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Social withdrawal and testosterone levels in early adolescent boys
Journal Pre-proof Social withdrawal and testosterone levels in early adolescent boys Noriyuki Hayashi, Shuntaro Ando, Seiichiro Jinde, Shinya Fujikawa, Naohiro Okada, Rie Toriyama, Mio Masaoka, Hiroshi Sugiyama, Toru Shirakawa, Tomoko Yagi, Masaya Morita, Ryo Morishima, Tomoki Kiyono, Syudo Yamasaki, Atsushi Nishida, Kiyoto Kasai
PII:
S0306-4530(20)30015-9
DOI:
https://doi.org/10.1016/j.psyneuen.2020.104596
Reference:
PNEC 104596
To appear in:
Psychoneuroendocrinology
Received Date:
20 March 2019
Revised Date:
29 October 2019
Accepted Date:
16 January 2020
Please cite this article as: Hayashi N, Ando S, Jinde S, Fujikawa S, Okada N, Toriyama R, Masaoka M, Sugiyama H, Shirakawa T, Yagi T, Morita M, Morishima R, Kiyono T, Yamasaki S, Nishida A, Kasai K, Social withdrawal and testosterone levels in early adolescent boys, Psychoneuroendocrinology (2020), doi: https://doi.org/10.1016/j.psyneuen.2020.104596
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Noriyuki Hayashi
Social withdrawal and testosterone levels in early adolescent boys
Noriyuki Hayashia, Shuntaro Andoa,b, Seiichiro Jindea, Shinya Fujikawaa, Naohiro Okadaa,c, Rie Toriyamaa, Mio Masaokaa, Hiroshi Sugiyamaa,d, Toru Shirakawaa, Tomoko Yagia, Masaya Moritaa, Ryo
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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a
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Morishimaa, Tomoki Kiyonoa, Syudo Yamasakib, Atsushi Nishidab, Kiyoto Kasaia,c.
Bunkyo-ku, Tokyo 113-8655, Japan.
Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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b
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes
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c
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Department of Integrated Educational Sciences, Graduate School of Education, The University of
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d
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Japan.
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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Noriyuki Hayashi
Noriyuki Hayashi Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
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Shuntaro Ando *
Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. +81-3-3815-5411
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[email protected]
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Seiichiro Jinde
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
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Noriyuki Hayashi
Shinya Fujikawa Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
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Naohiro Okada Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan.
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes
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for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
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Rie Toriyama
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[email protected]
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Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Mio Masaoka
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Noriyuki Hayashi
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Hiroshi Sugiyama
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Integrated Educational Sciences, Graduate School of Education, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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[email protected]
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Toru Shirakawa
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
Tomoko Yagi
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
Masaya Morita Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
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Ryo Morishima
Bunkyo-ku, Tokyo 113-8655, Japan.
Tomoki Kiyono
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[email protected]
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Syudo Yamasaki Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. [email protected]
Atsushi Nishida Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
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[email protected]
Kiyoto Kasai
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Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
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Japan.
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[email protected]
* corresponding author
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Noriyuki Hayashi Highlights
Social withdrawal in general early adolescent boys was evaluated by primary parents
Salivary testosterone was measured by liquid chromatography-tandem mass spectrometry
A higher risk of boy’s social withdrawal was related to a lower testosterone level
The association remained after adjusting for confounders such as pubertal stage
Negative relationship between social withdrawal and testosterone levels was found
ABSTRACT
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Social withdrawal may lead to mental health problems and can have a large impact on a life course, particularly among boys. To support adolescents with social withdrawal, an integrative understanding
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of the biological bases would be helpful. Social dominance, a possible opposite of social withdrawal, is known to have positive associations with testosterone levels. A previous study suggested that social
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withdrawal has a negative relationship with sexual maturity among adolescent boys. However, the
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relationship between social withdrawal and testosterone in adolescence is unknown. This study aimed
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to examine whether social withdrawal was negatively associated with testosterone levels in early adolescent boys. Salivary samples were collected from 159 healthy early adolescent boys (mean age
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[standard deviation]: 11.5 [0.73]) selected from participants of the “population-neuroscience study of
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the Tokyo Teen Cohort” (pn-TTC). Social withdrawal and confounding factors, such as the secondary sexual characteristics and their age in months, were evaluated by self-administered questionnaires completed by the primary parents. The degree of social withdrawal was assessed with the Child Behaviour Checklist (CBCL). Levels of salivary testosterone, and cortisol as a control, were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Logistic regression was conducted
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to examine the association between social withdrawal and testosterone levels. A higher risk of social withdrawal was associated with a lower salivary testosterone level after adjustment for age in months (odds ratio 0.55, 95% confidence interval 0.33-0.94), and the association remained significant after adjusting for body mass index, the degree of anxiety/depression and pubertal stage. Thus, we found a negative relationship between social withdrawal and testosterone levels in early adolescent boys. These
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findings may help to clarify the biological foundations of and to develop support for social withdrawal.
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Keywords: social withdrawal, testosterone, puberty, LC-MS/MS, the population-neuroscience study of
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the Tokyo Teen Cohort
1. Introduction
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Social withdrawal is one of the internalizing problems, and defined as “the child’s isolating
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himself/herself from the peer group” (Rubin et al., 2009). Social withdrawal might lead to other
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psychiatric problems. Children aged 8 years with a higher degree of social withdrawal had higher risks of depression and anxiety disorders in adolescence (Goodwin et al., 2004). Adolescents aged 13-16 years with a higher degree of social withdrawal had higher risks of suicide attempts several years later (Ferdinand and Verhulst, 1995). Moreover, social withdrawal among boys seems to have a much larger impact on their life courses than girls. Males who had been shy (a construct of social withdrawal) in
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childhood married, became fathers and established stable carriers at later ages in life than other males, while no such disadvantage was found in female (Caspi et al., 1988; Kerr et al., 1996). This sex difference is thought to be attributed to the norm of the traditional gender roles (men as breadwinner and women as caregiver) (Asendorpf et al., 2008), from which recent Japanese society has not shifted away (Kato, 2018). Thus, social withdrawal in boys might be more problematic than in girls in Japanese
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society. A form of severe social withdrawal called “hikikomori” (a Japanese word which can be translated as “withdrawal”), first described in Japan, usually occurs in ages 15-19 years during
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adolescence (Li and Wong, 2015a, 2015b). Although there is lack of consensus on its definition, hikikomori is recognized as social withdrawal not going to school or work and less communicating with
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people during more than 3 or 6 months (Li and Wong, 2015a, 2015b). It was experienced by as many
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as 1-2% of the community sample in Japan and Hong Kong (Koyama et al., 2010; Wong et al., 2015).
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The lifetime prevalence of hikikomori in males was approximately five times as high as in females (Koyama et al., 2010). This is in contrast with anxiety/depression, one of the main internalizing
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problems, for which adolescent girls have the higher risks than boys (Merrell, 2008). A competitive but
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rapidly changing society and family relationship in modern-day Japanese society is considered to contribute to the development of hikikomori to some extent especially in boys (Koyama et al., 2010). Based on these facts, early support would be needed, especially among boys with social withdrawal.
Current support methods of social withdrawal are formed mainly from psycho-social standpoints (Li and Wong, 2015b; Rubin et al., 2009), probably because studies of this behaviour have been developed
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from these standpoints traditionally. Although the biological foundations of social withdrawal have also been studied (Rubin et al., 2009), integrative understanding has been insufficient. Revealing the biological foundations of social withdrawal in childhood or adolescence is a challenge to be addressed. New knowledge about biological foundations could provide helpful suggestions to current various supports for social withdrawal.
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In the framework of the approach-avoidance behaviors, social withdrawal is classified as an avoidance behavior (Lopez et al., 2004). On the other hand, social dominance means acquiring and
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maintaining higher status in social hierarchy to have a power and affect conspecifics (Mazur and Booth, 1998), and is classified as an approach behavior (Terburg et al., 2013). Therefore, social
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withdrawal could be opposed to social dominance in the spectrum of approach-avoidant behaviors. A
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significant negative correlation was found between the degree of social withdrawal and social
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dominance in 9- to 13-year-old boys (Lease et al., 2002). Biological studies of social dominance have focused on the role of sexual hormones (Qu et al., 2017; Stanton and Schultheiss, 2009). Cross-
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sectional data show that adolescent boys aged 13 years who were regarded as socially dominant by
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their peers had significantly higher levels of testosterone than boys who were regarded as less socially dominant (Schaal et al., 1996). A different study shows that a degree of social dominance had a significant positive relationship with testosterone level in boys aged 9-15 years (Rowe et al., 2004). Therefore, one might predict that social withdrawal, a possible opposite to social dominance, has a negative relationship with testosterone level among adolescent boys.
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On the other hand, there have been few studies examining associations between social withdrawal and sexual maturation. A retrospective study of healthy college students showed that males with extremely late sexual maturation had significantly higher degrees of social withdrawal than males with extremely early maturation (Gruzelier and Kaiser, 1996). This result and a positive correlation between sexual maturity and testosterone level (Granger et al., 2004; Shirtcliff et al., 2009) also suggest that
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higher degrees of social withdrawal may be associated with lower testosterone levels in adolescent males.
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However, the relationship between social withdrawal and testosterone levels among adolescent boys remains unclear. The aim of this study was to test our hypothesis that a higher risk of social withdrawal
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2. METHOD
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was associated with a lower level of testosterone in early adolescent boys.
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2.1. Study design and participants The data in this study are based on the population-neuroscience study of the Tokyo Teen Cohort (pn-
TTC) (Okada et al., 2019). In this longitudinal cohort study, physical, socioeconomic, psychiatric and psychological data as well as hormonal and deoxyribonucleic acid (DNA) data derived from saliva and magnetic resonance imaging (MRI) data were obtained from adolescents and their primary parents for
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the exploration of the neurobiological substrates of development during adolescence. The targeted number of participants in the pn-TTC study was approximately 300 pairs of early adolescents and their primary parents. The participants of the pn-TTC were chosen from the 3,171 pairs participating in the Tokyo Teen Cohort (TTC) study (Ando et al., 2019) who had been recruited from the 4,478 pairs participating in the Tokyo Early Adolescence Survey (T-EAS). The data acquired in the T-EAS were
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used as wave 1 data of the longitudinal studies (the TTC and the pn-TTC). The T-EAS is a multidisciplinary cross-sectional survey of teen’s health and development carried out
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from Oct. 2012 to Jan. 2015. Briefly, adolescents born between September 1st, 2002, and August 31st, 2004, and recorded on the basic resident register of Setagaya-ku, Chohu-city and Mitaka-city, Tokyo,
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were randomly selected and regarded as candidates for the survey. Among the 10,234 contactable early
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adolescents aged 10 and their primary parents, 4,478 pairs cooperated with the survey (cooperation rate:
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43.8%). In this survey, physical, socioeconomic, psychiatric and psychological data were acquired through interviews by trained interviewers and various questionnaires (including the Child Behaviour
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Checklist [CBCL]) (Achenbach, 1991; Itani et al., 2001).
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Among 3,171 pairs who participated in the TTC, which was the subsequent longitudinal study, invitation letters to the pn-TTC were sent to 980 pairs who showed possibilities of cooperation with the study. The exclusion criteria were as follows: (1) current problems in mental health, interpersonal relationships or behaviours, (2) visual or hearing disabilities, (3) past history of head injury accompanied by more than five minutes of loss of consciousness, (4) current chronic endocrine or
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metabolic diseases, and (5) current use of medications that affect the central nervous system. A total of 176 adolescents who had first menstruation or were in the stage 3 or more advanced stages of Tanner pubertal stage (Marshall and Tanner, 1969, 1970) were oversampled, and the remaining 804 adolescents were randomly sampled. Out of 980 pairs, a total of 301 pairs (boys; 162, girls; 139) agreed and participated in the pn-TTC study. The participants were asked to visit our laboratory from September
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2013 to February 2016. The adolescent’s height, body weight and secondary sexual characteristics were assessed there. The adolescents collected their salivary samples at home by the method described in the
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next section. Of the participating adolescent boys, 159 boys collected their salivary samples properly. Salivary testosterone and cortisol levels of these samples were measured (the latter as a control). Finally,
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the data of 159 boys were analysed in this study.
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This study was approved by ethics committees of the University of Tokyo, Faculty of Medicine,
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Tokyo Metropolitan Institute of Medical Science, and the Graduate University for Advanced Studies (SOKENDAI), according to the Declaration of Helsinki. The adolescents and their primary parents were
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orally informed of the contents of this research and provided written consent forms.
2.2. Measurement
2.2.1.
Social withdrawal
In this study, early adolescents’ behaviours and emotions at age 10 were evaluated by primary parents
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with the CBCL/4-18 Japanese version (Itani et al., 2001). The CBCL (Achenbach, 1991) is composed of 118 questions about problems of child behaviours and emotions within the past six months, which include scales of social withdrawal and anxiety/depression. The questions about social withdrawal included the following 9 items: “Would rather be alone”, “Refuses to talk”, “Secretive”, “Shy”, “Stares blankly”, “Sulks a lot”, “Lacks energy”, “Unhappy, sad,
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depressed”, and “Withdrawn”. The primary parents chose the best answers to the questions among 0 = “not true”, 1 = “somewhat or sometimes true”, and 2 = “very true or often true”. The sum score of the
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questions is referred to as the raw score of social withdrawal, which could range from 0 to 18.
Raw score can be converted to a normalized T score, which was calculated by sex and ranges of ages
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of standard samples. After calculation of the midpoint percentile of the cumulative frequency
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distribution of the raw score in the standard samples, the T score was determined by the procedure of
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Abramowitz and Stegun (Abramowitz and Stegun, 1968). Raw scores of 50 midpoint percentiles and under were assigned a T score of 50. The raw score of the
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97.7th percentile was assigned a T score of 70, and the remaining raw scores were assigned T scores
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from 71 to 100 in equal intervals. T scores of less than 67 (95th percentile), 67 to 70, and over 70 are referred to as “normal range”, “borderline range”, and “clinical range”, respectively. For a specific categorical cutoff point, a T score of 67 has been commonly used to represent the bottom of a deviant range (including the borderline and the clinical range) (Lien et al., 2015; Wolak et al., 2007a, 2007b) as the original textbook of the CBCL had instructed (Achenbach, 1991). The reliability and validity of the
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CBCL have been well established (Achenbach, 1991).
2.2.2.
Salivary testosterone level
The adolescents collected their salivary samples at home early in the morning. In advance, both the adolescents and their primary parents were informed of how to collect the
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adolescents’ saliva using sample tubes. The adolescents tried it under the guidance of the survey staff for practice. They were instructed not to collect the saliva within a week after a tooth extraction or
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immediately after dental treatment to avoid contamination with blood. They were also asked not to eat food after brushing their teeth on the night before the saliva collection. They were instructed to rinse
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their mouth soon after getting up and to make sure they were at their normal body temperature, and not
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to have breakfast and not to brush teeth before the collection. Furthermore, they were asked to wait for 20 minutes after the rinse and then to collect 4.5 ml of their saliva by passive drool in sterilized tubes
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(1.5 ml/tube * 3 tubes) made of polypropylene (NalgeneTM General Long-Term Storage Cryogenic
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Tubes, Thermo Fisher SCIENTIFIC, U.S. A) within 60 minutes. Salivary samples were collected in
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only one day, since high correlation among morning salivary testosterone levels across days in adolescents was reported (Granger et al., 2004). Salivary samples were kept in household refrigerator freezers, delivered frozen to our laboratory, where the weights were measured and tubes stored at minus 80 degrees C until the testosterone levels were measured. The concentration of salivary testosterone was measured once by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which has
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become the current standard (Ketha et al., 2014). The lower limit of quantification (LLOQ) of testosterone was 0.7 pg/ml. There were 3 boys (1.9%) who showed testosterone levels below the LLOQ (BLOQ). Such data were incorporated in our analysis (Keizer et al., 2015). The significance of the following results remained even after exclusion of these 3
2.2.3.
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boys’ data.
Salivary cortisol level
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Salivary cortisol levels in the same saliva sample were simultaneously measured as a control hormone by the LC-MS/MS method to exclude the possibility that any hormone was negatively associated with
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social withdrawal. The LLOQ of cortisol was 6.7 pg/ml, and cortisol levels in all samples were
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above this limit.
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2.2.4. Potential confounders
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2.2.4.1. Secondary sexual characteristics The primary parents were presented drawings of pubic hair and genitalia, which were classified into
the five Tanner stages (from Stage 1: pre-pubertal to Stage 5: post-pubertal). They were asked which pictures fit their children (Marshall and Tanner, 1970). The higher of the pubic hair stage or the genital stage measures represented the Tanner pubertal stage in this study. A previous study showed that
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evaluation of children’s sexual development by their parents using the drawings or similar pictures had a strong correlation with the evaluation with physical examination by medical practitioners (the gold standard method) (Coleman and Coleman, 2002).
2.2.4.2. Body mass index (BMI)
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BMI was calculated from the adolescents’ height and body weight measured by trained interviewers. A previous study showed that children aged 10-12 years with a BMI at the 95th percentile or more
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had higher total problem scores (including social withdrawal score) on the CBCL than other children (Hwang et al., 2006). A meta-analysis showed that testosterone levels in males had a negative
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excluded (MacDonald et al., 2010).
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relationship with BMI, although the studies in which the mean ages were under 12 years old were
2.2.4.3. Anxiety/depression
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Previous studies have reported significant association of anxiety/depression with social withdrawal
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and testosterone. A positive relationship between degree of social withdrawal and degree of depressive symptoms had been reported (Barragan et al., 2011). Negative relationships between degrees of anxiety/depression and testosterone levels in adolescent males have been reported in other studies (Granger et al., 2003; Susman et al., 1991). In this study, the degree of anxiety/depression was assessed by the CBCL anxiety/depression scale. This scale comprised 14 questions, and the raw score could
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range from 0 to 28. The T score and normal/borderline/clinical ranges of that scale were defined in a similar manner as the CBCL social withdrawal scale mentioned above.
2.3. Data analysis In terms of age in months, the data at the day of the saliva collection were used. Medians and
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interquartile ranges (IQR) were calculated for salivary testosterone levels and the CBCL raw scores because of their positively skewed distributions. For the following analyses, natural logarithms of
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salivary testosterone levels (lnT) were used. For the correlation coefficients between two observed variables with a nonnormal distribution, Spearman’s rho was calculated. Similarly, for comparison of
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an observed variable among two or more groups, the Mann-Whitney U test or Kruskal-Wallis test were
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performed.
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The adolescents were placed into one of five groups, from the lowest lnT group to the highest lnT group, created by dividing the range of lnT into 5 equal-width bins. The ratios of adolescents with the
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borderline or clinical ranges of social withdrawal in each group were calculated. The ratios were
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compared between the five groups by the chi-squared test. The linearity between the order of the five groups and the rates of social withdrawal was tested by the Cochran-Armitage test. A similar analysis was performed for cortisol levels. In the logistic regression of this study, the binominal variable of 1 was assigned for the borderline or clinical range of social withdrawal (T score 67 or above), and 0 was assigned for the normal range (T
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score < 67). LnT (and cortisol) and other variables were treated as the independent variables in the regression analyses. The effect of testosterone (and cortisol) levels on social withdrawal was evaluated after adjustment for age in months (demographic-adjusted model). After evaluation of the potential confounders in our actual data, we put all these confounders in the regression one by one. Besides the logistic regressions, we conducted linear regressions using raw scores of social
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withdrawal as dependent variable, and age in months, lnT, BMI, and depression/anxiety as independent variables. However, since normal P-P plots of regression standardized residuals were not linear, we
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judged linear regressions as inappropriate in this study. A lot of zeros of social withdrawal’ raw scores
in this section) seemed to cause this result.
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(median 1.0, IQR 2.0 as shown in Table 1) and positively skewed distribution of raw scores (mentioned
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IBM SPSS ver 24 and EZR (Kanda, 2013) were used for the analyses (the latter used only for the
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Cochran-Armitage test), and the level of significance was set at p < 0.05.
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3. RESULTS
The mean age on the day of saliva collection was 138.0 months (standard deviation [S.D.]: 8.7; range:
from 127 to 161) (Table 1). Among the 159 boys who participated in this study, 18 boys (11.3%) were classified in the borderline or clinical range of social withdrawal. There were missing values only in the Tanner stage (n = 2; data were missing for both the pubic hair stage and genital stage). The mean time
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when boys started their salivary collections was 7:46 (S.D. 1:03) A.M. There was no significant association between the time of saliva collection and testosterone level (lnT) (Spearman’s rho = 0.08, p = 0.33). There was a significant negative correlation with the collection time and cortisol level (Spearman’s rho = - 0.23, p = 0.004). Rates of children in the borderline or clinical range of social withdrawal in the five equal-width bins
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across the range of lnT are shown in Figure 1. There were no significant difference in the prevalence rates of social withdrawal between five groups, according to the chi-squared test (degree of freedom:4,
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p = 0.78). Although there was no significant linearity across the rates of five groups according to the Cochran-Armitage test (p = 0.23), groups with higher lnT tended to have lower rates of borderline or
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clinical range of social withdrawal. There was no such trend among the five groups divided by cortisol
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level (Cochran-Armitage test p = 0.37). There were no significant correlations between cortisol levels
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and the degree of social withdrawal or between cortisol levels and lnT. Based on these results, binominal logistic regression analyses were performed treating social
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withdrawal as a dependent variable and lnT as an independent variable (Table 2). There was a significant
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association between a decrease in the odds of social withdrawal and lnT (odds ratio 0.55, 95% C.I. [confidence interval] 0.33-0.94, p = 0.028) in the demographic adjusted model where age in months was adjusted. On the other hand, there were no significant associations between the odds and cortisol levels after adjustment for age in months (odds ratio 0.85, 95% C.I. 0.59-1.22, p = 0.37) or after adjustment for age in months together with the time of saliva collection (odds ratio 0.82, 95% C.I. 0.57-
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1.20, p = 0.32). We examined confounding factors for the association between social withdrawal and lnT. The borderline and clinical range of the social withdrawal group showed a non-significant but higher BMI (Mann-Whitney test, p = 0.12) and a significantly higher raw score for anxiety/depression (MannWhitney test, p < 0.001) than the normal range group. On the other hand, there were positive significant
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correlations between lnT and age in months (Spearman’s rho = 0.62, p < 0.001), between lnT and BMI (Spearman’s rho = 0.31, p < 0.001), and between lnT and Tanner stage (Spearman’s rho = 0.47, p <
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0.001). When boys were divided into three groups by the percentile of the CBCL anxiety/depression raw score, the middle group had a lower lnT than the other groups (Kruskal-Wallis test, p = 0.052).
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From these results and previous reports, BMI (Hwang et al., 2006; MacDonald et al., 2010),
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anxiety/depression (Barragan et al., 2011; Granger et al., 2003) and Tanner stage (Granger et al., 2004;
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Gruzelier and Kaiser, 1996; Shirtcliff et al., 2009) were considered potential confounders. These confounders were put into multivariate analysis. The association between decreased odds of
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social withdrawal and lnT remained significant after additional adjustments for BMI (Model 1 in Table
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2), for the CBCL anxiety/depression raw score (Model 2) and for Tanner stage (Model 3).
4. DISCUSSION This was the first study showing the inverse association between social withdrawal and salivary
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testosterone levels in early pubertal boys. Furthermore, this was also the first study to examine the relationship between social withdrawal and testosterone taking into account Tanner stage.
Several explanations could be considered for the negative association of social withdrawal with testosterone level. First, lower testosterone level may be one of the risk factors of social withdrawal. This assumption accords with the previous findings on testosterone. Several studies in both nonhuman
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animals and humans have shown that a rise in testosterone levels before and after winning a
competition enhances the motivation to compete (Losecaat Vermeer, et al., 2016), which could lead to
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a rise in a hierarchy of social dominance. Another study showed that adolescent males with decreased
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testosterone after loss of competition had lower motivation to compete again (Mehta and Josephs, 2006). There was only one study which examined the relationship between a level of avoidance and
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the morning salivary testosterone level measured by LC-MS/MS. The study showed that higher levels
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of age-standardized testosterone in boys tended to relate to lower levels of avoidance personality (Peper et al., 2018). Second, social withdrawal may induce psychological distress, which could
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decrease testosterone levels. Although there are no studies of adolescents examining the relationship
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between testosterone and psychological distress, previous studies of adults showed decreases in testosterone levels by mental stress (Zitzmann and Nieschlag, 2001). However, in the present study, boys in the borderline and over range of social withdrawal did not have significantly higher levels of cortisol, which psychological distress might increase, than boys in the normal range of social withdrawal (Mann-Whitney U test, p = 0.38).
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Noriyuki Hayashi
The results of our study are consistent with the previous studies on clinical samples. Avoidance tendencies were alleviated by testosterone in women with social anxiety disorder as well as in healthy women (Enter et al., 2014: Enter et al., 2016), while these studies were limited only in female due to lack of established protocol for male (Enter et al., 2014). Further, congenital hypogonadotrophic hypogonadism, which could be an extreme and rare example of the disability of testosterone production,
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is thought to be one of the risks for social withdrawal (Swee and Quinton, 2019). This study has several strengths. First, testosterone levels were measured by the LC-MS/MS method.
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The measurement of a sex steroid at low levels by the typical immune assay method has problems of cross reactions with other hormones. The LC-MS/MS method overcame this problem and made
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measurement of steroid hormones, even at a low level, trustable (Handelsman and Wartofsky, 2013;
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Ketha et al., 2014). Second, we utilized a large sample of early adolescents. Third, we focused on the
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relationship between social withdrawal and testosterone on which little work had been done. This study has some limitations. First, each participant had a different time interval between the
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acquisition of the CBCL score and the saliva collection. Therefore, we conducted an additional
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adjustment for age in months at the time of acquiring the CBCL score and found that the association between social withdrawal and testosterone remained significant (Supplementary table 1). Second, this cross-sectional study cannot refer to a causal relationship between social withdrawal and testosterone, for which further longitudinal studies are expected. Third, we cannot deny a selection bias, because we used data on selected participants’ from whom those with evident mental health problems were excluded
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Noriyuki Hayashi
due to the protocol of the pn-TTC. Fourth, we assessed Tanner stage with questionnaires answered by the primary parents, but not with physical examination by medical practitioners. Neither this study nor previous studies examined the relationship between hikikomori and the CBCL social withdrawal score. Considering the general definition of hikikomori (social withdrawal not going to school or work and less communicating with people during more than 3 or 6 months), adolescents
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with hikikomori should be evaluated as social withdrawal by the CBCL. Because it has been unclear whether adolescents with the tendency of social withdrawal incline to result in hikikomori in the future,
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future longitudinal studies are expected to address this issue.
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5. Conclusion
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Social withdrawal in early adolescent boys had an inverse association with salivary testosterone
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levels. To elucidate the causal relationship between social withdrawal and testosterone, further
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longitudinal studies are needed.
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Conflict of interests
K. Kasai reports grants from JSPS during the conduct of the study; personal fees from Otsuka,
Yoshitomi, Astellas, Dainippon-Sumitomo, Meiji Seika Pharma, Eisai and Fuji Film RI Pharma, and grants from Novartis, MSD, Astellas, Eli Lilly, Takeda, Tanabe-Mitsubishi, Dainippon-Sumitomo, and Eisai outside the submitted work.
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Noriyuki Hayashi
The other authors declare that there is no conflict of interest.
Contributors Noriyuki Hayashi—conception and design of the study, data acquisition, analysis and interpretation of data, drafting the manuscript, critical revision and final approval of the version to be published.
critical revision and final approval of the version to be published.
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Shuntaro Ando—conception and design of the study, data acquisition, interpretation of the data,
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Seiichiro Jinde, Tomoki Kiyono, Syudo Yamasaki, Atsushi Nishida and Kiyoto Kasai—interpretation of the data, critical revision and final approval of the version to be published.
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Shinya Fujikawa, Naohiro Okada, Rie Toriyama, Mio Masaoka, Hiroshi Sugiyama, Toru Shirakawa,
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Tomoko Yagi, Masaya Morita and Ryo Morishima—data acquisition, interpretation of the data, critical
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revision and final approval of the version to be published.
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Role of funding source
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The funding source had no role in this study.
Acknowledgment
This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (23118001, 23118002, and 23118004; Adolescent Mind & Self-Regulation) from the Ministry of Education,
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Noriyuki Hayashi
Culture, Sports, Science and Technology of Japan, and JSPS KAKENHI Grant Numbers JP16H06395, 16H06398, 16H06399, 16K21720 & 17H05931. This work was also supported in part by UTokyo Center for Integrative Science of Human Behavior (CiSHuB), and by the International Research Center for Neurointelligence (WPI-IRCN) at The
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University of Tokyo Institutes for Advanced Study (UTIAS).
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Prevalence rate of social withdrawal 25 20 15 10 5 0 1
2
3
4
5
LnT levels divided into 5 equal-width bins
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Rate of social withdrawal (%)
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Fig. 1 Prevalence rate of borderline and clinical range of social withdrawal assessed with the Child Behaviour Checklist.
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lnT: natural logarithms of testosterone level. Horizontal axis: lnT levels divided into 5 equal-width bins. LnT was lowest in group 1 and higher in group 5. N = 33 (group 1), 50 (group 2), 30 (group 3), 26 (group 4), and 20 (group 5). Error bar: 95% confidence intervals
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Noriyuki Hayashi Table 1 Demographic characteristics of the participants (n = 159)
Age in months
138.0(8.7)
Height (cm)
145.4(8.4)
Body weight (kg)
36.8(8.2)
BMI (kg/m2)
17.2(2.3)
Tanner stage (1-5)
2.3(1.1)
n (%)
1
32(20.1)
2
61(38.4)
3
50(31.4)
4
13(8.2)
5
1(0.6)
missing
2(1.3)
Testosterone (pg/ml)
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mean (SD)/ median(IQR)
14.8(22.5)
Cortisol (ng/ml)
3.0(1.5)
Social withdrawal (CBCL) 1.5(1.6)
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Raw score
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3.9(15.8)
1.0(2.0)
Anxiety/depression (CBCL) Raw score
18(11.3)
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Borderline/clinical range,n (%)
2.7(2.8)
2.0(4.0)
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Borderline/clinical range,n (%)
12(7.5)
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SD:standard deviation, IQR:interquartile range, BMI:body mass index, CBCL:the Child Behaviour Checklist Borderline/clinical range:T score of 67 or more on the CBCL.
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Table 2 Multivariate logistic regression on the effects of salivary testosterone level on social withdrawal Model1
Demographic adjusted model
Model2
Model3
OR
95%CI
OR
95%CI
OR
95%CI
OR
95%CI
lnT
0.55*
0.33-0.94
0.54*
0.32-0.92
0.38*
0.17-0.84
0.33*
0.14-0.77
Age in months
1.09*
1.01-1.18
1.08*
1.00-1.17
1.11
1.00-1.24
1.10
0.99-1.23
1.14
0.93-1.39
1.39*
1.02-1.90
1.44*
1.05-1.99
1.79**
1.41-2.28
1.82**
1.41-2.34
1.74
0.78-3.91
BMI Anxiety/depression Tanner stage 159
159
159
157
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n
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na
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OR: odds ratio, CI: confidence interval, lnT: natural logarithms of salivary testosterone concentrations, BMI: body mass index. Anxiety/depression: raw score from the anxiety/depression scale (the Child Behaviour Checklist). N ranged between 157 and 159 due to missing values in the Tanner stage measures. * p< 0.05 ** p< 0.001
35
PII:
S0306-4530(20)30015-9
DOI:
https://doi.org/10.1016/j.psyneuen.2020.104596
Reference:
PNEC 104596
To appear in:
Psychoneuroendocrinology
Received Date:
20 March 2019
Revised Date:
29 October 2019
Accepted Date:
16 January 2020
Please cite this article as: Hayashi N, Ando S, Jinde S, Fujikawa S, Okada N, Toriyama R, Masaoka M, Sugiyama H, Shirakawa T, Yagi T, Morita M, Morishima R, Kiyono T, Yamasaki S, Nishida A, Kasai K, Social withdrawal and testosterone levels in early adolescent boys, Psychoneuroendocrinology (2020), doi: https://doi.org/10.1016/j.psyneuen.2020.104596
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Noriyuki Hayashi
Social withdrawal and testosterone levels in early adolescent boys
Noriyuki Hayashia, Shuntaro Andoa,b, Seiichiro Jindea, Shinya Fujikawaa, Naohiro Okadaa,c, Rie Toriyamaa, Mio Masaokaa, Hiroshi Sugiyamaa,d, Toru Shirakawaa, Tomoko Yagia, Masaya Moritaa, Ryo
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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a
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Morishimaa, Tomoki Kiyonoa, Syudo Yamasakib, Atsushi Nishidab, Kiyoto Kasaia,c.
Bunkyo-ku, Tokyo 113-8655, Japan.
Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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b
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes
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c
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Department of Integrated Educational Sciences, Graduate School of Education, The University of
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d
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Japan.
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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Noriyuki Hayashi
Noriyuki Hayashi Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
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Shuntaro Ando *
Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. +81-3-3815-5411
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[email protected]
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Seiichiro Jinde
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
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Noriyuki Hayashi
Shinya Fujikawa Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
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Naohiro Okada Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan.
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes
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for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
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Rie Toriyama
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[email protected]
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Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Mio Masaoka
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Noriyuki Hayashi
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Hiroshi Sugiyama
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Integrated Educational Sciences, Graduate School of Education, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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[email protected]
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Toru Shirakawa
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
Tomoko Yagi
Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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Noriyuki Hayashi
[email protected]
Masaya Morita Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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[email protected]
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Ryo Morishima
Bunkyo-ku, Tokyo 113-8655, Japan.
Tomoki Kiyono
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[email protected]
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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Bunkyo-ku, Tokyo 113-8655, Japan. [email protected]
Syudo Yamasaki Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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Noriyuki Hayashi
2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. [email protected]
Atsushi Nishida Department of Psychiatry and Behavioural Sciences, Tokyo Metropolitan Institute of Medical Science,
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2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
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[email protected]
Kiyoto Kasai
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Bunkyo-ku, Tokyo 113-8655, Japan.
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Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
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International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
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Japan.
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[email protected]
* corresponding author
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Noriyuki Hayashi Highlights
Social withdrawal in general early adolescent boys was evaluated by primary parents
Salivary testosterone was measured by liquid chromatography-tandem mass spectrometry
A higher risk of boy’s social withdrawal was related to a lower testosterone level
The association remained after adjusting for confounders such as pubertal stage
Negative relationship between social withdrawal and testosterone levels was found
ABSTRACT
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Social withdrawal may lead to mental health problems and can have a large impact on a life course, particularly among boys. To support adolescents with social withdrawal, an integrative understanding
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of the biological bases would be helpful. Social dominance, a possible opposite of social withdrawal, is known to have positive associations with testosterone levels. A previous study suggested that social
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withdrawal has a negative relationship with sexual maturity among adolescent boys. However, the
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relationship between social withdrawal and testosterone in adolescence is unknown. This study aimed
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to examine whether social withdrawal was negatively associated with testosterone levels in early adolescent boys. Salivary samples were collected from 159 healthy early adolescent boys (mean age
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[standard deviation]: 11.5 [0.73]) selected from participants of the “population-neuroscience study of
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the Tokyo Teen Cohort” (pn-TTC). Social withdrawal and confounding factors, such as the secondary sexual characteristics and their age in months, were evaluated by self-administered questionnaires completed by the primary parents. The degree of social withdrawal was assessed with the Child Behaviour Checklist (CBCL). Levels of salivary testosterone, and cortisol as a control, were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Logistic regression was conducted
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to examine the association between social withdrawal and testosterone levels. A higher risk of social withdrawal was associated with a lower salivary testosterone level after adjustment for age in months (odds ratio 0.55, 95% confidence interval 0.33-0.94), and the association remained significant after adjusting for body mass index, the degree of anxiety/depression and pubertal stage. Thus, we found a negative relationship between social withdrawal and testosterone levels in early adolescent boys. These
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findings may help to clarify the biological foundations of and to develop support for social withdrawal.
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Keywords: social withdrawal, testosterone, puberty, LC-MS/MS, the population-neuroscience study of
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the Tokyo Teen Cohort
1. Introduction
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Social withdrawal is one of the internalizing problems, and defined as “the child’s isolating
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himself/herself from the peer group” (Rubin et al., 2009). Social withdrawal might lead to other
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psychiatric problems. Children aged 8 years with a higher degree of social withdrawal had higher risks of depression and anxiety disorders in adolescence (Goodwin et al., 2004). Adolescents aged 13-16 years with a higher degree of social withdrawal had higher risks of suicide attempts several years later (Ferdinand and Verhulst, 1995). Moreover, social withdrawal among boys seems to have a much larger impact on their life courses than girls. Males who had been shy (a construct of social withdrawal) in
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childhood married, became fathers and established stable carriers at later ages in life than other males, while no such disadvantage was found in female (Caspi et al., 1988; Kerr et al., 1996). This sex difference is thought to be attributed to the norm of the traditional gender roles (men as breadwinner and women as caregiver) (Asendorpf et al., 2008), from which recent Japanese society has not shifted away (Kato, 2018). Thus, social withdrawal in boys might be more problematic than in girls in Japanese
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society. A form of severe social withdrawal called “hikikomori” (a Japanese word which can be translated as “withdrawal”), first described in Japan, usually occurs in ages 15-19 years during
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adolescence (Li and Wong, 2015a, 2015b). Although there is lack of consensus on its definition, hikikomori is recognized as social withdrawal not going to school or work and less communicating with
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people during more than 3 or 6 months (Li and Wong, 2015a, 2015b). It was experienced by as many
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as 1-2% of the community sample in Japan and Hong Kong (Koyama et al., 2010; Wong et al., 2015).
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The lifetime prevalence of hikikomori in males was approximately five times as high as in females (Koyama et al., 2010). This is in contrast with anxiety/depression, one of the main internalizing
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problems, for which adolescent girls have the higher risks than boys (Merrell, 2008). A competitive but
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rapidly changing society and family relationship in modern-day Japanese society is considered to contribute to the development of hikikomori to some extent especially in boys (Koyama et al., 2010). Based on these facts, early support would be needed, especially among boys with social withdrawal.
Current support methods of social withdrawal are formed mainly from psycho-social standpoints (Li and Wong, 2015b; Rubin et al., 2009), probably because studies of this behaviour have been developed
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from these standpoints traditionally. Although the biological foundations of social withdrawal have also been studied (Rubin et al., 2009), integrative understanding has been insufficient. Revealing the biological foundations of social withdrawal in childhood or adolescence is a challenge to be addressed. New knowledge about biological foundations could provide helpful suggestions to current various supports for social withdrawal.
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In the framework of the approach-avoidance behaviors, social withdrawal is classified as an avoidance behavior (Lopez et al., 2004). On the other hand, social dominance means acquiring and
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maintaining higher status in social hierarchy to have a power and affect conspecifics (Mazur and Booth, 1998), and is classified as an approach behavior (Terburg et al., 2013). Therefore, social
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withdrawal could be opposed to social dominance in the spectrum of approach-avoidant behaviors. A
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significant negative correlation was found between the degree of social withdrawal and social
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dominance in 9- to 13-year-old boys (Lease et al., 2002). Biological studies of social dominance have focused on the role of sexual hormones (Qu et al., 2017; Stanton and Schultheiss, 2009). Cross-
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sectional data show that adolescent boys aged 13 years who were regarded as socially dominant by
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their peers had significantly higher levels of testosterone than boys who were regarded as less socially dominant (Schaal et al., 1996). A different study shows that a degree of social dominance had a significant positive relationship with testosterone level in boys aged 9-15 years (Rowe et al., 2004). Therefore, one might predict that social withdrawal, a possible opposite to social dominance, has a negative relationship with testosterone level among adolescent boys.
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On the other hand, there have been few studies examining associations between social withdrawal and sexual maturation. A retrospective study of healthy college students showed that males with extremely late sexual maturation had significantly higher degrees of social withdrawal than males with extremely early maturation (Gruzelier and Kaiser, 1996). This result and a positive correlation between sexual maturity and testosterone level (Granger et al., 2004; Shirtcliff et al., 2009) also suggest that
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higher degrees of social withdrawal may be associated with lower testosterone levels in adolescent males.
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However, the relationship between social withdrawal and testosterone levels among adolescent boys remains unclear. The aim of this study was to test our hypothesis that a higher risk of social withdrawal
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2. METHOD
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was associated with a lower level of testosterone in early adolescent boys.
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2.1. Study design and participants The data in this study are based on the population-neuroscience study of the Tokyo Teen Cohort (pn-
TTC) (Okada et al., 2019). In this longitudinal cohort study, physical, socioeconomic, psychiatric and psychological data as well as hormonal and deoxyribonucleic acid (DNA) data derived from saliva and magnetic resonance imaging (MRI) data were obtained from adolescents and their primary parents for
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the exploration of the neurobiological substrates of development during adolescence. The targeted number of participants in the pn-TTC study was approximately 300 pairs of early adolescents and their primary parents. The participants of the pn-TTC were chosen from the 3,171 pairs participating in the Tokyo Teen Cohort (TTC) study (Ando et al., 2019) who had been recruited from the 4,478 pairs participating in the Tokyo Early Adolescence Survey (T-EAS). The data acquired in the T-EAS were
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used as wave 1 data of the longitudinal studies (the TTC and the pn-TTC). The T-EAS is a multidisciplinary cross-sectional survey of teen’s health and development carried out
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from Oct. 2012 to Jan. 2015. Briefly, adolescents born between September 1st, 2002, and August 31st, 2004, and recorded on the basic resident register of Setagaya-ku, Chohu-city and Mitaka-city, Tokyo,
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were randomly selected and regarded as candidates for the survey. Among the 10,234 contactable early
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adolescents aged 10 and their primary parents, 4,478 pairs cooperated with the survey (cooperation rate:
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43.8%). In this survey, physical, socioeconomic, psychiatric and psychological data were acquired through interviews by trained interviewers and various questionnaires (including the Child Behaviour
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Checklist [CBCL]) (Achenbach, 1991; Itani et al., 2001).
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Among 3,171 pairs who participated in the TTC, which was the subsequent longitudinal study, invitation letters to the pn-TTC were sent to 980 pairs who showed possibilities of cooperation with the study. The exclusion criteria were as follows: (1) current problems in mental health, interpersonal relationships or behaviours, (2) visual or hearing disabilities, (3) past history of head injury accompanied by more than five minutes of loss of consciousness, (4) current chronic endocrine or
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metabolic diseases, and (5) current use of medications that affect the central nervous system. A total of 176 adolescents who had first menstruation or were in the stage 3 or more advanced stages of Tanner pubertal stage (Marshall and Tanner, 1969, 1970) were oversampled, and the remaining 804 adolescents were randomly sampled. Out of 980 pairs, a total of 301 pairs (boys; 162, girls; 139) agreed and participated in the pn-TTC study. The participants were asked to visit our laboratory from September
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2013 to February 2016. The adolescent’s height, body weight and secondary sexual characteristics were assessed there. The adolescents collected their salivary samples at home by the method described in the
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next section. Of the participating adolescent boys, 159 boys collected their salivary samples properly. Salivary testosterone and cortisol levels of these samples were measured (the latter as a control). Finally,
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the data of 159 boys were analysed in this study.
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This study was approved by ethics committees of the University of Tokyo, Faculty of Medicine,
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Tokyo Metropolitan Institute of Medical Science, and the Graduate University for Advanced Studies (SOKENDAI), according to the Declaration of Helsinki. The adolescents and their primary parents were
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orally informed of the contents of this research and provided written consent forms.
2.2. Measurement
2.2.1.
Social withdrawal
In this study, early adolescents’ behaviours and emotions at age 10 were evaluated by primary parents
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with the CBCL/4-18 Japanese version (Itani et al., 2001). The CBCL (Achenbach, 1991) is composed of 118 questions about problems of child behaviours and emotions within the past six months, which include scales of social withdrawal and anxiety/depression. The questions about social withdrawal included the following 9 items: “Would rather be alone”, “Refuses to talk”, “Secretive”, “Shy”, “Stares blankly”, “Sulks a lot”, “Lacks energy”, “Unhappy, sad,
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depressed”, and “Withdrawn”. The primary parents chose the best answers to the questions among 0 = “not true”, 1 = “somewhat or sometimes true”, and 2 = “very true or often true”. The sum score of the
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questions is referred to as the raw score of social withdrawal, which could range from 0 to 18.
Raw score can be converted to a normalized T score, which was calculated by sex and ranges of ages
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of standard samples. After calculation of the midpoint percentile of the cumulative frequency
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distribution of the raw score in the standard samples, the T score was determined by the procedure of
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Abramowitz and Stegun (Abramowitz and Stegun, 1968). Raw scores of 50 midpoint percentiles and under were assigned a T score of 50. The raw score of the
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97.7th percentile was assigned a T score of 70, and the remaining raw scores were assigned T scores
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from 71 to 100 in equal intervals. T scores of less than 67 (95th percentile), 67 to 70, and over 70 are referred to as “normal range”, “borderline range”, and “clinical range”, respectively. For a specific categorical cutoff point, a T score of 67 has been commonly used to represent the bottom of a deviant range (including the borderline and the clinical range) (Lien et al., 2015; Wolak et al., 2007a, 2007b) as the original textbook of the CBCL had instructed (Achenbach, 1991). The reliability and validity of the
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CBCL have been well established (Achenbach, 1991).
2.2.2.
Salivary testosterone level
The adolescents collected their salivary samples at home early in the morning. In advance, both the adolescents and their primary parents were informed of how to collect the
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adolescents’ saliva using sample tubes. The adolescents tried it under the guidance of the survey staff for practice. They were instructed not to collect the saliva within a week after a tooth extraction or
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immediately after dental treatment to avoid contamination with blood. They were also asked not to eat food after brushing their teeth on the night before the saliva collection. They were instructed to rinse
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their mouth soon after getting up and to make sure they were at their normal body temperature, and not
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to have breakfast and not to brush teeth before the collection. Furthermore, they were asked to wait for 20 minutes after the rinse and then to collect 4.5 ml of their saliva by passive drool in sterilized tubes
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(1.5 ml/tube * 3 tubes) made of polypropylene (NalgeneTM General Long-Term Storage Cryogenic
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Tubes, Thermo Fisher SCIENTIFIC, U.S. A) within 60 minutes. Salivary samples were collected in
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only one day, since high correlation among morning salivary testosterone levels across days in adolescents was reported (Granger et al., 2004). Salivary samples were kept in household refrigerator freezers, delivered frozen to our laboratory, where the weights were measured and tubes stored at minus 80 degrees C until the testosterone levels were measured. The concentration of salivary testosterone was measured once by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which has
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become the current standard (Ketha et al., 2014). The lower limit of quantification (LLOQ) of testosterone was 0.7 pg/ml. There were 3 boys (1.9%) who showed testosterone levels below the LLOQ (BLOQ). Such data were incorporated in our analysis (Keizer et al., 2015). The significance of the following results remained even after exclusion of these 3
2.2.3.
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boys’ data.
Salivary cortisol level
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Salivary cortisol levels in the same saliva sample were simultaneously measured as a control hormone by the LC-MS/MS method to exclude the possibility that any hormone was negatively associated with
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social withdrawal. The LLOQ of cortisol was 6.7 pg/ml, and cortisol levels in all samples were
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above this limit.
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2.2.4. Potential confounders
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2.2.4.1. Secondary sexual characteristics The primary parents were presented drawings of pubic hair and genitalia, which were classified into
the five Tanner stages (from Stage 1: pre-pubertal to Stage 5: post-pubertal). They were asked which pictures fit their children (Marshall and Tanner, 1970). The higher of the pubic hair stage or the genital stage measures represented the Tanner pubertal stage in this study. A previous study showed that
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evaluation of children’s sexual development by their parents using the drawings or similar pictures had a strong correlation with the evaluation with physical examination by medical practitioners (the gold standard method) (Coleman and Coleman, 2002).
2.2.4.2. Body mass index (BMI)
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BMI was calculated from the adolescents’ height and body weight measured by trained interviewers. A previous study showed that children aged 10-12 years with a BMI at the 95th percentile or more
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had higher total problem scores (including social withdrawal score) on the CBCL than other children (Hwang et al., 2006). A meta-analysis showed that testosterone levels in males had a negative
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excluded (MacDonald et al., 2010).
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relationship with BMI, although the studies in which the mean ages were under 12 years old were
2.2.4.3. Anxiety/depression
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Previous studies have reported significant association of anxiety/depression with social withdrawal
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and testosterone. A positive relationship between degree of social withdrawal and degree of depressive symptoms had been reported (Barragan et al., 2011). Negative relationships between degrees of anxiety/depression and testosterone levels in adolescent males have been reported in other studies (Granger et al., 2003; Susman et al., 1991). In this study, the degree of anxiety/depression was assessed by the CBCL anxiety/depression scale. This scale comprised 14 questions, and the raw score could
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range from 0 to 28. The T score and normal/borderline/clinical ranges of that scale were defined in a similar manner as the CBCL social withdrawal scale mentioned above.
2.3. Data analysis In terms of age in months, the data at the day of the saliva collection were used. Medians and
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interquartile ranges (IQR) were calculated for salivary testosterone levels and the CBCL raw scores because of their positively skewed distributions. For the following analyses, natural logarithms of
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salivary testosterone levels (lnT) were used. For the correlation coefficients between two observed variables with a nonnormal distribution, Spearman’s rho was calculated. Similarly, for comparison of
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an observed variable among two or more groups, the Mann-Whitney U test or Kruskal-Wallis test were
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performed.
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The adolescents were placed into one of five groups, from the lowest lnT group to the highest lnT group, created by dividing the range of lnT into 5 equal-width bins. The ratios of adolescents with the
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borderline or clinical ranges of social withdrawal in each group were calculated. The ratios were
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compared between the five groups by the chi-squared test. The linearity between the order of the five groups and the rates of social withdrawal was tested by the Cochran-Armitage test. A similar analysis was performed for cortisol levels. In the logistic regression of this study, the binominal variable of 1 was assigned for the borderline or clinical range of social withdrawal (T score 67 or above), and 0 was assigned for the normal range (T
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score < 67). LnT (and cortisol) and other variables were treated as the independent variables in the regression analyses. The effect of testosterone (and cortisol) levels on social withdrawal was evaluated after adjustment for age in months (demographic-adjusted model). After evaluation of the potential confounders in our actual data, we put all these confounders in the regression one by one. Besides the logistic regressions, we conducted linear regressions using raw scores of social
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withdrawal as dependent variable, and age in months, lnT, BMI, and depression/anxiety as independent variables. However, since normal P-P plots of regression standardized residuals were not linear, we
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judged linear regressions as inappropriate in this study. A lot of zeros of social withdrawal’ raw scores
in this section) seemed to cause this result.
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(median 1.0, IQR 2.0 as shown in Table 1) and positively skewed distribution of raw scores (mentioned
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IBM SPSS ver 24 and EZR (Kanda, 2013) were used for the analyses (the latter used only for the
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Cochran-Armitage test), and the level of significance was set at p < 0.05.
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3. RESULTS
The mean age on the day of saliva collection was 138.0 months (standard deviation [S.D.]: 8.7; range:
from 127 to 161) (Table 1). Among the 159 boys who participated in this study, 18 boys (11.3%) were classified in the borderline or clinical range of social withdrawal. There were missing values only in the Tanner stage (n = 2; data were missing for both the pubic hair stage and genital stage). The mean time
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when boys started their salivary collections was 7:46 (S.D. 1:03) A.M. There was no significant association between the time of saliva collection and testosterone level (lnT) (Spearman’s rho = 0.08, p = 0.33). There was a significant negative correlation with the collection time and cortisol level (Spearman’s rho = - 0.23, p = 0.004). Rates of children in the borderline or clinical range of social withdrawal in the five equal-width bins
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across the range of lnT are shown in Figure 1. There were no significant difference in the prevalence rates of social withdrawal between five groups, according to the chi-squared test (degree of freedom:4,
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p = 0.78). Although there was no significant linearity across the rates of five groups according to the Cochran-Armitage test (p = 0.23), groups with higher lnT tended to have lower rates of borderline or
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clinical range of social withdrawal. There was no such trend among the five groups divided by cortisol
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level (Cochran-Armitage test p = 0.37). There were no significant correlations between cortisol levels
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and the degree of social withdrawal or between cortisol levels and lnT. Based on these results, binominal logistic regression analyses were performed treating social
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withdrawal as a dependent variable and lnT as an independent variable (Table 2). There was a significant
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association between a decrease in the odds of social withdrawal and lnT (odds ratio 0.55, 95% C.I. [confidence interval] 0.33-0.94, p = 0.028) in the demographic adjusted model where age in months was adjusted. On the other hand, there were no significant associations between the odds and cortisol levels after adjustment for age in months (odds ratio 0.85, 95% C.I. 0.59-1.22, p = 0.37) or after adjustment for age in months together with the time of saliva collection (odds ratio 0.82, 95% C.I. 0.57-
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1.20, p = 0.32). We examined confounding factors for the association between social withdrawal and lnT. The borderline and clinical range of the social withdrawal group showed a non-significant but higher BMI (Mann-Whitney test, p = 0.12) and a significantly higher raw score for anxiety/depression (MannWhitney test, p < 0.001) than the normal range group. On the other hand, there were positive significant
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correlations between lnT and age in months (Spearman’s rho = 0.62, p < 0.001), between lnT and BMI (Spearman’s rho = 0.31, p < 0.001), and between lnT and Tanner stage (Spearman’s rho = 0.47, p <
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0.001). When boys were divided into three groups by the percentile of the CBCL anxiety/depression raw score, the middle group had a lower lnT than the other groups (Kruskal-Wallis test, p = 0.052).
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From these results and previous reports, BMI (Hwang et al., 2006; MacDonald et al., 2010),
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anxiety/depression (Barragan et al., 2011; Granger et al., 2003) and Tanner stage (Granger et al., 2004;
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Gruzelier and Kaiser, 1996; Shirtcliff et al., 2009) were considered potential confounders. These confounders were put into multivariate analysis. The association between decreased odds of
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social withdrawal and lnT remained significant after additional adjustments for BMI (Model 1 in Table
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2), for the CBCL anxiety/depression raw score (Model 2) and for Tanner stage (Model 3).
4. DISCUSSION This was the first study showing the inverse association between social withdrawal and salivary
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testosterone levels in early pubertal boys. Furthermore, this was also the first study to examine the relationship between social withdrawal and testosterone taking into account Tanner stage.
Several explanations could be considered for the negative association of social withdrawal with testosterone level. First, lower testosterone level may be one of the risk factors of social withdrawal. This assumption accords with the previous findings on testosterone. Several studies in both nonhuman
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animals and humans have shown that a rise in testosterone levels before and after winning a
competition enhances the motivation to compete (Losecaat Vermeer, et al., 2016), which could lead to
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a rise in a hierarchy of social dominance. Another study showed that adolescent males with decreased
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testosterone after loss of competition had lower motivation to compete again (Mehta and Josephs, 2006). There was only one study which examined the relationship between a level of avoidance and
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the morning salivary testosterone level measured by LC-MS/MS. The study showed that higher levels
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of age-standardized testosterone in boys tended to relate to lower levels of avoidance personality (Peper et al., 2018). Second, social withdrawal may induce psychological distress, which could
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decrease testosterone levels. Although there are no studies of adolescents examining the relationship
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between testosterone and psychological distress, previous studies of adults showed decreases in testosterone levels by mental stress (Zitzmann and Nieschlag, 2001). However, in the present study, boys in the borderline and over range of social withdrawal did not have significantly higher levels of cortisol, which psychological distress might increase, than boys in the normal range of social withdrawal (Mann-Whitney U test, p = 0.38).
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The results of our study are consistent with the previous studies on clinical samples. Avoidance tendencies were alleviated by testosterone in women with social anxiety disorder as well as in healthy women (Enter et al., 2014: Enter et al., 2016), while these studies were limited only in female due to lack of established protocol for male (Enter et al., 2014). Further, congenital hypogonadotrophic hypogonadism, which could be an extreme and rare example of the disability of testosterone production,
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is thought to be one of the risks for social withdrawal (Swee and Quinton, 2019). This study has several strengths. First, testosterone levels were measured by the LC-MS/MS method.
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The measurement of a sex steroid at low levels by the typical immune assay method has problems of cross reactions with other hormones. The LC-MS/MS method overcame this problem and made
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measurement of steroid hormones, even at a low level, trustable (Handelsman and Wartofsky, 2013;
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Ketha et al., 2014). Second, we utilized a large sample of early adolescents. Third, we focused on the
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relationship between social withdrawal and testosterone on which little work had been done. This study has some limitations. First, each participant had a different time interval between the
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acquisition of the CBCL score and the saliva collection. Therefore, we conducted an additional
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adjustment for age in months at the time of acquiring the CBCL score and found that the association between social withdrawal and testosterone remained significant (Supplementary table 1). Second, this cross-sectional study cannot refer to a causal relationship between social withdrawal and testosterone, for which further longitudinal studies are expected. Third, we cannot deny a selection bias, because we used data on selected participants’ from whom those with evident mental health problems were excluded
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due to the protocol of the pn-TTC. Fourth, we assessed Tanner stage with questionnaires answered by the primary parents, but not with physical examination by medical practitioners. Neither this study nor previous studies examined the relationship between hikikomori and the CBCL social withdrawal score. Considering the general definition of hikikomori (social withdrawal not going to school or work and less communicating with people during more than 3 or 6 months), adolescents
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with hikikomori should be evaluated as social withdrawal by the CBCL. Because it has been unclear whether adolescents with the tendency of social withdrawal incline to result in hikikomori in the future,
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future longitudinal studies are expected to address this issue.
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5. Conclusion
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Social withdrawal in early adolescent boys had an inverse association with salivary testosterone
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levels. To elucidate the causal relationship between social withdrawal and testosterone, further
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longitudinal studies are needed.
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Conflict of interests
K. Kasai reports grants from JSPS during the conduct of the study; personal fees from Otsuka,
Yoshitomi, Astellas, Dainippon-Sumitomo, Meiji Seika Pharma, Eisai and Fuji Film RI Pharma, and grants from Novartis, MSD, Astellas, Eli Lilly, Takeda, Tanabe-Mitsubishi, Dainippon-Sumitomo, and Eisai outside the submitted work.
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The other authors declare that there is no conflict of interest.
Contributors Noriyuki Hayashi—conception and design of the study, data acquisition, analysis and interpretation of data, drafting the manuscript, critical revision and final approval of the version to be published.
critical revision and final approval of the version to be published.
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Shuntaro Ando—conception and design of the study, data acquisition, interpretation of the data,
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Seiichiro Jinde, Tomoki Kiyono, Syudo Yamasaki, Atsushi Nishida and Kiyoto Kasai—interpretation of the data, critical revision and final approval of the version to be published.
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Shinya Fujikawa, Naohiro Okada, Rie Toriyama, Mio Masaoka, Hiroshi Sugiyama, Toru Shirakawa,
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Tomoko Yagi, Masaya Morita and Ryo Morishima—data acquisition, interpretation of the data, critical
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revision and final approval of the version to be published.
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Role of funding source
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The funding source had no role in this study.
Acknowledgment
This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (23118001, 23118002, and 23118004; Adolescent Mind & Self-Regulation) from the Ministry of Education,
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Culture, Sports, Science and Technology of Japan, and JSPS KAKENHI Grant Numbers JP16H06395, 16H06398, 16H06399, 16K21720 & 17H05931. This work was also supported in part by UTokyo Center for Integrative Science of Human Behavior (CiSHuB), and by the International Research Center for Neurointelligence (WPI-IRCN) at The
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University of Tokyo Institutes for Advanced Study (UTIAS).
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Prevalence rate of social withdrawal 25 20 15 10 5 0 1
2
3
4
5
LnT levels divided into 5 equal-width bins
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Rate of social withdrawal (%)
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Fig. 1 Prevalence rate of borderline and clinical range of social withdrawal assessed with the Child Behaviour Checklist.
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lnT: natural logarithms of testosterone level. Horizontal axis: lnT levels divided into 5 equal-width bins. LnT was lowest in group 1 and higher in group 5. N = 33 (group 1), 50 (group 2), 30 (group 3), 26 (group 4), and 20 (group 5). Error bar: 95% confidence intervals
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Noriyuki Hayashi Table 1 Demographic characteristics of the participants (n = 159)
Age in months
138.0(8.7)
Height (cm)
145.4(8.4)
Body weight (kg)
36.8(8.2)
BMI (kg/m2)
17.2(2.3)
Tanner stage (1-5)
2.3(1.1)
n (%)
1
32(20.1)
2
61(38.4)
3
50(31.4)
4
13(8.2)
5
1(0.6)
missing
2(1.3)
Testosterone (pg/ml)
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mean (SD)/ median(IQR)
14.8(22.5)
Cortisol (ng/ml)
3.0(1.5)
Social withdrawal (CBCL) 1.5(1.6)
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Raw score
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3.9(15.8)
1.0(2.0)
Anxiety/depression (CBCL) Raw score
18(11.3)
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Borderline/clinical range,n (%)
2.7(2.8)
2.0(4.0)
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Borderline/clinical range,n (%)
12(7.5)
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SD:standard deviation, IQR:interquartile range, BMI:body mass index, CBCL:the Child Behaviour Checklist Borderline/clinical range:T score of 67 or more on the CBCL.
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Table 2 Multivariate logistic regression on the effects of salivary testosterone level on social withdrawal Model1
Demographic adjusted model
Model2
Model3
OR
95%CI
OR
95%CI
OR
95%CI
OR
95%CI
lnT
0.55*
0.33-0.94
0.54*
0.32-0.92
0.38*
0.17-0.84
0.33*
0.14-0.77
Age in months
1.09*
1.01-1.18
1.08*
1.00-1.17
1.11
1.00-1.24
1.10
0.99-1.23
1.14
0.93-1.39
1.39*
1.02-1.90
1.44*
1.05-1.99
1.79**
1.41-2.28
1.82**
1.41-2.34
1.74
0.78-3.91
BMI Anxiety/depression Tanner stage 159
159
159
157
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n
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OR: odds ratio, CI: confidence interval, lnT: natural logarithms of salivary testosterone concentrations, BMI: body mass index. Anxiety/depression: raw score from the anxiety/depression scale (the Child Behaviour Checklist). N ranged between 157 and 159 due to missing values in the Tanner stage measures. * p< 0.05 ** p< 0.001
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