Bloch, Jennings-White, and Berliner, 2000) do not truly address a report of change in women’s behavior.
The general failure to find a behavioral affect in human females that is elicited by individual constituents of natural body odor (e.g., described above) may be due to the likelihood that olfactory/pheromonal input is typically present in mixtures that rarely, if ever, would allow a woman to be exposed to any individual constituent. DHEA (Labows et al., 1979) and androsterone (Toth and Faredin, 1985) are present in the axillary secretions of men, which alters LH and mood in human females, as noted earlier. From a neuroendocrine approach that links olfactory/pheromonal input to hormones and behavior, recent evidence suggests that reinforcement of the downstream effect of GnRH on LH and behavior might occur when one olfactory/pheromonal constituent converts a secondary olfactory/pheromonal constituent into a conditioned stimulus (Coureaud, Moncomble, Montigny, Dewas, Perrier, and Schaal, 2006). Kohl (2006) details the species-specific and sexually dimorphic characteristics of androsterone production, and quotes Margolese and Janiger (1973). They proposed “. . . that the metabolic pathway which results in a relatively high androsterone value [in either males or females] is associated with sexual preference for females by either sex, whereas a relatively low androsterone value is associated with sexual preference for males by either sex (p. 210).” We posited that androsterone is a species-specific pro-sex steroid hormone-derived putative human pheromone that acts in concert with the effect of androstenol on LH (Shinohara et al., 2000) to elicit behaviors associated with the conditioned sexual preferences of women.
Specifically, in Experiment 1 we hypothesized that female mood would improve, and the frequency of non-verbal courtship behaviors toward a male confederate would increase during a cooperative task when the confederate wore an olfactory/pheromonal mixture (containing androsterone and androstenol diluted in propylene glycol) as opposed to when he wore only the vehicle (propylene glycol). In Experiment Two, we repeated Experiment 1 with the same goals of finding mood improvement and non-verbal courtship behavior in female subjects. However, any consciously perceived odor of androsterone and androstenol was masked with sandalwood essential oil to keep the confederate blind to the condition (and thereby eliminating some of the potential confounds in Experiment 1).
Experiment 1
Method
Participants

Eighteen female college students (mean age= 20 years; 14 Caucasian, 4 Asian; self-identified as “attracted to men”) were recruited from a small, Midwestern, liberal arts college via a campus-wide email solicitation asking for women who were not using hormonal birth control to participate in a study on the menstrual cycle and cooperation. Subjects who met the criteria participated in the experiment while ovulating (assessed through ovulation testing kit, Walgreens Brand). A 21-year-old Caucasian male served as the confederate subject.
Materials
Subject Sheet. Subjects completed a questionnaire asking age, relationship status, whether subject is sexually attracted to men, whether subject is taking SSRIs, first day of last menstrual period, and enjoyment in and experience with completing mind puzzles.
Mood Assessment. A five-point Likert scale (from “not at all” to “extremely”) Profile of Mood States (POMS) questionnaire was administered that asked subjects to “Describe how you feel right now”. There were 65 emotions listed which were categorized for scoring purposes including: “positive social feelings” (friendly, considerate, sympathetic); “positive general feelings” (lively, energetic, relaxed); “anxious” (tense, on edge, panicky, uneasy, restless); “angry” (angry, peeved, grouchy, spiteful); and “depressed” (unhappy, sad, blue, hopeless, unworthy). Sample emotions that were not scored included: “clear-headed”, “shaky”, “fatigued”, etc.
Post-interaction Questionnaire. Subjects were asked to rate the confederate on a ten-point Post-Interaction Likert-scale (from “not at all” to “extremely”) questionnaire. The questions were separated into three categories for scoring purposes: confederate’s personality (“How intelligent was your task partner?”; “How comfortable were you with your task partner?”; “How funny was your task partner?”; “If you had to do the task again, would you opt to have the same partner?”), confederate’s physical appearance (“How “good-looking” do you think your task partner was?”), and attraction to the confederate (“How attracted to your task partner were you?”). Non-relevant questions that were excluded from scoring included: “How difficult was the task?”, “Did you feel confined by the time limit?” etc.
Activity. A “Mind Trap” mind puzzle game (Pressman Toy Corp., New York) was used as a cooperative activity that encouraged constant conversation and close physical proximity (to ensure that the subject would be close enough to the confederate to potentially detect, either consciously or subconsciously, the pheromonal/olfactory mixture or vehicle).
Olfactory/pheromonal stimuli. A combination of androsterone (1 mg/ml) and androstenol (4 mg/mL ) (Applied Pheromones Research, Laguna Niguel, CA), were dissolved in propylene glycol. Propylene glycol was the control.
Scent Recognition Evaluations. After completing the task, subjects sniffed 10 ml of the propylene glycol-olfactory/pheromone mixture from a vial and were asked “Did you smell anything in the vial?”; “What did it smell like to you?” and “Did you like the scent?”. A positive answer to the latter question was scored as a three, a statement of indifference was scored as a two, and a negative statement was scored as a one.
A separate group of males and females were asked to rate the hedonic value of both the olfactory/pheromonal mixture and the vehicle. That questionnaire was similar to the worksheet used for the subjects, except the question “Did you like the scent?” was replaced with “On a scale of 1-5, with 1 being the worst and 5 being the best, how much did you like the scent?”.
Procedure
Approximately 10-15 minutes before arriving at the test site, the confederate applied 1 ml of either propylene glycol or the propylene glycol containing the androstenol/androsterone mixture to a cotton ball, which he then applied to his throat/neck (actual application amount was approximately 0.11 ml) in a separate room from the test site. The subject and confederate were introduced and left alone for approximately 1-2 minutes during which the confederate asked the subject questions (i.e. year in school, major) to put her at ease. Before beginning the activity, the subject and confederate filled out a subject sheet. The subject and confederate were then given instructions to work together on answering as many “mind” puzzles as possible in 15 minutes, told they would be filmed and asked to decide who would read a puzzle question aloud and who would record answers. The confederate had prior instructions to always read the questions aloud, maintain close physical proximity to subject, correctly answer the majority of puzzles, and appear flirtatious and outgoing. The 15 minute interaction was filmed and afterwards the confederate and subject were separated and each completed a post-interaction questionnaire and POMs questionnaire. The subject was also asked to smell a vial containing the olfactory/pheromonal/propylene glycol mixture and complete a corresponding worksheet. At the conclusion of the experiment, the subject was paid and de-briefed.
    Video Analysis. The frequency of several non-verbal courtship behaviors made by female subjects was recorded from the videotaped interaction. These behavioral categories were selected from a study that gathered information on female flirting by describing courtship behaviors initiated by women in bars that result in male responses (e.g., male approach, invitation to dance, touching, kissing, etc.; Moore, 2002). The specific courtship displays that were selected from the Moore study were chosen for ease of operationalization and included: making eye contact with the confederate and nodding, hair flipping (consisting of a woman raising one hand and pushing her fingers through her hair or running her palm along the surface of her hair), touching body or clothing (“primping behavior”), laughing, drawing attention to lips/breasts, and “dancing” (i.e. being overtly animated with lots of upper body movement) in their chair (Moore, 2002). A tally was made for each behavior when that act took place. The frequency of response or flirting behavior by the confederate was also recorded. These included: making eye contact with the subject, touching the subject, and increasing physical proximity to the subject. The video analysis was completed independently by both the experimenter and a second rater (20 year old Caucasian female student). Both individuals were blind to the condition of the subjects.
Results
             All statistical analyses were performed using SPSS 14.0 (p <.05). Independent t-tests were used to determine differences between the olfactory/pheromonal mix and control groups with respect to the female subject’s rating of the confederate, the female’s behavior, the female’s mood, and the female’s hedonic rating of the olfactory/pheromonal mix. Pearson’s correlation coefficients were used to determine inter-rater reliability (IRR) as well as other relationships among dependent measures. A one-way analysis of variance (ANOVA) was used to assess differences in hedonic rating of the olfactory/pheromonal mix among men and women who were and were not using hormonal birth control.
Female’s Rating of Confederate
As shown in Figure 1, when the confederate was wearing the androstenol/androsterone mixture, he was rated as being funnier (t(16) = 2.33, p = .03) and more comfortable to be around (t(16) = 3.42, p = .01). When he was wearing the androstenol/androsterone mixture, there was also a trend suggesting that subjects were more likely to want to work with the confederate again in a future interaction when he was wearing the androstenol/androsterone mixture (t(16) = 1.42, p = .18). There was no significant effect of the androstenol/androsterone mixture in the subject’s rating of the confederate as being more intelligent (t(16) = .76, p = .46). The score for the four personality questions combined was higher when the confederate was wearing the androstenol/androsterone mixture than when he was not (combined scores: t(15) = 2.57, p = .02).   No significant effects of the androstenol/androsterone mixture were found in the rating of the confederate’s physical appearance (t(16) = .63, p =.54) or in how attracted the subject was to the confederate (t(16) = .78, p = .449), as shown in Figure 1.
Female’s Behavior
As seen in Figure 2, female subjects were more likely to play with her hair and/or toss her head when the confederate was wearing the androstenol/androsterone mixture than when he wore the vehicle, (t(15) = 2.34, p = .03). For this measure, inter-rater reliability (IRR) was high, r = .954, p <.01. The presence of the androstenol/androsterone mixture did not have a significant effect on the subject’s frequency of touching her clothing/body (t(16) = .83, p = .42; IRR: r = .854, p <.01), making eye contact with the confederate and nodding (t(16) = .62, p = .55; IRR: r = .234, p = .34), laughing (t(16) = 0, p = 1; IRR: r = .486, p = .04), drawing attention to her lips and/or breasts (t(16) = 0, p = 1; IRR: r = .586, p = .01), or “dancing” in her seat (t(16) = .19, p = .85; IRR: r = .650, p <.01), as displayed in Figure 2.
Female’s Mood
There was no significant effect on the subject’s mood related to the presence of the androstenol/androsterone mixture with respect to a general positive feeling (t(16) = .91, p = .38), a social positive feeling (t(16) = .76, p = .46), depressed feelings (t(16) = .65, p = .53), angry feelings (t(16) = .1, p = .92), or anxious feelings (t(16) = .27, p = .80).
Hedonic Rating of Pheromones
There was no significant correlation between any subject’s hedonic rating of the androstenol/androsterone mixture and their frequency of courtship behaviors toward the confederate versus control (r = .023, p = .93). There was no significant difference in the hedonic value of the scent of the androstenol/androsterone mixture as rated by a separate group of male subjects, female subjects not using a hormonal birth control, and female subjects on a hormonal birth control (F(2) = .325, p = .72) versus control, as shown in Figure 3.
Confederate’s Behavior
    The confederate exhibited a higher frequency of combined courtship behaviors: making eye contact with the subject, increasing his physical proximity to the subject, and touching the subject when he was wearing the androstenol/androsterone mixture (t(16) = 2.69, p = .02). Individually, none of these behaviors differed significantly when the confederate was wearing the androstenol/androsterone mixture versus when he was not, (eye contact: t(16) = 2.01, p = .06; physical proximity: t(16) = .94, p = .36; touching: t(16) = 0, p = 1) indicating that that the effect of the androstenol/androsterone mixture on the confederate’s behavior was most likely minimal.
Discussion
Female exposure to the androstenol/androsterone mixture, which was the variable stimulus in our study, appeared to influence hair flipping/head tossing behavior. Additionally, when the confederate was wearing the androstenol/androsterone stimulus, he was given higher ratings in being “funnier” and “more comfortable to be around”. The confederate showed a tendency to behave differently toward female subjects when he was wearing the androstenol/androsterone mixture. Whether his behavior was reactionary to the courtship behaviors of the female subject, or perhaps a subconscious response to wearing the androstenol/androsterone mixture, is not clear, and seems somewhat unlikely to be clarified. Masking the androstenol/androsterone mixture with an odor to conceal it, as done in Experiment 2, attempted to rule out any confounds associated with the confederate knowing the treatment condition.
Experiment 2
Method
Participants
Fourteen female college students (mean age= 20 years; 11 Caucasian, 1 African-American, 2 Other) participated in this study. Methods of recruitment and identification of ovulation were identical to those in Experiment 1. The same confederate was also used as in Experiment 1.
Materials
            The materials in this study were identical to those used in Experiment 1 with the inclusion of sandalwood essential oil (Lotus Brands, Twin Lakes, WI) as the masking odor. We included the masking odor in the “scent recognition evaluations”. A shorter and more applicable mood evaluation questionnaire was substituted for the POMs.
            Mood Assessment. Subjects completed a questionnaire both before and after the cooperative task asking them to rate their feelings according to how they feel at that exact moment on a scale from -4 to 4 that correlates with two emotional extremes (i.e. happy and unhappy). The list of extreme emotions include: happy and unhappy, pleased and annoyed, satisfied and unsatisfied, contented and melancholic, hopeful and despairing, relaxed and bored, stimulated and relaxed, excited and calm, frenzied and sluggish, jittery and dull, wide-awake and sleepy, aroused and unaroused, controlling and controlled, influential and influenced, in control and cared-for, important and awed, dominant and submissive, and autonomous and guided (Mehrabian and Russell, 1974).
Scent Recognition Evaluations. After completing the task, subjects sniffed a cotton ball in a vial with 0.1 ml of the propylene glycol containing the androstenol/androsterone mixture and a cotton ball in a vial with 0.1 ml of propylene glycol.   Three drops (approximately .088 ml) of sandalwood essential oil were added to both cotton balls to mask any consciously perceived odor of the androstenol/androsterone mixture. Subjects responded to the following questions: “Did you smell anything in the vial?”, “What did it smell like to you?” and “On a scale of 1-5, with 1 being the worst and 5 being the best, how much did you like the scent?”
Procedure
Before the start of the experiment, the experimenter applied 1 ml of either propylene glycol or the propylene glycol containing the androstenol/androsterone mixture to a cotton ball and added three drops (approximately .088 ml) of sandalwood essential oil to the cotton ball in a separate room from the confederate (as to withhold information about which condition he was running in). The experimenter swabbed the confederate’s throat/neck with the cotton ball (actual application amount was approximately 0.11 ml). The remainder of the procedure was identical to Experiment 1, with the inclusion and substitution of the aforementioned materials. Additionally, in the video analysis, the observed female behaviors were pared down to only include the frequencies of: touching lips (with fingers or pen), hair flipping (consisting of a woman raising one hand and pushing her fingers through her hair or running her palm along the surface of her hair), eye contact, and laughing.
Results
Female’s Rating of Confederate
As shown in Figure 4, when the confederate was wearing the androstenol/androsterone mixture, the females rated themselves as being more attracted to him (t(12) = 2.786, p = .016). There was no significant effect of the androstenol/androsterone mixture in the subject’s rating of the confederate as being more intelligent (t(12) = .965, p = .35), more comfortable to be around (t(12) = .106, p = .92), funnier (t(12) = .079, p = .94), more “good-looking” (t(12) = .1.38, p = .19), or in having the confederate as a task partner again (t(12) = 1.02, p = .33).
Female’s Behavior
As seen in Figure 5, female subjects were overall more likely to display non-verbal courtship behaviors when the confederate was wearing the androstenol/androsterone mixture than when he wore the vehicle (t(12) = 4.38, p <.01; IRR: r =.914, p = .01). Specifically, they were more likely to make eye contact with the confederate (t(12) = 3.43, p = .01; IRR: r = .964, p = .01) and laughed more during the interaction (t(12) = 5.20, p <.01; IRR: r = .810, p = .01) . The presence of the androstenol/androsterone mixture did not have a significant effect on the subject’s frequency of touching her lips (t(12) = 1.96, p = .07; IRR: r = .894, p = .01) or “hair flipping” (t(12) = 1.57, p = .14; IRR: r = .794, p = .01).
Female’s Mood
There was no significant effect on the subject’s mood related to the presence of the androstenol/androsterone mixture (t(12) = 0, p = 1).
Hedonic Rating of Pheromones
There was no significant correlation between any subject’s hedonic rating of the androstenol/androsterone mixture and their frequency of courtship behaviors toward the confederate (r = .174, p = .55).
Confederate’s Behavior
    The confederate exhibited a higher frequency of combined courtship behaviors: making eye contact with the subject, increasing his physical proximity to the subject, and touching the subject when wearing the androstenol/androsterone mixture (t(12) = 3.08, p = .01; IRR: r = .725, p = .01). Specifically, the confederate made eye contact more often when he was wearing the androstenol/androsterone mixture versus when he was not, (t(12) = 2.71, p = .02; IRR: r = .749, p = .01). Physical proximity (t(12) = 1.20, p = .25; IRR: r = .616, p = .05) and touching (t(12) = 2.12, p = .06; IRR: r = .826, p = .01) were not individually significant between the two conditions.
Discussion
In Experiment 2, we found that female subjects were more likely to make eye contact with the confederate and laugh more frequently during the cooperative task when the confederate was wearing the androstenol/androsterone mixture. Additionally, when the confederate was wearing the androstenol/androsterone mixture, subjects gave a higher rating in response to the question “How attracted to your task partner were you?”. Just as in Experiment 1, the confederate showed a tendency to display more flirting behaviors toward female subjects when he was wearing the androstenol/androsterone mixture, even though he was blind to the condition in this experiment. The two experiments differed in that Experiment 1 took place in the winter and spring months, whereas Experiment 2 took place in the fall. Additionally, having taken part as the confederate in the first study, the confederate was more familiar and comfortable with his role in Experiment 2, than he may have been in Experiment 1 and subsequently have interacted with the female subjects in Experiment 2 in a more confident and natural manner resulting in more pronounced reactions from the subjects. In Experiment 2, the trend for hair flipping was favorable toward the androstenol/androsterone mixture condition, although it was not significant as it was in Experiment 1. However, if the subjects from the two experiments are combined, hair flipping remains significant (t(30) = 2.66, p = .01).
Overall, it appears that female subjects displayed more subtle reactions to the olfactory/pheromonal stimuli in Experiment 1 by rating the confederate higher on indirect measures of affinity (e.g., funnier, more comfortable to be around) and displaying a more subtle courtship behavior (hair flipping). Given that the confederate may have been more at ease with the interaction in Experiment 2, his behavior may have been more natural and female subjects, consequently, may have felt more comfortable identifying their attraction toward the confederate and displaying more overt signs of interest (eye contact and laughing) in the androstenol/androsterone mixture condition. The fact that the confederate’s frequency of eye contact was significantly greater in the androstenol/androsterone mixture condition, appears to support our assumption that his flirting behavior was reactionary and complementary to the flirting behavior of the female subjects. Although we had also originally posited an improvement in mood among the female subjects exposed to the androstenol/androsterone mixture condition, the reason any specific behavioral affect might not be paired with mood change is because the behavior is conditioned and preceded by a rapid hormone change–one that is perhaps too rapid to be concurrently measured in mood.
In our study, we did not measure hormone levels, which may have indicated increased LH. Thus, we lack data that would support the androstenol/androsterone mixture as a proximate source that effected a hormonal change. We did observe behavioral changes within the context of a rather brief interaction that might best approximate an uncontrolled social situation.
Ultimately, “There is no proof as yet that any compound is a human pheromone” (Kohl, 2006, 324). This does not mean that there is no such thing as human pheromones, and does not mean that a androstenol/androsterone mixture, or any mixture of olfactory/pheromonal stimuli might best be used to test the existence of human pheromones. Because there is no direct (e.g., neuroendocrine) link from non-olfactory social-environmental sensory input to sex differences in behavior, we chose to test the likelihood that species specific human olfactory/pheromonal input fits the traditional definition of pheromones, and that women might be conditioned to respond to them.
In other species, sexual behavior is subject to vary in response to hormone-derived, ever-changing, complex, species-specific chemical blends. Exposure to any one of several proposed putative human pheromones (e.g. androstadienone, androstenol, androsterone) is unlikely compared to the possibility of exposure to all human pheromones. Additional focus on putative human pheromones derived from adrenal hormone metabolism, like androsterone, may more fully address the complexity of genetically predisposed olfactory/pheromonal conditioning of human sexual behavior.
It is generally agreed that sex steroid hormones influence mammalian behavior and that olfactory/pheromonal input from the social environment influence mammalian behavior. It is not known how visual or other non-olfactory/pheromonal sensory input from the social environment might have any direct effect either on mammalian sex steroid hormones or – in the absence of a direct connection to hormonal changes – how non-olfactory/pheromonal sensory input might influence either avian or mammalian behavior. Thus, if not for additional studies of putative human pheromones, and olfactory/pheromonal mixtures, we might be left with only a hypothetical innate releasing mechanism (IRM), like the one that supposedly offers the only explanation for how visual input altered the sexual behavior of avian species (Tinbergen, 1948).
Given what is known about mammalian olfactory/pheromonal conditioning, we hypothesized that an olfactory/pheromonal stimulus may play a role in a woman’s altered perception of a man (e.g., funnier, more intelligent, more comfortable to be around, more attractive) who is wearing an androstenol/androsterone mix. This role may extend to a difference in a behavior associated with women’s sexual interest (e.g., hair flipping, eye contact, laughing). We are aware that perception of a man may be altered by fragrance use alone. We are not likely to be aware when either perception of a man, or behaviors associated with women’s sexual interest change unless these behaviors are observed to change. Future studies may include either more or fewer measures of behavioral change, with different controls, to arrive at greater statistical significance, or to dispute the likely association between putative human pheromones and behavior. We may be the first to use a mixture of putative human pheromones; the first to incorporate species-specificity into the mixture; and the first to show change in women’s rating of personality characteristics of our confederate. We are decidedly the first to show the predictable behavioral affects of putative human pheromones.
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Figure Captions
Figure 1. Mean scores from ten-point Likert-scale Post-interaction questionnaire (n= 18). Personality questions asked about confederate’s intelligence, sense of humor, whether he was comfortable to be around, and whether subject would opt to be partners with him again. Physical appearance question asked how “good-looking” confederate was. Attraction question asked subject how attracted she was to confederate. Personality was significantly different (p= .03).
Figure 2. Mean scores from video analysis of subject-confederate interaction (n= 18). Key flirting behaviors included touching own body and/or clothing, playing with hair/head toss, eye contact with nodding, laughing, drawing attention to lips or breasts, and “dancing” in seat. Playing with hair/head toss was significantly different (p= .03).
Figure 3. Mean hedonic ratings of olfactory/pheromonal stimuli and vehicle among men (n= 20), women not on hormonal birth control (n= 22), and women on hormonal birth control (n= 7). There were no significant differences in hedonic ratings between the groups with respect to olfactory/pheromonal stimuli and vehicle.
Figure 4. Mean scores from ten-point Likert-scale Post-interaction questionnaire (n= 14). Questions included: confederate’s intelligence, how funny he was, whether he was comfortable to be around, whether subject would opt to be partners with him again, how “good-looking” confederate was, and how attracted subject was to the confederate. The question concerning how subject’s attraction to the confederate was significantly different (p = .02).
Figure 5. Mean scores from video analysis of subject-confederate interaction (n= 14). Key flirting behaviors included playing with hair, eye contact, laughing, and touching lips. Eye contact (p < .01) and laughing were significantly different (p< .01).
 
 
 
 
 
 

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