the Microbiome: The Relationship between Baseline Gut Mi‐ Composition, Gender and Pain in Healthy Individuals

41 Background and Aim. Relative to men, women present with pain conditions more commonly. Although consistent differences exist between men and women in terms of physiological pain sensitivity, the underlying mechanisms are incompletely understood and yet could inform the development of effective sex specific treatments for pain. The gut microbiota can modulate nervous 45 system functioning, including pain signaling pathways. We hypothesized that the gut microbiota and 46 critical components of the gut-brain axis might influence electrical pain thresholds. Further, we 47 hypothesized that sex, menstrual cycle, and hormonal contraceptive use might account for inter-sex 48 differences in pain perception . salivary cortisol levels levels of lipopolysaccharide-binding protein (LBP), soluble (sCD14), pro-inflammatory cytokines assessed microbiota composition and short-chain fatty acids (SCFAs) levels were determined in fecal


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Pain is described as an unpleasant sensory and emotional event, often associated with underlying 74 tissue injury. Acute pain usually lasts for a short period of time following injury, allowing for tissue 75 healing and usually resolves on its own (Fong and Schug, 2014). demonstrated no significant sex differences in pain perception; however, for those of age >12y, 87 females report higher pain intensity in response to the cold pressor test compared to males (Boerner 88 et al., 2014). These data suggest that the onset of puberty and the associated hormonal changes may 89 contribute to sex bias in pain sensitivity in teenagers and possibly adults (Boerner et al., 2014).

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The general pathway of pain sensation is regulated by the peripheral nervous system, which senses 91 the variations of sensory perceptions in the peripheral organs and conveys this information to the brain plastic container with an ice pack prior to refrigerator placement. Additional notes included instructions not to brush teeth throughout the collection process, drink or eat anything prior to Sample

Women display a lesser ratio of pain tolerance threshold (PTT) to pain sensation threshold (PST)
315 compared to men.

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The assessment of sensory pain did not show any significant differences in PST or PTT between males 317 and females nor in females using hormonal contraceptives and non-users (Figure 2A and B). In 318 categorizing women as contraceptive users or non-users, significantly lesser PTT/PST ratios were 319 demonstrated for the EF phase of contraceptive users (p≤0.05) and the ML phase of non-users (p≤0.05) 320 compared to males ( Figure 2C). However, when we combined all women, we did observe a significantly 321 lesser PTT/PST ratios in all phases of the menstrual cycle (EF, ML = p≤0.01; LF= p≤0.05) compared to 322 males ( Figure 2D).     Figure 1). This profile was not observed in the separate groups, except for the EF phase of females 533 using hormonal contraceptives ( Table 2). In addition, no significant differences were observed 534 between groups/menstrual cycle phases for 1) cortisol levels at a specific time point, 2) cortisol 535 increase during the first 30 minutes after awakening and 3) cortisol output during the first hour after 536 awakening (area under the curve) ( Table 2). Of special interest, for the LF phase of normally 537 menstruating females, a mean negative cortisol awakening response was observed (  Lastly, we investigated potential associations between pain sensitivity thresholds, cortisol awakening 616 responses and plasma levels of LBP, sCD14, and inflammatory cytokines or stool levels of SCFAs for 617 males ( Figure 9A) and Supplementary Table 7) and females ( Figure 9B) and Supplementary Tables 8)   618 apart. In males, we found three significant (FDR-corrected) correlations within the SCFAs data which 619 we do not deem overly interesting (Figure 9A). In females, all SCFAs data, a great part of the cortisol 620 data, some inflammatory markers, and PTT and PST significantly correlated within each dataset (FDR-621 corrected p-value <0.05, <0.01 Figure 9B). Potentially the most interesting finding was that IL-8 622 correlated positively with PST in females (FDR-corrected p-value <0.05; Figure 8B).  represents the proportionate increase in electric current that can be tolerated relative to that at which 705 pain is first perceived (Kanner, 2009

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The main limitation of the present study is the small number of subjects enrolled in the trial. For 786 instance, the lack of differences in PTT or PST across the menstrual cycle observed in our study is most likely due to the low sample size.

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26% of the participants failed to collect at least 1 out of 6 saliva samples at the correct time of the day, 801 and this had a significant impact on the circadian cortisol profile (Kudielka et al., 2003).

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We acknowledge that the low sample number may have obstructed us from showing significant 803 correlations between the gut microbiota and host stress or pain outcomes; however, our results 804 demonstrate a relationship between somatic sensory pain and stress response in healthy women, in 805 which the baseline gut microbiota seems to play a role.

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This is the first study to highlight the connections between the gut microbiota and l sex differences in 808 physiological pain perception. Further studies will help to elucidate how sex interacts with host factors 809 and the gut microbiota and how these relationships modulate somatic pain sensitivity, for example, by The authors declare that they have no known competing financial interests or personal relationships 828 that could have appeared to influence the work reported in this paper.