Sex Differences in Brain Function and Dysfunction 2023 ALGRAWANY

Current Topics in Behavioral Neurosciences 62
Claire Gibson
Liisa A. M. Galea Editors
Sex Differences
in Brain
Function and
Dysfunction
Current Topics in Behavioral Neurosciences
Volume 62
Series Editors
Mark A. Geyer, Department of Psychiatry, University of California San Diego,
La Jolla, CA, USA
Charles A. Marsden, Queen’s Medical Centre, University of Nottingham,
Nottingham, UK
Bart A. Ellenbroek, School of Psychology, Victoria University of Wellington,
Wellington, New Zealand
Thomas R. E. Barnes, The Centre for Mental Health, Imperial College London,
London, UK
Susan L. Andersen, Medfield, MA, USA
Martin P. Paulus, Laureate Institute for Brain Research, Tulsa, OK, USA
Jocelien Olivier, GELIFES, University of Groningen, Groningen, The Netherlands
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Claire Gibson • Liisa A. M. Galea
Editors
Sex Differences in Brain

Function and Dysfunction
Editors
Claire Gibson Liisa A. M. Galea
School of Psychology Centre for Addiction and Mental Health
University of Nottingham University of Toronto
Nottingham, UK Toronto, ON, Canada
ISSN 1866-3370 ISSN 1866-3389 (electronic)

Current Topics in Behavioral Neurosciences
ISBN 978-3-031-26722-2 ISBN 978-3-031-26723-9 (eBook)
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Preface
Until recently the topic of sex differences generated very little interest in researchers
beyond those working in fields related to reproductive behaviours. Over the last couple
of decades, it has become clear that sex influences can be found in almost every aspect
of brain function. National funding bodies including the National Institute of Health
(NIH) in the USA, the Canadian Institutes of Health Research (CIHR) and various
European and UK agencies have mandated, albeit to varying extents, the inclusion of
both sexes in clinical and/or biomedical grants [1, 2].
However, across many research fields, hypotheses are developed and treatments
identified for investigation based on a wealth of, preclinical and clinical, research
data largely based on the exclusive use of males which is no longer scientifically
defensible or ethologically valid. Even when both sexes are included in research
studies they typically don’t analyse the data with sex as a discovery variable [3, 4].
This lack of attention to appropriate analyses and consideration of sex/gender
restricts our understanding of how sex and gender can influence health and disease.
The importance of studying sex and gender differences in biology and medicine is
crucial to our understanding of why women are more typically misdiagnosed and
experience delays in diagnosis and experience more side effects from new therapeu-
tics than men [5–8]. Early treatment is at the core for better prognosis and thus, these
delays in diagnosis could lead to worse health outcomes for women. Although
women live longer than men, they are more likely to suffer from chronic disease
[9]. The understanding of how sex and gender influence our biology and psychology
will lead to improved diagnoses, and treatment efficacy for brain diseases. Best
practices in sex and gender science will lead to better health for men, women and
gender diverse individuals.
This volume covers three main themes of research into sex differences in basic
neurobiology, psychology, preclinical research and clinical research. It begins by
exploring our understanding of sex and gender in relation to both animal and human
behaviours and discusses the relevance, and importance, of considering sex and
gender when conducting research into brain function and behaviours. The second
v
vi Preface
theme focuses on how sex and gender influence mental health and considers the
impact of our immune system and the changes that occur with ageing. Finally, the
third theme focuses on neurological disorders which show sex differences in terms
of their aetiology and/or symptomology and considers the relevance in the develop-
ment of treatment for various disorders including stroke, Alzheimer’s disease, and
multiple sclerosis.
Sub-theme 1: Our first sub-theme has papers on considerations and history of the
study of gender differences in Europe and how the funding agency, Horizon’s
Europe started to incorporate the idea of sex and gender into research (Klinge,
2022). Brown et al. (2022) write about the importance of studying the interaction
between sex and gender in research. They include several case studies to illustrate
their point that it is rarely one factor driving effects in humans. The next chapter is on
the interesting species, the naked mole rat that does not show many of the sex
differences in the brain or in social hierarchy that is seen in most species. Edwards et
al. (2022) take us on a journey of what we can learn from studying this interesting
species on how sex and social roles may shape our brain and behaviour. Finally,
Hodgetts and Hausmann (2022) outline some fascinating sex differences in connec-
tivity in the human brain and how this may or may not contribute to behaviour.
Sub-theme 2: In this part of the book, the authors explore cognitive and mental
health throughout the lifespan. The paper from Pavlidi et al (2022) explores sex and
gender differences that are seen in the prevalence and manifestation of major
depressive disorder and anxiety. They outline a number of biological mechanisms
including transcriptomics, immune signalling and stress hormones may be contrib-
uting to these sex differences. Gogos and van den Buuse (2022) discuss sex
differences in psychosis, and although schizophrenia does not show sex differences
in the prevalence of disease, there are many sex differences in the timing and
manifestation of disease. Breach and Lenz (2022) discuss sex differences in the
neurodevelopmental disorders, autism and attention deficit and hyperactivity disor-
der. They discuss how immune signalling during the perinatal time period is
important to understand how these disorders result in greater risk for males and
they discuss differences in manifestation of symptoms between males and females.
Paletta et al (2022) dive into sex differences in social cognition. They use social
cognition to illustrate that numerous sex and sex hormone influences exist to drive
social cognitive behaviours in males and females. Lee et al. (2022) discuss sex
differences in cognition across aging, why this is important to study in the context of
normal aging and with disease. They outline examples of sex differences in
neuroplasticity and how this may contribute to differences in cognition.
Sub-theme 3: Finally, in the last theme we consider the influence of sex on disease
focusing on two of the most prevalent diseases worldwide, dementia and cerebro-
vascular disease along with multiple sclerosis. Whilst evidence supports the notion
that sex differences occur in the aetiology, pathology, symptoms, and response to
treatments for many diseases to cover them all is beyond the remit of a single book.
As the population ages and healthcare interventions improve, there are an increasing
number of stroke survivors living with long-term consequences of stroke. Stewart et
Preface vii
al (2022) describe the impact of stroke on mobility, cognition, mental health and
mood whilst considering known sex differences in such effects. They argue that
there is a need to better understand the long-term consequences of stroke in each sex
and discuss the challenges of using preclinical models to focus on the long-term
outcomes. Hogervorst et al. (2023) discuss the possibility that menopausal decline in
steroid hormones increases the susceptibility of human females to known risk factors
for dementia such as obesity, reduced exercise, etc. which if reversed or prevented
could be effective, possibly in combination with short-term hormone therapy, at
reducing dementia risk. Multiple sclerosis, of the relapse-remitting presentation, also
shows higher female risk with such a sex difference only becoming apparent post-
puberty implicating a role of sex hormones in this regulation. However, very little is
known of the biological mechanisms underpinning sex difference in onset of mul-
tiple sclerosis and Alvarez-Sanchez and Dunn (2022) discuss the usefulness of an
experimental model of chronic autoimmunity in researching such sex differences
underlying immune mechanisms.
We must of course acknowledge that the majority of this book was written during
the COVID-19 global pandemic when all of us faced unique professional and
personal challenges. We’d like to thank all the contributing authors, series editors
Susanne Dathe and Alamelu Damodharan and the rest of the production team at
Springer for their commitment and patience with this project. We hope this will give
a broad overview of this exciting and complex field that will encourage all of us to
consider the influences of sex on brain function and behaviour.
Nottingham, UK Claire Gibson

Toronto, ON, Canada Liisa A. M. Galea
References
1. Clayton JA, Collins Fs (2014) Policy: NIH to balance sex in cell and animal studies. Nature
509:282–283
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Rev 35:565–572
4. Rechlin RK, Splinter TFL, Hodges TE, Albert AY, Galea LAM (2022) An analysis of neuro-
science and psychiatry papers published from 2009 and 2019 outlines opportunities for increas-
ing discovery of sex differences. Nat Commun 19:2137
5. Marzo-Ortega H, Navarro-Compán V, Servet A, Kiltz U, Clark Z, Nikiphorou E (2022) The
impact of gender and sex on diagnosis, treatment outcomes and health-related quality of life in
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6. Hideki W, Miyauchi K, Daida H (2019) Gender differences in the clinical features and outcomes
of patients with coronary artery disease. Expert Rev Cardiovasc Ther 17:127–133
7. Westergaard D, Moseley P, Sørup FKH, Baldi P, Brunak S (2019) Population-wide analysis of
differences in disease progression patterns in men and women. Nat Commun 8:666
viii Preface
8. Zucker I, Prendergast BJ (2020) Sex differences in pharmacokinetics predict adverse drug

reactions in women. Biol Sex Differ 5:32
9. OECD/European Union (2020) Health at a Glance: Europe 2020: State of Health in the EU Cycle.
OECD Publishing, Paris. https://doi.org/10.1787/82129230-en
Contents
Part I Considering Sex and Gender in Research Studies

Sex and Gender Science: The World Writes on the Body . . . . . . . . . . . . 3
Alana Brown, Laurice Karkaby, Mateja Perovic, Reema Shafi,
and Gillian Einstein
Best Practices in the Study of Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Ineke Klinge
The Curious Case of the Naked Mole-Rat: How Extreme Social
and Reproductive Adaptations Might Influence Sex Differences
in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Phoebe D. Edwards, Ilapreet Toor, and Melissa M. Holmes
Sex/Gender Differences in Brain Lateralisation and Connectivity . . . . . . 71
Sophie Hodgetts and Markus Hausmann
Part II Sex Differences in Cognitive and Mental Health Across the

Lifespan
Sex Differences in Depression and Anxiety . . . . . . . . . . . . . . . . . . . . . . . 103
Pavlina Pavlidi, Nikolaos Kokras, and Christina Dalla
Sex Differences in Psychosis: Focus on Animal Models . . . . . . . . . . . . . . 133
Andrea Gogos and Maarten van den Buuse
Sex Differences in Neurodevelopmental Disorders: A Key Role
for the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Michaela R. Breach and Kathryn M. Lenz
Sex Differences in Social Cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Pietro Paletta, Noah Bass, Dario Aspesi, and Elena Choleris
ix
x Contents
Sex Differences in Cognition Across Aging . . . . . . . . . . . . . . . . . . . . . . . 235

Bonnie H. Lee, Jennifer E. Richard, Romina Garcia de Leon, Shunya Yagi,
and Liisa A. M. Galea
Part III Diseases of the Brain and Relevance of Sex

Sex Differences in the Long-Term Consequences of Stroke . . . . . . . . . . . 287
Courtney E. Stewart, Taylor E. Branyan, Dayalan Sampath,
and Farida Sohrabji
Sex Differences in Dementia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Eef Hogervorst, Sophie Temple, and Emma O’Donnell
Immune Cell Contributors to the Female Sex Bias in Multiple Sclerosis
and Experimental Autoimmune Encephalomyelitis . . . . . . . . . . . . . . . . . 333
Nuria Alvarez-Sanchez and Shannon E. Dunn
Part I
Considering Sex and Gender in Research
Studies
Sex and Gender Science: The World Writes
on the Body
Alana Brown, Laurice Karkaby, Mateja Perovic, Reema Shafi,

and Gillian Einstein
Contents
1 Sex and Gender Science: The World Writes on the Body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Integrating Objective and Subjective Sleep Measures with Sex and Gender Science . . . . . . 7
2.1 The Role of Sex and Gender in Explaining Misalignment of PSG and Self-Reports 7
2.2 Considering Gender Might Explain Objective and Subjective Sleep Misalignment . 8
3 Integrating Subjective Experiences in Memory Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4 Neuropathic Pain and Very Mixed Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5 Sex and Gender in Concussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
5.1 Gender in Repetitive Brain Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
5.2 Contextualizing Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5.3 The Intersection of Sex and Gender in Concussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Abstract Sex and Gender Science seeks to better acknowledge that the body cannot
be removed from the world it inhabits. We believe that to best answer any neuro-
science question, the biological and the social need to be addressed through both
objective means to learn, “how it is like” and subjective means to learn, “what it is
like.” We call bringing the biological and social together, “Situated Neuroscience”
and the mixing of approaches to do so, Very Mixed Methods. Taken together, they
constitute an approach to Sex and Gender Science. In this chapter, we describe
neural phenomena for which considering sex and gender together produces a fuller
knowledge base: sleep, pain, memory, and concussion. For these brain phenomena
Laurice Karkaby, Mateja Perovic, and Reema Shafi contributed equally to this work.
A. Brown (*), L. Karkaby, M. Perovic, and R. Shafi

Department of Psychology, University of Toronto, Toronto, ON, Canada
e-mail: alana.brown@mail.utoronto.ca
G. Einstein
Department of Psychology, University of Toronto, Toronto, ON, Canada
Rotman Research Institute, Baycrest Health Sciences, North York, ON, Canada
Linköping University, Linköping, Sweden
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

Curr Topics Behav Neurosci (2023) 62: 3–26
https://doi.org/10.1007/7854_2022_304
Published Online: 7 March 2022
4 A. Brown et al.
examples, studying only quantitative measures does not reveal the full impact of
these lived experiences on the brain but studying only the qualitative would not
reveal how the brain responds. We discuss how Sex and Gender Science allows us to
begin to bring together biology and its social context and acknowledge where
context can contribute to resolving ignorance to offer more expansive, complemen-
tary, and interrelating pictures of an intricate neuro-landscape.
Keywords Gender · Qualitative · Quantitative · Research methods · Science · Sex ·

Very mixed methods
1 Sex and Gender Science: The World Writes on the Body
In the biomedical literature, the terms “sex” and “gender” are often used interchange-
ably, despite having different meanings: Sex refers to biological attributes and the
variation in how they are expressed. Gender refers to socially constructed attributes
and experiences, including roles, behaviors, identities, traits, attitudes, stereotypes,
and socialization practices (https://cihr-irsc.gc.ca/e/48642.html). There has been a
surge of neuroscientific interest in sex and in gender, yet an intellectual divide seems
to persist; one side concentrates on sex differences without considering the influence
of gender on biology, and another side maintains that sex differences in the brain are
either non-existent or driven entirely by experiential factors (Hankivsky 2012;
Springer et al. 2012; Eliot et al. 2021).
There have been efforts to reunite sex with gender, reflected in both the recent
establishment in Canada of Sex and Gender Chairs (CIHR; https://cihr-irsc.gc.ca/e/
51596.html) and the Canadian Organization for Gender and Sex Research (2020)
(COGS; https://cogsresearch.ca/). This bringing together of sex and gender in
research is called “Sex and Gender Science.” Sex and Gender Science is “the field
in which sex differences and gender are studied with transdisciplinary methodolo-
gies, to advance our understanding that neither is independent of the other, and that
sex and gender differences happen in the context of environment and social inter-
actions” (https://cogsresearch.ca/). Sex and Gender Science posits that to fully
understand human biology, we should assume that biology interacts with influences
and is influenced by multiple systems at different times and levels (Oyama 2000;
Fausto-Sterling 2005). Its premise is that it is impossible to remove the body from
the world it inhabits, and no bodily system is “discrete” or independent of others or
the world “outside” (Einstein 2012). For example, the world via stress affects
allostatic load, resulting in individual habits and developmental experiences having
a cumulative impact on the nervous, cardiovascular, immune, and endocrine systems
involved with coping and adaptation (McEwen and Seeman 1999). Furthermore,
social context via behavior influences sex steroid hormone expression (van Anders
and Watson 2006). Influenced by dark and light, circadian rhythms also affect
ovarian hormone production and vice versa (Toffol et al. 2014; Stock et al. 2019).
Sex and Gender Science: The World Writes on the Body 5
Sex and gender are like a Möbius strip, a seamless surface with only one side and
one boundary curve, illustrating the inseparability of sex and gender. Even though
the “sides” of the strip appear to be above or below each other, they are really the
same side. This metaphor captures the interwoven relationship between the outside
(sociocultural context) and inside (the corporeal body; Grosz 1994). Sex and Gender
Science acknowledges the dynamic interactions between sex and gender, with the
two concepts inextricably tied, as the interweaving of the world, body, and subjec-
tivity. To understand the brain, this interweaving requires integrating approaches
that exist at very different epistemic levels, the objective (third person) or, “how it is
like” and the subjective (first person), “what it is like.”
Bringing together these different epistemic stances, third person and first person,
may be key to reuniting sex and gender research. Quantitative methods, as used in
neuroscience, seek to numerically describe the relationship between variables that
are often seen as distinct. Quantitative methods are rooted in a tradition that assumes
knowledge is unambiguous and certain (Illing 2010). In contrast, qualitative
methods involve a variety of practices describing various paradigms of inquiry,
types/sources of data (e.g., stories, photographs, field notes, etc.), and data collection
(e.g., interviewing), analysis, and interpretation techniques (Sandelowski 1997).
Importantly, qualitative methods can incorporate unstructured interviewing to pro-
vide in-depth descriptions of phenomena from the perspective of the person
experiencing them. Qualitative methods also assume that knowledge is situated
and particular to an individual (Sandelowski 1997). These methods from different
ontological traditions can be seen as overlapping and complementary, with each
approach on its own illuminating differing aspects of participants’ experiences
(Einstein 2012).
Our approach has been to combine quantitative and physiological methods with
qualitative methods, naming the approach, after Haraway, Situated Neuroscience,
which emphasizes that research findings must be interpreted within their social
context and contextualized within lived experiences (Haraway 1988; Einstein
2012). We call this mixing of approaches, Very Mixed Methods. Very Mixed
Methods is an approach that combines qualitative, quantitative, and physiological
methods to integrate the biological and the social, treating the brain as an important
way station for combining external and internal worlds. Use of Very Mixed Methods
in Sex and Gender Science allows for the study of the corporeal body situated in its
sociocultural context (Einstein 2012).
Situating our knowledge with Very Mixed Methods requires following the field
standards for individual methodologies, collaborating, and ensuring one approach is
not subordinated or used in service of the other. It may require publishing qualitative
and quantitative data separately, but also permits them to be combined as multiple,
multidisciplinary “case” reports (Einstein 2012; Perović et al. 2021). This allows the
observation of where sex and gender align, where they diverge, and how they
interact. This combination of methods is especially important when accounting for
influences of interacting factors that may be difficult to study using a single
approach; while quantitative methods are important in examining effects of biolog-
ical sex, qualitative methods may tap into gender. While it is widely recognized that
6 A. Brown et al.
qualitative research can generate questions to be probed further by quantitative tools

(McKenna et al. 2011; Ricci et al. 2019), Very Mixed Methods allow us to also first
identify issues through quantitative research and then delve deeper into them to
learn, “what it is like” through qualitative methods.
Historically, gender-related factors in neuroscience have been understudied and
undervalued, preventing comprehensive understanding of neural phenomena. We
propose that increased focus on subjective experience using Very Mixed Methods
can facilitate multifaceted understandings of such neural phenomena; the gender
component of Sex and Gender Science (including but not limited to gendered
socioeconomic status, education, stigma, and personal identity) must be further
emphasized in research to achieve this. Fortunately, the study and understanding
of gender in biomedical research over the last few decades has evolved and research
on the development of gender-related behaviors and processes has grown consider-
ably (Johnson et al. 2009; Bauer et al. 2017; Schiebinger et al. 2020; Birze et al.
2021). However, sex and gender have not been fully integrated in brain research. In
this chapter we describe neural phenomena for which a combined study of sex and
gender might clarify some gaps in understanding and increase our knowledge base:
sleep, pain, memory, and concussion. We demonstrate how ignoring gender and the
subjective in neuroscience ultimately affects health and sense of self and show that a
more balanced ensemble of biological (sex) and social (gender) in neuroscience
research may deliver a fuller and richer understanding of neuroscientific phenomena.
Key Terms
SEX: Biological attributes, including physical features, anatomy, gene expres-
sion, chromosomes, hormones, and anatomy. Sex is usually categorized as
female or male but there is variation in the biological attributes that comprise
sex and how they are expressed (https://cihr-irsc.gc.ca/e/48642.html).
GENDER: Socially-constructed attributes and experiences, including
norms, roles, behaviors, identities, traits, attitudes, stereotypes, and socializa-
tion practices expressions of girls, women, boys, men, and gender-diverse
individuals (https://cihr-irsc.gc.ca/e/48642.html).
SITUATED NEUROSCIENCE: Emphasizes that research findings must be
interpreted within their social context and contextualized within lived
experiences.
SEX AND GENDER SCIENCE: The field in which sex differences and
gender are studied with transdisciplinary methodologies, to advance our
understanding that neither is independent of the other and that sex and gender
differences happen in the context of environment and social interactions. A
key question in Sex and Gender Science is: How does this combination of
different biologies as well as social outcomes and cultural influences lead to
unique health outcomes, economies, and technological advances (https://
cogsresearch.ca/)?
VERY MIXED METHODS: A research approach that combines qualitative,
quantitative, and physiological methods (the biological and the social),
enabling the study of the corporeal body situated in its sociocultural context.
2 Integrating Objective and Subjective Sleep Measures

with Sex and Gender Science
Sleep research has long combined objective and self-report measures to understand
neuroscientific phenomena of sleep disruption. However, self-report measures are
not really first-person accounts as they often make assumptions about what needs to
be known. In sleep research, they can involve sleep diaries and questionnaires (such
as the frequently used assessment of sleep quality and disturbance, the Pittsburgh
Sleep Quality Index (PSQI); Buysse et al. 1989). Answers to self-report question-
naires focused on sleep quality and daytime functioning are often the decisive factor
of whether an insomniac seeks and obtains therapeutic intervention (Stepanski et al.
1989). Objective measures of sleep can include polysomnography (PSG), which
combines electroencephalography (EEG), electromyography, and electrooculogra-
phy to classify sleep stages based on brain, muscle, and eye activity. Sleep stages
include rapid eye movement (REM, characterized by rapid movement of the eyes,
and low muscle tone throughout the body) and non-REM (NREM, further
subdivided into three stages, with increasing sleep depth from N1-N3). Quantitative
sleep measures may be used to estimate macrostructural sleep staging, sleep latency
(duration to first sleep stage), sleep efficiency (percentage of time spent asleep in
bed), breathing measures, and microstructural spectral power, while the self-report is
what initiates the quantitative inquiry.
When self-reports of poor sleep are coupled with objective measures, the mix
often reveals a confusing picture because they do not always overlap. This is called,
“paradoxical insomnia” – when self-reported difficulty falling and/or staying asleep
is not supported by PSG. Individuals with paradoxical insomnia tend to overestimate
the time it takes to go to sleep and underestimate total sleep time, relative to objective
measures (Harvey and Tang 2012; Bianchi et al. 2013). Considering sex with gender
might aid in explaining the lack of alignment between these measures, enabling a
deeper understanding of sleep mechanisms.
2.1 The Role of Sex and Gender in Explaining Misalignment

of PSG and Self-Reports
Among insomniacs, men, but not women, have high frequency beta EEG power
(indicative of increased cortical arousal) correlating with their misperception of
awakenings in the middle of the night; the lower the beta power, the greater the
misperception (Buysse et al. 2008). This suggests that beta power may be a good
predictor of sleep misperception for men, but not women. Further, while healthy
older men with high PSQI scores show longer PSG-measured sleep latency, lower
sleep efficiency, and decreased total sleep time compared to men with low PSQI
scores, women do not demonstrate this pattern (Vitiello et al. 2004). Thus, men and
women show different relationships between objective and subjective sleep
8 A. Brown et al.
measures, with men demonstrating greater correspondence. Therefore, what we

consider to be objective measures of good-quality sleep may align with men’s but
not women’s self-assessment. While the sense of what a good sleep is like matches
for men, it does not for women; neither self-report instruments nor PSG measures
capture what it is like for women.
In terms of objective sleep, across age groups, women spend more time in slow-
wave N3 sleep compared to men, especially in postmenopause (Ehlers and Kupfer
1997; Carrier et al. 2001; Hachul et al. 2015). Since N3 is thought of as restorative,
this objective measure might lead one to think that women’s sleep is better than
men’s, despite women having a higher prevalence of insomnia and sleep complaints
(Suh et al. 2018, 2020). Given this contrast between objective and subjective sleep
for women, it may be important to treat women according to their subjective sense of
sleep, rather than disregarding sleep complaints based on currently used objective
sleep measures. This could facilitate advanced diagnosis, treatment, and ultimately
prevention of sleep disorders, leading to women’s improved brain health (Lemola
et al. 2013).
2.2 Considering Gender Might Explain Objective

and Subjective Sleep Misalignment
In tandem with sex-related factors, gender-related factors – often disregarded – also

influence sleep quality. For example, while men may be more likely than women to
have greater neck circumference and susceptibility to disordered breathing (e.g.,
obstructive sleep apnea, OSA; Subramanian et al. 2012), women may express more
“atypical” OSA symptoms, and even embarrassment when reporting symptoms such
as snoring (Quintana-Gallego et al. 2004). As well, women may have greater
exposure to socioeconomic risk factors and gendered activities such as increased
caregiving responsibilities, which could result in increased stress and allostatic load,
compounding issues that may contribute to insomnia (Chen et al. 2005). Insomnia
symptoms may also be influenced by gender-related factors. For example, among
women, higher educational attainment was associated with better self-reported
nighttime sleep quality. Meanwhile, men with higher educational attainment were
more likely to self-report nighttime sleep disturbances (Chen et al. 2005). Thus,
gender-related factors may mediate some of the sex differences observed in sleep
quality.
In a large actigraphy study of middle-aged adults, gendered lifestyle factors
differentially correlated with women’s and men’s objective and subjective sleep
misalignment; living with a spouse was associated with less sleep underestimation in
women but not in men. The perceived presence of intimate social relationships was
associated with overestimation of subjective sleep in both men and women, while
economic satisfaction and support from a spouse were associated with
overestimation of objective sleep only in women (Park et al. 2020). Thus, economic
satisfaction and living with a spouse protected women from underestimating their
sleep quality, but not men. In men, discussing concerns with a spouse protected
against sleep underestimation. Further, stressful social network-related events may
have a greater influence on women’s reported sleep quality compared to men’s
(Kawachi 2001; Troxel et al. 2010). Taken together, these results suggest that social
relationships may disproportionately affect subjective sleep perception in women,
underscoring how sociocultural factors surrounding sleep play an important role in
the subjective sleep experience. Accounting for gender-related factors in self-report
instruments may provide a more nuanced understanding of how sex differences in
sleep disturbance might arise from multiple psychosocial risk factors. This could
involve incorporating gender scales geared to biomedical research (Nielsen et al.
2021), which are currently seldom used in tandem with objective measures.
Situated Neuroscience emphasizes that research findings must be interpreted
within their social context. The interplay of objective and the subjective sleep
depends on both sex and gender. For example, with increased family and unpaid
work responsibilities, leisure time declines more for women than for men. Conse-
quently, even though women across age groups have shown increased total sleep
time and time spent in N3 sleep compared to men, it is possible that 30 additional
minutes of sleep for mothers does not compensate for their greater likelihood of
waking to provide care (Burgard and Ailshire 2013). Thus, work and family roles
can differentially influence their sleep quality, and sleep quality needs to be under-
stood in those contexts.
Sex differences in sleep misperception, therefore, tell us that there is an important
gap between the objective sleep measures and self-reports for women, in particular.
If sleep questionnaires are not asking women the right questions, they will not
accurately represent their experiences. Asking women, themselves, what their
sleep is like through qualitative interviewing may best explain their experience.
For instance, the qualitative interviews conducted by Climent-Sanz et al. (2021)
uncovered a lack of satisfactory effective sleep management strategies provided to
women with fibromyalgia by health care professionals. Questionnaires miss the
nuance of these descriptions, including reports of improvised self-management
strategies to improve sleep, which were ineffective or even detrimental to women’s
sleep.
Qualitative interviewing of postmenopausal women has also revealed that their
sleep quality depended on a multitude of psychosocial factors, such as grief related
to the loss of a loved one, divorce, and even concerns regarding hysterectomy
(Vigeta et al. 2012). This underscores the possibility that gendered life factors, rarely
considered in sleep self-report questionnaires, may underlie the discrepancy between
women’s objective and self-reported sleep, serving as a guide to what factors should
be included in self-report questionnaires. Hence, focusing on the role of first-person
accounts in sleep research may elucidate major gaps in calibrating women’s sleep
experiences with objective measures.
In summary, both sex and gender influence sleep. Without recognizing the critical
impact of gendered factors on sleep, we lack nuanced understanding of the subjec-
tive and objective misalignment in understanding women’s sleep. Combining
10 A. Brown et al.
experiential accounts with objective measures through qualitative interviewing

might produce a more comprehensive understanding of what produces poor sleep,
particularly for women, as well as sex differences in sleep mechanisms, with
implications for the development of optimal sleep interventions.
3 Integrating Subjective Experiences in Memory Research
Like sleep, memory has strongly subjective components that are not often studied
from a first-person perspective in tandem with objective measures like neuropsy-
chological testing and brain imaging. The gradual nod toward subjective cognitive
decline (SCD) – the experience of memory loss and/or decline in other cognitive
abilities without concomitant decline beyond neuropsychological norms (Jessen
et al. 2014) – is an important realization by dementia researchers that subjective
experiences may presage more severe memory problems. Alzheimer’s disease, the
most common form of dementia, can be conceptualized as a continuum: patients
progress from normal cognition to mild cognitive impairment (MCI), followed by
increasing severity of Alzheimer’s disease (Albert et al. 2011). Annual transition
probabilities from MCI to Alzheimer’s disease are approximately 22% (Davis et al.
2018). Importantly, women have a higher prevalence of non-amnestic MCI (Au et al.
2017) and experience a greater Alzheimer’s disease burden compared to men, such
that approximately two-thirds of affected persons are women (Alzheimer’s Associ-
ation 2019). Yet, there have been no memory studies to our knowledge that combine
quantitative measures with qualitative interviewing to better understand the texture
of the memory loss experience.
Integrating subjective accounts therefore becomes an opportunity to gain a deeper
understanding of subtle memory changes which occur, by definition, on the level of
subjective and internal experience. While traditional measurements of SCD – ques-
tionnaires and fixed-response Y/N questions – have good predictive power for
conversion to MCI, and later, dementia, it has been suggested that they are unable
to capture the true experiential nature of self-experienced cognitive decline (Buckley
et al. 2015). Qualitative methods can “reach where other methods cannot” (Pope and
Mays 1995) and perhaps one of the areas where this methodological toolset could be
especially important is in memory research.
To understand the influence of ovarian hormone loss on memory, we are studying
memory in women who experienced the gendered practice of bilateral salpingo-
oophorectomy, involving the removal of both ovaries and fallopian tubes (Einstein
et al. 2012). Subtle declines in episodic and spatial working memory compared to
age-matched women with intact ovaries (Gervais et al. 2020) and declines in
hippocampal activity during associative memory tasks (Brown et al. 2020) suggest
that these women might experience SCD; our qualitative interviews underscore it in
rich detail.
I did feel that cognitively things just weren’t happening as fast as they were before. My
ability to manage as much as I did before just wasn’t there anymore. I don’t know if you
would call it like brain fog—and nothing so severe that it was debilitating or it impacted my
ability to function or manage my . . . but I did notice that things like multitasking or quick
decisions, memory, just like cognitive or mental stamina, I felt like those were impacted.
This participant mentioned unexpected cognitive impacts of oophorectomy,

including “brain fog,” “multitasking,” her ability to make “quick decisions,” and
“cognitive or mental stamina.” These all suggest problems with frontal cortical
function and, as we analyze our brain images, her descriptions direct us to the frontal
cortex.
The rich detail of the interview response can also lead to new research directions.
Another participant revealed:
I have joked that I feel like when they do an oophorectomy it’s a lobotomy. I felt so stupid—
it’s a word-finding problem—I’m like (sigh) “you know the icon with the bird?”. I can’t
think of the name, I can’t retrieve it. I just feel like the quintessential dumb blonde so often
and it’s not a comfortable place for me to be.
Here we learn that she feels as if she lost the front portion of her brain; she feels
stupid and self-conscious. She is highly stressed about remembering what she needs
to know for her job and, in particular, she highlights word-finding and remembering
future happenings. These also point to frontal cortical and hippocampal losses, as
well as potential anxiety due to stigma. When evaluating possibly declining brain
functions (e.g., inability to remember people’s names) and stigma, they point to
possible interventions and strategies for her.
In interviews, participants describe a rich landscape in which to integrate their
memory problems: stress, anxiety, and physical changes that accompany rapid,
surgical menopause and a new regimen of hormone therapy. Amid a sea of stresses
in the lives of these women, these cognitive complaints are interwoven with other
experiences and are difficult, if not impossible, to disentangle. Ultimately, the
interviews can confirm and extend what we learn from neuropsychological tests
and imaging, giving us insight into strategies that women themselves choose,
helping us further develop empowering instead of demoralizing interventions.
Finally, the interview allows us to contextualize these memory changes amid the
backdrop of the cultural baggage of being a woman. One woman’s decline in word-
finding is described as a “menopause moment,” perhaps reflecting the stigma of
menopause (Nosek et al. 2010; Chrisler 2013). Moreover, she identifies worries
about how these changes intersect with her appearance feeding into the stereotype of
a “dumb blonde,” a media trope almost exclusively associated with women (Poole
2010).
Prior to interviews, we ask about memory concerns on our demographic form.
Sometimes there is an affirmative response and other times, not. When yes, there is
very little elaboration. How can this be when we get such rich descriptions in
interview? As with sleep research, perhaps when we devise questionnaires, we are
guided by what we want to know and not what our participants want us to know
(Barroso and Sandelowski 2001). The phrasing of the question itself might not
12 A. Brown et al.
resonate, a problem exacerbated by the types of prompts chosen by the research team
(e.g., forgetting phone numbers). In our study, many minimize their complaints,
stating that they are not “severe” or “debilitating,” and thus, perhaps not important
enough to report formally. Given that concerns with this self-perceived decline
predict conversion to dementia (Jessen et al. 2014), it is important to use qualitative
methods to understand the nature of memory concerns beyond their mere presence.
Using Very Mixed Methods and including neuropsychology and brain imaging
helps to pinpoint participant’s problems as well as provide them with confirmation
that there are problems to which they are finding solutions.
In sum, interviews open a whole world on the experience of this decline and bring
sex (ovarian hormone loss) together with gender (family caregiver, aging, concerns
about appearance). By providing a more nuanced and experiential understanding of
SCD, this may inform our understanding of how sex and gender affect memory as
well as which brain regions are affected, what interventions might be most effective,
and ultimately better long-term care outcomes.
4 Neuropathic Pain and Very Mixed Methods
In addition to ovarian removal, there are other gendered practices that can have
whole body effects. Female genital cutting/mutilation/circumcision (FGC) is one of
them. FGC is the traditional practice of cutting, and often removing, portions of the
vulvar region. Approximately two million girls have the procedure every year and
about 200 million women globally have FGC (World Health Organization 2008).
There are four types of FGC, with the least extensive type including cutting the
clitoral hood and the most extensive type involving removal of the labia minora,
external clitoris, the medial portion of the labia majora and stitching together the
remaining skin leaving a small opening for exit of menstrual blood and urine
(“infibulation”; WHO, 2008).
FGC is clearly sexed, altering the female phenotype, but it is also a highly
gendered practice. Not only does the procedure itself mark the transition from
girlhood into womanhood, but this early experience of pain reinforces cultural
expectations around gendered pain expression (Jacobson et al. 2018). Pain – during
FGC, intercourse, and childbirth – is considered an expected part of womanhood, not
to be dwelled on (Abdalla 2006). However, the intersection of gender (Grace and
MacBride-Stewart 2007), culture (Bates 1987; Mustafa et al. 2020), and pain
(International Association for the Study of Pain 2020) is widely understudied,
necessitating development of more comprehensive methods and increased research
efforts.
The use of Very Mixed Methods is uniquely poised to address such multifaceted
research questions in Sex and Gender Science. While reproductive health of women
with FGC has been studied extensively (Obermeyer 2005; Einstein et al. 2018),
effects of FGC on the nervous system are not well understood. As FGC involves
cutting multiple major nerves innervating the vulva, chronic neuropathic pain is a
likely outcome of the procedure (Einstein 2008). In fact, chronic vulvar pain
resulting from a surgery that involves cutting much of the same tissue as FGC –
radical vulvectomy for treatment of vulvar cancer – is well documented
(Wesselmann et al. 1997). Further evidence for more generalized neuropathic pain
in women with FGC comes from studies documenting indicators of chronic pain,
such as fatigue and guarded gait (Walker 1993; Walker and Parmar 1993; Johansen
2002; Jacobson et al. 2018). An important consideration when studying pain is that it
is a phenomenon affected by sociocultural factors (Bates 1987), and the subjective
experience of pain is taken into account during assessment (Wideman et al. 2019).
We used Very Mixed Methods to study neuropathic pain in women with FGC by
conducting qualitative interviews focusing on experiences of pain and pleasure in
daily life, administering quantitative pain questionnaires, including the short form of
the McGill Pain Questionnaire (Dworkin et al. 2009), and quantitative sensory
testing of the vulvar region (Perović et al. 2021). The latter involved applying
pressure with specialized devices to four regions of the vulva. After each increase
in pressure, women were asked if they felt pressure or pain, and the testing stopped
as soon as they identified the sensation as pain with the pressure at which pain was
reported being recorded. If participants were still reporting pressure at 500 g, the
testing stopped and “500+ g” was reported (Pukall et al. 2004, 2007). The quanti-
tative sensory testing was carried out at a local community center with a Somali
woman doctor present.
Results revealed complex, multifaceted, and seemingly contradictory pain expe-
riences. The women reported good overall health and very low pain levels on the
pain questionnaire, with the average pain score being approximately 1.42/5. How-
ever, as with sleep, the subjective did not align with the objective measures, as all
participants had extremely low vulvar pain thresholds – consistent with those
reported by women with chronic vulvar pain (Pukall et al. 2007). Interestingly,
when identifying body regions that were painful as a part of the pain questionnaire,
none of the women circled the vulvar region. Finally, when asked directly about pain
in qualitative interviews, the women denied being in pain, but later in the interviews,
when talking about daily experiences of pain or pleasure, or when asked about how
different parts of their body felt, reported considerable pain in everyday life (Fig. 1).
A possible explanation for the disagreement between qualitative and quantitative
sensory testing results may lie in gendered cultural norms. Somali culture in general
values stoicism in the face of pain, considering it a sign of strength (Finnstrom and
Soderhamn 2006; Isman et al. 2013). The expectation may be especially important
for Somali women with FGC, who report pride in their resilience (Jacobson et al.
2018) and note that expressing pain, either during the FGC procedure or later in life,
would cause shame (Isman et al. 2013). This has implications for clinical practice as
it suggests that, like in the cases of sleep and memory, standardized assessment tools
may fall short of capturing the full range of patients’ bodily experiences.
Understanding how culture and gender intersect to affect experiences and reports
of pain is vital for meeting needs of all patients, including those whose conceptual-
ization of pain might differ from that of the Western healthcare system. While
standardized pain questionnaires are efficient, they rely on a predetermined set of
14 A. Brown et al.
Qualitave Short-Form of Sensory Tesng

Interviews McGill Pain of Vulvar
Quesonnaire Region
When directly asked about pain:
“I feel everything good and I feel, Pain sensivity threshold
like, happy [in my body]” Low self-reported pain
Average score 1.42/5 indicave of chronic pain
When asked about daily life:
Average score 103g
“In the morning, I am not even
Lowest rang: 40g*
able to get down off the bed…It’s
my whole body…it’s ache. It’s like *Studies of women with
aching and pain. That is what I chronic vulvar pain show
feel in the morning” mean scores of a165g
(Pukall et al., 2007)
Fig. 1 Example of findings for one participant across different methods, demonstrating the
complex manifestation of pain in women with female genital cutting/mutilation/circumcision
(Perović et al. 2021)
criteria that may not take into account various sociocultural norms that affect
patients’ willingness and ability to report pain. Open-ended questions and unstruc-
tured interviews in which the patient is encouraged to express cultural idioms and
give descriptive accounts are advisable in conjunction with standard pain question-
naires (Power 2013). Physiological tests may also be needed in cases when patients
do not explicitly report pain, despite bodily expressions that suggest otherwise.
Thus, Very Mixed Methods approaches to clinical care can effectively give the
body “voice.”
In summary, the use of Very Mixed Methods in the context of neuropathic pain in
FGC demonstrates how different research paradigms can build on each other to
provide holistic, rich datasets. Diverse approaches – qualitative, quantitative,
physiological – combine to illuminate different aspects of participants’ experiences.
In the case of neuropathic pain in FGC, tapping into culture and gender through
qualitative interviews illuminates the dearth of acknowledgement on validated
questionnaires in the face of strong physiological response. Together, these
approaches help explain the lack of alignment between subjective and objective
pain reports and offer a more nuanced account of women’s pain.
5 Sex and Gender in Concussion
Both sex and gender are heavily implicated in traumatic brain injury (TBI),
encompassing who is studied, etiologies, and course of brain damage. However, it
is an area in which the influences of sex and gender, especially ensemble, remain
understudied. Traditionally, TBI has been highly gendered, with primarily males
studied within the context of sports and risk-taking activities (Costello et al. 2014).
Due to primarily studying men/males, some important but unexplored research
questions remain: (1) do sex-specific and biologically divergent brain responses to
trauma lead to different long-term consequences and post injury outcomes for
women and men and (2) do gender-based stereotypes play a role in further exacer-
bating these post-injury outcomes?
Brain injuries, even mild, can have long-term consequences that manifest as
physical, cognitive, psychological, and/or social impairments. When persistent,
these impairments have the potential to negatively influence recovery and resump-
tion of life roles, leading to poor functional outcomes. Studies of sex differences in
the impact of a brain injury are relatively absent in the traditional concussion
literature, which focuses primarily on men/males (Gupte et al. 2019), failing to
acknowledge the extent to which women suffer TBI through intimate partner
violence (IPV; Valera and Berenbaum 2003; Iverson et al. 2017). Furthermore,
there is now converging evidence that points to a paradoxical trend within the sports
context: even though men generally sustain more concussions, women carry the
greater symptom burden, show more deficits across certain cognitive domains, and
take longer to recover (O’Connor et al. 2017; Snook et al. 2017; La Fountaine et al.
2019). In the past, this disparity was attributed to the gendered bias that women
simply report more symptoms than their male counterparts (Colvin et al. 2009).
However, current evidence reveals that a sex-difference in neural architecture –
axonal structure – may lead to a greater risk of axonal injury in women after sports
injuries (Rubin et al. 2018; Sollmann et al. 2018) despite equivalent mechanical
damage (Dolle et al. 2018).
5.1 Gender in Repetitive Brain Injuries
While sex-based biological variations can lead to different cascades of damage

following mild TBI (mTBI), gender biases influence not only who gets studied but
also the protocols designed to protect from brain injury. For example, women in
sports undergo less extensive strengthening and endurance training compared to
their male counterparts, leading to compromised isometric neck musculature
strength. This is a particularly important omission, since women have reduced
head-neck mass and neck girth at baseline (Mansell et al. 2005; Tierney et al.
2008). These gendered training protocols may play an active role in creating sex
differences in biological outcomes within the sports context.
As well, to date, there has been little to no exploration of the long-term influence
of repetitive mTBI on women’s health risk, perhaps because our notion of who has
TBI is gendered. Women and men who sustain a single concussion generally recover
without any measurable lasting effects; however, studies of mixed-sex cohorts
suggest multiple concussions have an accumulative effect (Vynorius et al. 2016).
In studies of men-only cohorts, repetitive mTBIs and concussions have been shown
to ultimately contribute to the development of neurodegenerative disease, such as
chronic traumatic encephalopathy (Mckee et al. 2018). However, we know little
about women because of the assumptions about who gets mTBIs. Women also
engage in competitive sports and need to be studied. As well, a highly gendered
life experience, IPV, is only now being recognized as a high risk for mTBIs
16 A. Brown et al.
(St. Ivany and Schminkey 2016). The number of women suffering IPV is an
alarming average of 736 million women globally and it is estimated that one in
four women will experience IPV at least once in their lifetime (WHO 2018), making
IPV more prevalent in women than breast cancer and diabetes combined. It is
estimated that 30–74% of women sustain a TBI during IPV (Kwako et al. 2011),
primarily due to repetitive physical assault and attempted strangulation leading to
head, neck, and face injuries (Roberts and Kim 2005; Sheridan and Nash 2007).
Despite these epidemiological estimates, the life-long impact of repetitive concus-
sions due to IPV, on women’s brain health remains largely unknown. To date, only a
handful of studies have characterized changes in cognitive function as well as
alterations in neural organization post IPV-induced TBI (Flegar et al. 2011;
(Maldonado-Rodriguez et al. 2021; Valera and Kucyi 2016).
5.2 Contextualizing Differences
While the mechanisms by which brain injuries are sustained vary considerably, in
general the “impact to the head” is often mistakenly thought to be quite similar in
women who experience IPV and men who sustain sports injury. Even if the
biological processes were similar (which they might not be; see above), biological
processes are not the only factors dictating disparate outcomes. The world writes
differently on different bodies, and gender-biased experiences affect who recovers –
or has been given the opportunities to recover – from TBI. Inequitable access to
health care resources can seriously affect recovery. The inadequate funding structure
available for shelters has also led to glaring gaps in service delivery and essentially
no access to rehabilitation opportunities for women (Goldin et al. 2016; Haag et al.
2016). In addition, stigma and fear associated with acknowledgement of IPV has led
to a pattern of some women not seeking healthcare or leaving healthcare facilities
before receiving treatment (Corrigan et al. 2003). TBI recovery is further compli-
cated by cognitive disruptions from other resulting factors, such as Post Traumatic
Stress Disorder, depression, physiological stress, and drug addiction (Hayes et al.
2012). Women with IPV are typically of childrearing ages and face mounting
pressures associated with housing uncertainties, financial dependences, and voca-
tional disruptions, not to mention living in fear of encountering the perpetrator.
Gender thus predisposes survivors toward health disparity and inequity (Moss
2002). Over the course of a woman’s lifetime, sex and gender influences may
have a larger role to play in long-term outcomes than the physical insults themselves.
5.3 The Intersection of Sex and Gender in Concussion
One often-overlooked biological player that contributes to sex differences in recov-

ery from concussion is the bioavailability of 17β-estradiol at the time of injury
effecting post-injury recovery outcomes, including cognition and quality of life

(Wunderle et al. 2014). Reproductive hormones can directly influence neuronal
recovery and function through synaptic remodeling, especially in the hippocampus
and prefrontal cortex (McEwen and Milner 2017). The relationship between 17-
β-estradiol levels and TBI is reciprocal; TBI has been shown to have a long-term
impact on endocrine aging (Biscardi et al. 2021). Hormones in turn influence sex
differences in cerebrovascular function and pathology and these vascular/hormonal
linkages are critical to explore given the early vascular origins of dementia, the role
of 17β-estradiol in modulating vascular tone and hemisphere-dependent cyclic
vasodilation, the promising neuroprotective role of progesterone as a potential
treatment for TBI given its capacity to reduce excitotoxicity and inflammation
after injury, as well as the high accuracy of vascular metrics in classifying concus-
sion at the single subject level (Shafi et al. 2021; Stein 2011).
Gender enters when sex differences are considered; post-concussion, men and
women show altered connectivity across large scale brain networks when compared
to age-matched controls, with sex-specific alteration in regional connectivity at the
lateral prefrontal cortex for women but not men (Shafi et al. 2020). Since the lateral
prefrontal cortex has been implicated in goal-oriented processing and alterations to
this region can have repercussions for network efficiency related to planning and
goal attainment (Zanto and Gazzaley 2013; Markett et al. 2014), the difficulties
survivors have in leaving an abusive environment/relationship may partly be attrib-
uted to TBI-induced deficits in executive function (Horne et al. 2020).
Lack of awareness regarding the coexistence of TBI and IPV among frontline
shelter staff, as well as the judicial system, leads to the perception of women as being
“noncompliant” and “failing to follow instructions.” The persistent tension of being
perceived as “broken” or “uncooperative” may be stressful, and this is particularly
relevant given that the psychological impact of trauma is a strong predictor of post-
traumatic stress disorder among women who experience IPV (Pico-Alfonso 2005).
In summary, both sex and gender affect recovery following concussion(s).
Without advancing the understanding of the interactions between the social con-
structs and the biology, we not only lack the scientific rigor required to appreciate the
full complement of factors modifying outcomes post injury, but indeed limit our
capacity to provide evidence-informed care.
6 Discussion
In this chapter, we argue for the importance using Sex and Gender Science to probe
brain phenomena and describe four phenomena in which their merger would either
help solve discontinuities or add to the knowledge base. We described one approach
to merging sex and gender which is to situate the brain in the world or, “Situated
Neuroscience.” We suggest that a combination of the quantitative, qualitative, and
physiological methods which we call, Very Mixed Methods, enables a deeper
understanding of the brain phenomenon – both how it is like and what it is like.
18 A. Brown et al.
The examples presented exemplify the need for Sex and Gender Science in different
ways. Comparing sleep, memory, pain, and TBI reveals different benefits to the
approach.
For sleep, Very Mixed Methods might inform objective and self-report sleep
measure “misalignment,” especially in women; a Sex and Gender Science lens leads
to an increased focus on psychosocial factors, such as social networks, caregiving
responsibilities, marital status, and employment. For memory, the unstructured
revelation of memory problems provides a rich texture in which to analyze neuro-
psychological and brain changes and provide a rich experience of mild memory
decline. For pain, Very Mixed Methods provides insight into how culture and gender
might skew pain reports and why objective and subjective pain reports do not always
align in women with FGC. For TBI, while Very Mixed Methods have yet to be
applied, Sex and Gender Science spotlights both ignorance about women’s TBI and
the gendered social structures and assumptions that make women’s recovery that
much more difficult to achieve. Currently, objective measures “silence” the sleep
experience; they corroborate the memory experience; and give “voice” to the body
for women with FGC, telling a different story than subjective accounts reveal.
These examples show that the physiological, often deemed quantitative, becomes
qualitative when looked at in a given individual as in Very Mixed Methods, giving
the body “voice” (Einstein 2012; Perović et al. 2021). For TBI, aligning the objective
with the subjective may provide insights into how women and men are differentially
affected to inform interventions. It is at the intersection of qualitative, quantitative,
and physiological factors that we can best understand and develop unique and
improved health outcomes. Sex and Gender Science is necessary for the full picture
of a brain phenomenon; investigating sex without gender and vice versa can provide
only a piece of the story, and, in many cases, will lead to less rigorous explanations.
Here, we have described one approach, Very Mixed Methods, to bringing sex and
gender together through Sex and Gender Science.
It is worth considering that although biomedical research has produced valuable
treatments and cures, this has largely been possible for acute conditions, through the
study of one body system in isolation (Einstein and Shildrick 2009). We submit that
if we are striving to seek similar outcomes for chronic disease, we need a paradigm
shift in both our conceptualization of the problem and the approach to the problem.
The examples provided herein highlight how contextualizing the complexities of
multiple biological systems and their intersection with societal practices and the
phenomenological experience might be a step toward such a paradigm shift. We
have especially stressed where our ignorance has accumulated (e.g., concussion),
demonstrating how women’s bodies can provide a new model for elucidating
research gaps (Tuana 2006).
Sex and Gender Science and Very Mixed Methods allow us to begin to bring
together biology and its social context and, as is the case with all the neuroscientific
areas discussed, acknowledge where context can contribute to resolving ignorance.
These linked approaches further provide richer understanding of individual variance
that may appear subtle, but still has important individual effects worthy of study and
intervention. Both contexts offer complementary, interrelating pictures of an
intricate neuro-landscape. We will never understand this complexity in its totality,

but Sex and Gender Science and Very Mixed Methods allow us to glimpse it from
various vantage points to gain meaning from individual differences as well as gaps
and contradictions, leading to a truly personalized medicine.
Acknowledgements This work was supported by the Wilfred and Joyce Posluns Chair in
Women’s Brain Health and Aging (GE; WJP-150643), Canadian Institutes of Health Research
(CIHR, GE; CAN 163902), Canadian Cancer Society (GE; 310336), Ontario Brain Institute (GE),
Alzheimer’s Society of Canada (GE), Women’s Brain Health Initiative (GE), Jacqueline Ford
Gender and Health Fund (GE), Natural Sciences and Engineering Research Council of Canada
(NSERC, AB; PGSD3-546667-2020), CIHR Master’s Award (AB; LK), General Motors Women
in Science and Mathematics Award (AB), Ontario Graduate Scholarship (MP), CIHR Sex and
Gender Science Chair in Immunity (RS; GS7-171367).
Conflict of Interest: We have no known conflict of interest to disclose.
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Best Practices in the Study of Gender
Ineke Klinge
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2 Exposing the Gender Bias in Traditional Biomedical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3 Introducing Sex and Gender into Research as Determinants of Health . . . . . . . . . . . . . . . . . . . . . 30
4 EU Research Policy 2000–2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.1 Gender as a Multidimensional Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
5 EU Research Policy 2010–2020 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.1 Comparing Research Policy Initiatives of International Science Funding Bodies . . . . 36
6 Best Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.1 Gender Medicine as a Scientific Field of Its Own . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.2 Measuring Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6.3 Curriculum Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.4 Dissemination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.5 (Online) Trainings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
6.6 Editorial Policies of Journals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7 Current Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.1 Diversity and Inclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.2 Evaluation Criteria and Monitoring of Policies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Abstract Integrating sex- and gender-related aspects into research and innovation
content has become an urgent requirement of major science funding bodies as f.e. the
European Commission, the Canadian Institutes of Health Research and the US
NIH/Office of Women’s Health Research.
It was the only right response in reaction to the documented failure of the ‘one
size fits all’ approach in traditional biomedical research practices. Attention to sex
differences seems to be taken up by researchers quite well, however integrating a
I. Klinge (*)
Gendered Innovations 2 at European Commission, Brussels, Belgium
Dutch Society for Gender and Health, Amsterdam, The Netherlands
Maastricht University, Maastricht, The Netherlands
e-mail: i.klinge@sylvahome.nl

https://doi.org/10.1007/7854_2022_306
28 I. Klinge
gender analysis has met with difficulties. Of prime importance here is to consider
gender as a multidimensional concept, covering gender norms, gender identities and
gender relations. A research design should clarify from the outset whether gender
norms, identities or relations are of relevance to the research question at hand. This
chapter provides an overview of international policies insisting on integrating a sex,
gender and intersectional analysis into research and innovation with a focus on the
the gender equality policy of the European Union. Next, more in depth, a collection
of best practices, to be understood as a coherent set of processes and activities,
corresponding to the starting points, theories and evidence of the field concerned of
which it can be expected to contribute to the intended results in a particular context is
described. Finally some challenges ahead are identified.
Keywords Biomedical and health research · EU gender equality policy · Gender

analysis · Intersectional analysis · Science funding policies · Sex analysis
1 Introduction
This chapter will focus on the concept of gender and its relevance for biomedical and
health research. Traditional medicine and biomedicine were not particularly inter-
ested in differences between women and men, except from the obvious reproductive
differences.
As a result, biomedical students for a long time did not receive proper training in
the relevance of sex or gender differences in health and disease. However, inspired
by the women’s health movement, feminist biomedical researchers developed an
interest in exactly those differences as they could demonstrate that the ‘one size fits
all paradigm’ was no longer valid. This interest became fuelled by looking at the
social sciences where the concept of gender had spurred a wealth of theories. As
such, gender was a new concept for biomedical researchers, yet very interesting to
consider for an explanation of differences in health and disease between women and
men. Gender theory offered a possibility to counter critiques of biological essential-
ism that a woman’s destiny was determined by biology. For many the concept of
gender – as the socially constructed roles and behaviours of women and men – came
as a useful tool for theorizing and understanding the actual lives, health and disease
of women and men. However, it took a long time before the concepts of sex,
referring to biological characteristics and gender, referring to socio-cultural atti-
tudes, behaviours and identities of humans were considered as relevant determinants
of health. Despite growing awareness of its relevance and implementation in
research practices, the process of acceptance is still evolving.
In what follows I will briefly review the bias in traditional biomedical research,
followed by a discussion on the relevance of sex and in particular gender for
biomedical research. Next, I will move to policy initiatives – taking policies in the
European Union (EU) as an example – to stimulate innovative ways of doing
Best Practices in the Study of Gender 29
biomedicine. Finally I will highlight best practices in the study of gender-related

aspects of health and disease and end with an outlook on further developments.
2 Exposing the Gender Bias in Traditional Biomedical

Research
How was the gender bias in biomedicine exposed? Women’s health researchers and
feminist biologists realized that a ‘male norm’ had influenced the development of
much biomedical knowledge. Biomedical science was conducted on males and on
the male body, that was taken as norm for the human body. Early publications by the
biologists Anne Fausto Sterling Myths of Gender and Lynda Birke Women feminism
and biology created an awareness among biomedical researchers of what was wrong
with traditional biomedicine and the resulting disadvantages for women and
women’s health (Fausto-Sterling 1985; Birke 1986). A later and most telling exam-
ple of gender bias harm came from drug research: from 10 drugs taken from the
market between 1997 and 2000, 8 had more and more severe adverse effects in
women (US General Accounting Office 2001). The reason was simple: drugs had
only been tested in male animals and men. But all fields of biomedical research were
affected as documented by Beery and Zucker in 2011 for 10 biomedical subfields
where male animals had dominated the research populations with the exception of
the field of reproduction (Beery and Zucker 2011).
So, obviously, large gaps in research on women and women’s conditions existed.
In 2010 the European Commission (EC) commissioned a project called Meta-
analysis of gender & science that aimed at providing an overview of the knowledge
production by gender research over the past three decades with a focus on current
approaches (Schiebinger et al. 2010). In the second part of that report, addressing
biomedical and health research, the responses to the identified bias by feminist
biomedical scientists were classified into three theoretical frameworks reflecting
different epistemological positions.
1. Strong objectivity framework: modelled on the notion of strong objectivity coined
by Harding (1991). Focus was on gaps in research and methodological flaws.
These pioneers investigated what science had to say about women and in doing
so, they revealed how normative notions of gender have distorted scientific
research priorities, designs and interpretations (Hubbard 1990; Fausto-Sterling
1985; Keller 1985; Birke 1986, 2000; Bleier 1988).
2. Partial perspective framework: influenced by the field of Science & Technology
Studies (STS) and based on the notion of partial perspectives coined by Haraway
(1988). Central to this framework are textual critiques, in particular the language
used by biomedical and health researchers to access and observe the world, which
turned out to be embedded with cultural assumptions and normative ideas about
gender (Oudshoorn 1994; Martin 2001; Klinge 1997; Butler 2011).
30 I. Klinge
3. Gendered Innovations framework: coined as term by Londa Schiebinger in 2008

(Schiebinger 2008). Gendered innovations are defined as “transformations in the
[personnel, cultures and] content of science and engineering brought about by
efforts to remove gender bias from these fields”.
To expose the existing bias can be considered a first step; to invest in developing
new tools for doing research in a different way, a next one. This branch of
researchers sought to promote innovations in (bio)medical research, health policy
and practice, by constructing practical tools for sex and gender analysis (Bird and
Rieker 1999; Doyal 2001; Krieger 2003; Phillips 2005; Johnson et al. 2009;
Nieuwenhoven and Klinge 2010).
3 Introducing Sex and Gender into Research

as Determinants of Health
The introduction in the 1980s of the new concept of gender, stemming from the
humanities, as relevant for biomedical research did not go without a struggle. Efforts
were needed to provide a proper description – next to sex – of the concept of gender
and an illustration of how to understand their respective influence on health out-
comes. For biomedical research it is crucial to make the conceptual distinction
between sex, referring to biological characteristics and gender referring to socio-
cultural attitudes, behaviours and identities of humans. Important as well is to
acknowledge how sex and gender interact to determine a certain health outcome.
In 2003, the epidemiologist Nancy Krieger was one of the first to provide telling
examples illustrating how both sex and gender can influence a health outcome. She
describes sex and gender interactions as the “biologic expressions of gender” and
“gendered expressions of biology”. A great example in this respect is the underdi-
agnosis of women for heart disease; next to the sex-related differences of age and
non-traditional symptoms in women, gender norms can be seen as a determinant of
physician likelihood of referral for diagnostic and therapeutic interventions resulting
in women less likely to be referred, especially at younger ages (Krieger 2003). More
examples of sex–gender interactions as provided by Gendered Innovations 1 and
2 are discussed in the section case studies.
Yet for the biomedical research community at large, gender was a new concept
and initially not well understood. It often resulted in the neglect or a discrediting of
the concept of gender as identical with women or only pertaining to women’s health
and therefore not of interest to the health of men. As a result, traditional biomedical
researchers missed out on the innovative potential of considering sex differences
beyond reproduction and on the gender-related influences of gender on the health of
men. Moreover, early articles in the medical literature by those who did take an
interest in differences between women and men, often reported their findings as
gender differences, although it turned out they had only studied biological sex
differences. The reason why is still unclear; it was speculated that the term sex
differences was considered less fashionable for use in biomedical literature about
women and men.
But even today there is some disagreement about terminology. Because of the
inextricable interaction between sex and gender, some scholars prefer the term
sex/gender (Schmitz 2010). Yet for practical reasons in biomedical research, the
conceptual distinction between sex and gender is a guiding principle because it
provides different starting points for treatment and interventions, either in the
biological or in the socio-cultural domain.
And so, a two-pronged mission emerged for feminist biomedical researchers
aiming at an innovation of their field of research: (1) that sex differences, beyond
reproduction, are relevant and should be considered and (2) that gender-related
differences are important as well for a meaningful understanding of differences
between women and men.
4 EU Research Policy 2000–2010
By the early years of the twenty-first century, a wealth of evidence was available on
the lack of attention to sex and gender aspects in research (Hubbard 1990; Fausto-
Sterling 1985; Keller 1985; Birke 1986, 2000; Bleier 1988; Bird and Rieker 1999).
For virtually all health conditions, handbooks had appeared summarizing the avail-
able evidence and knowledge (Hurrelman and Kolip 2002; Legato 2004; Rieder and
Lohff 2008; Oertelt-Prigione and Regitz-Zagrosek 2012; Kuhlmann and Annandale
2012; Franconi and Campesi 2013).
The efforts to incorporate attention to sex and gender into research, that were
started by feminist biomedical researchers in the 80s, now became adopted by
science funding bodies. Take the example of the EU Framework programmes, the
largest multiannual research funding programmes of Europe. After the EC had
commissioned a Gender Impact Analysis of their Fifth Framework Programme
(FP5), in 2002 they started to require the integration of sex and gender in research
and innovation where relevant (European Commission 2001). For the 6th Frame-
work Programme that ran from 2002 to 2006, a new term was launched: applicants
(for several major funding instruments) had to consider Women’s Participation
(WP) and the Gender Dimension in the content of research (GD). Both parts were
brought together in the EU’s formula for Gender Equality (GE): GE ¼ WP + GD.
Initially this new concept of the ‘gender dimension’ desperately needed a clari-
fication: the term was meant to capture paying attention to sex and gender-related
influences in the content of research and innovation. For many researchers in Europe
this was a top-down policy requirement that few researchers understood in the way it
was intended. A large majority interpreted the gender dimension as gender balance
or women’s participation issues and the implementation of this policy lagged behind.
What was lacking and became addressed in later projects were practical tools for
researchers to incorporate sex and gender-related aspects into research.
32 I. Klinge
Evidence and tools needed to further support researchers with integrating the
gender dimension when applying to EU money were created by several ground
breaking projects. GenderBasic aimed to provide practical tools, relevant examples
and best practices. The project resulted in a collection of reviews on methodological,
technical (such as breeding of female animals), ethical and financial aspects of
incorporating sex and gender analysis into basic, translational, clinical and public
health research, and provided state-of-the-art reviews of relevant sex and gender
aspects of six important major conditions (anxiety disorders, asthma, metabolic
syndrome, nutrigenomics, osteoporosis and work-related health) (GenderBasic
2007).
The Meta-analysis of 30 years (1980–2010) gender & science literature was
another milestone in summarizing all available evidence on the relevance of sex
and gender for biomedical research (European Commission 2012).
The implementation of the EU policy of integration of the gender dimension was
evaluated in the 2010 publication Stocktaking 10 years of Women in Science policy
by the European Commission 1999–2009 (European Commission 2010). It offered a
comprehensive review of the EU’s GE policy and its effectiveness for increasing
women’s participation and for the integration of the gender dimension. The devel-
opment of the integration of the gender dimension into research content throughout
consecutive Framework Programmes was summarized as progressing from a modest
start to a strong thrust followed by an unexpected hold-up. A full integration of the
gender dimension was still hampered by a lack of understanding what the ‘gender
dimension’ entailed and by challenges in identifying practical measures that could
have been taken to address the gender aspects in research. The need for the
development of practical tools for biomedical researchers to integrate sex and gender
into their research had now become very urgent.
4.1 Gender as a Multidimensional Concept
By 2010 gender was further defined as referring to socio-cultural norms, identities,

and relations that: (1) structure societies and organizations; and (2) shape behav-
iours, products, technologies, environments, and knowledge. Gender attitudes and
behaviours are complex and change across time and place. Importantly, gender is
multidimensional and intersects with other social categories, such as sex, age,
socioeconomic status, sexual orientation and ethnicity. Gender is distinct from sex
and comprises three dimensions: gender norms, gender identities and gender rela-
tions (Term Gender Website n.d.).
Gender norms are produced through social institutions (such as families, schools,
workplaces, laboratories, universities or boardrooms), social interactions (such as
between romantic partners, work colleagues, or family members) and wider cultural
products (such as textbooks, literature, film and video games).
Gender identities refer to how individuals or groups perceive and present them-
selves in relation to gender norms. Gender identities may be context-specific and
interact with other identities, such as ethnicity, class or cultural heritage.
Gender relations refer to how we interact with people and institutions in the
world around us, based on our sex and our gender identity. Gender relations
encompass how gender shapes social interactions in families, schools, workplaces
and public settings, for instance, the power relation between a man patient and
woman physician.
These descriptions make it clear that gender is a pervasive concept deeply
engrained in any society, and affects roles and behaviours of women and men
alike. The challenge ahead was to provide biomedical researchers at large with
practical methods of analysis. These skills were lacking because it had not been
included in their training.
5 EU Research Policy 2010–2020
The EU GE policy became reinforced when preparations were made for the next
Framework Programme Horizon 2020. The GE policy by then consisted of three
objectives:
• To increase women’s participation at all levels
• To stimulate gender balance in decision making
• The integration of the gender dimension in Research and Innovation (R&I)
content
The EC – as a science funding body – had made their intentions clear: Integrating
the gender dimension in research and innovation is an added value in terms of
excellence, creativity and business opportunities. It helps researchers question gen-
der norms and stereotypes, to rethink standards and reference models. It leads to an
in-depth understanding of both genders’ needs, behaviours and attitudes. It enhances
the societal relevance of the knowledge, technologies and innovations produced. It
also contributes to the production of goods and services better suited to potential
markets (Gender Equality in Horizon 2020).
The EC had also realized the need of researchers for innovative methods of
analysis and so in 2011, they convened the Expert Group “Innovation through
Gender” to help develop the gender dimension in EU Research.
The goal of the Expert Group was to provide scientists and engineers with
practical methods for sex and gender analysis and to develop case studies as concrete
illustrations how sex and gender analysis leads to new ideas and excellence in
research. Gendered Innovations was initiated by Londa Schiebinger at Stanford
University in 2008, and the EC joined by co-financing the project from 2011 to
2013. Gendered Innovations aimed to fill in the needs of researchers. The underlying
principle was to trigger the interest of researchers to engage with the new methods
34 I. Klinge
rather than retelling the story of gender biases of the past. It aimed to offer hands-on
materials directly relevant to their own research.
The first edition of Gendered Innovations (2011–2013) was realized by a group of
more than 60 experts from Europe and the USA, through 7 workshops held in over
2 years; peer-reviewed case studies were developed that illustrated how – by
applying methods of sex and gender analysis – new knowledge could be developed.
The project aimed to provide:
• clear definition of terms,
• methods of sex and gender analysis and
• case studies to illustrate how the application of methods of sex and gender
analysis sparks creativity and leads to new knowledge.
The Report Gendered Innovations: How gender analysis contributes to research
was published in 2013 and launched in a special session of the European Parliament
(Gendered Innovations 2013). The Report was accompanied by a website with
additional portals summarizing relevant policies developed by important science
funding bodies and by journals (Schiebinger and Klinge 2018).
The case studies that GI produced were categorized into four domains: science,
health & medicine, engineering and environment as the EC – as a science funding
body – covers all domains of science. Some examples from the health & medicine
domain are Nutrigenomics which examines the epidemic of non-communicable
diseases such as heart disease, diabetes and cancers and Osteoporosis research in
men as a counterexample of heart disease in women; in this field, men are the
neglected sex.
Nutrigenomics: The focus of this case study is on understanding sex- and gender-
related variables in non-communicable disease risk factors. The relative influences
of sex- and gender-related factors determining a person’s disease risk over her or his
lifetime are reviewed. Importantly, gender-related social factors (such as obesity,
lack of exercise, etc.) interact with sex-related biological factors (such as genetic
predispositions, birth weight, and hormones) to determine how a person ages. For
instance, to understand differences in women’s and men’s obesity rates, we need to
analyse gender differences in lifestyle. Perhaps gender norms in society lead men to
exercise more than women; this can lead to greater disease among women. Or
perhaps gender norms in society lead men to eat less healthy food than women.
This gendered behaviour can lead to greater disease among men (Nutrigenomics:
Intersectional Approaches n.d.).
Osteoporosis research in men: Osteoporosis has long been defined as a disease
primarily of post-menopausal women – an assumption that has shaped its screening,
diagnosis and treatment. Why is this a problem? Men account for a third of
osteoporosis-related hip fractures after age 75 – and when they break their hips,
they die more often than women. Osteoporosis is a disease with both sex and gender
components: bones are formed by biology and also by culture, such as exercise rates,
nutrition and general lifestyle. Differences in lifestyle may explain differences in
osteoporosis rates across ethnic groups. Current studies are analysing cohorts of men
from China and Sweden, for example, to understand these types of differences
(Osteoporosis Research in Men: Rethinking Standards and Reference Models n.d.).
After 2013, the materials of the Gendered Innovations project have been spread to
many countries in the world and translations into German, Swedish, Korean, Spanish
and Taiwanese have been produced (http://genderedinnovations.stanford.edu/
translations.html). Numerous workshops, on invitation of Universities, scientific
societies, and science funding bodies were given by Londa Schiebinger and the
author all over Europe and the world to spread the contents of the report and to
stimulate the research community to harness the power of sex and gender analysis
for research and innovation.
With the launch in 2014 of Horizon 2020 and as a result of Gendered Innovations
1, it became clear that attention to integration of the gender dimension was
reinforced by the EC and that applicants became supported in doing so. They were
referred to the GI materials and particular topics of the Work Programmes were
‘gender flagged’, meaning to indicate topics for which sex and gender were relevant.
An interim analysis of Horizon 2020 did reveal that the number of flagged topics had
increased over the consecutive work programmes. Numbers of flagged topics
increased from 16% (19/610 topics) for work programme 2014–2015, to 19%
(108/508 topics) for work programme 2016–2017 and to 23% (110/473 topics) for
work programme 2018–2020 (Interim Evaluation 2017). Yet there was room for
improvement (Horizon 2020).
Therefore the Commission convened a second Expert Group to deliver Gendered
Innovations 2 (2018–2020), to prepare for Horizon Europe, the next framework
programme starting from 2021. The Report Gendered Innovations 2: How inclusive
analysis contributes to Research and Innovation has refined the methods and now
comprises five general methods and nine field specific methods (European Commis-
sion 2020). Increased efforts were made to provide a proper description of the
concept of intersectionality and to translate it into a methodological approach. The
resulting new method intersectional approaches has been incorporated into a num-
ber of case studies such as Facial Recognition and Virtual Assistants and Chatbots.
The 15 new case studies cover more comprehensively all research domains funded
by the European Commission; next to health, case studies addressed: climate
change, energy and agriculture; urban planning and transport; information and
communication technology (artificial intelligence, machine learning, robotics);
finance, taxation and economics; in early spring 2020 an ad hoc case study on the
coronavirus pandemic was created: The impact of sex and gender in the COVID-19
pandemic (Sabine Oertelt Prigione 2020). For the Health Cluster three new case
studies were developed: Prescription drugs: analysing sex and gender; Systems
Biology: collecting sex and gender specific data and Chronic Pain; analysing how
sex and gender interact. The latter case study highlights how women, men and
gender-diverse individuals perceive pain differently (Chronic Pain: Analysing How
Sex and Gender Interact n.d.). They often express pain differently and may receive
different treatments. A better understanding of sex and gender differences in pain
may lead to better health outcomes. Gender-related influences are striking in how
women and men are socialized to respond differently to pain, and this might
36 I. Klinge
influence their sensitivity to pain. Men, for example, may be less willing to report
pain than women because gender roles in Western cultures often associate male
identity with toughness and stoicism (Hoffmann and Tarzian 2001). Gender stereo-
types can also influence how pain is experienced, a patient’s willingness to report
pain, and how healthcare professionals manage pain. For example, some healthcare
providers are more likely to classify pain as psychological in women compared to
men (Samulowitz et al. 2018).
The publication of GI2 was launched by the EC in November 2020. It was
accompanied by the dissemination of factsheets and a media campaign on Twitter
by Commission representatives. When an agreement on Horizon Europe was final-
ized in Feb 2021 two new requirements for funding became apparent: “the integra-
tion of the gender dimension into research and innovation content is a requirement
by default, an award criterion evaluated under the excellence criterion, unless the
topic description explicitly specifies otherwise”; having a Gender Equality Plan
(GEP) in place becomes an eligibility criterion for certain categories of legal entities
from EU countries and associated (Gabriel n.d.).
5.1 Comparing Research Policy Initiatives of International

Science Funding Bodies
Policies similar to the EU GE policy to support the research community in incorpo-

rating attention to sex and gender into research were launched in 2010 by the
Canadian Institutes for Health Research (CIHR) with the Institute for Gender &
Health (IGH) as the main driver, when adopting their Sex and Gender Based
Analysis plus policy (SGBA+) (Government of Canada 2009) and in 2016 by the
US National Institutes of Health’s Office of Women’s Health Research (NIH/
OWHR) with their Sex As Biological Variable policy (SABV) (National Institutes
of Health 2015). A shared notion among these influential research funding bodies is
that a clear definition of both concepts is essential for a proper guidance or instruc-
tion on how to incorporate a sex and gender analysis into research design.
Definitions between science funding bodies may slightly differ and become
adapted over time. The IGH has developed the following definitions:
Gender refers to the socially constructed roles, behaviours, expressions and
identities of girls, women, boys, men and gender-diverse people. It influences how
people perceive themselves and each other, how they act and interact, and the
distribution of power and resources in society. Gender is usually conceptualized as
a binary (girl/woman and boy/man), yet there is considerable diversity in how
individuals and groups understand, experience and express it.
Sex refers to a set of biological attributes in humans and animals. It is primarily
associated with physical and physiological features including chromosomes, gene
expression, hormone levels and function, and reproductive/sexual anatomy. Sex is
usually categorized as female or male but there is variation in the biological
attributes that comprise sex and how those attributes are expressed (https://cihr-irsc.
gc.ca/e/50836.html).
It is beyond the scope of this article to dive into similarities and differences
between the policies of various science funding bodies. A recent article titled The
integration of sex and gender analysis into biomedical research: Lessons from
international funding agencies compares initiatives of major science funding bodies
(CIHR/IGH, NIH/OWHR and EC) and highlights the various ‘carrots and sticks’
(White et al. 2021).
6 Best Practices
Various initiatives can be considered as best practices. According to a definition for

the field of health promotion: Best practices consist of a coherent set of processes
and activities, corresponding to the starting points, theories and evidence of the field
concerned of which it can be expected to contribute to the intended results in a
particular context.
Contextual parameters further define good practices in sex and gender-sensitive
research such as field of study (basic, clinical or public health), rate of sophistication
of the field or available methods. Here I will highlight best practices in widely
different domains as for example funding a new research field, developing measures
of gender, curriculum development, dissemination, online trainings and editorial
policies of journals The collection of best practices below follows a more or less
chronological order.
6.1 Gender Medicine as a Scientific Field of Its Own
Parallel to the EU developments in the first decade of the twenty-first century,

Gender Medicine developed as a scientific field of its own in the USA, Canada,
Europe, and globally. The name Gender Medicine itself received some critique as
expressed at the first IGM congress in 2007 in Berlin. The founders of the field
wanted to have a short and fashionable name and the critique was countered by
pointing out that it was used as shorthand for ‘sex and gender medicine’.
Yet the critics had a point: despite good intentions, most research addressed just
sex differences but reported the results as gender differences. This confusion still
makes it not easy to perform a meta-analysis of scientific results in biomedical
literature. The other reason for mainly addressing sex differences can be found in
the fact that integration of a gender analysis into research was not well developed
during the initial rise of the field of Gender Medicine.
As a specialized field of research, Gender Medicine has continued to grow and
expand. From the beginning, 2-yearly congresses have been organized (starting
38 I. Klinge
2007), an international society and national societies have been launched, followed
by specialized journals (ISOGEM – IGM n.d.).
6.2 Measuring Gender
When science funding bodies launched their initiatives to incorporate attention to

sex differences and gender-related aspects into research and innovation content, an
initial step has been to create awareness of how gender norms based on gender
stereotypes can influence a research design: the research question, the methodology,
the analysis and interpretation. Checklists were developed to help researchers to
avoid pitfalls of that kind (Nieuwenhoven and Klinge 2010).
A further development of integrating gender-related influences in research took
place when researchers wanted to measure the influence of gender. At first sight this
may seem contradictory; how to ‘measure’ or quantify a process that is dependent
upon culture and time? Several groups took up this challenge. Roxanne Pelletier and
colleagues developed a ‘gender score’, a composite measure of gender-related
characteristics and correlated that with the recurrence of Acute Coronary Syndrome
(ACS). A score could be more ‘masculine’ or ‘feminine’ and could be applied to
predict the recurrence of ACS in a particular population. The outcome suggests that
personality traits and social roles traditionally ascribed to women, and not sex, are
associated with adverse outcomes in young patients with ACS (Pelletier et al. 2015).
According to another group of researchers at Stanford University, the operational
definition of gender in the work by Pelletier deserved further consideration. They
claimed that it is no longer sufficient to reduce gender to a two-dimensional spectrum
stretching from ‘masculinity’ and ‘femininity’, i.e., concepts which were historically
construed as complementary oppositions. These concepts are too broad and impre-
cise to be useful in health research. Given that the goal is to provide physicians and
policy makers with gender- related health interventions, measures of gender-related
behaviours, such as caregiving or risk-taking, should be labelled as such and not as
‘feminine‘ or ‘masculine’ behaviours. And so they invested in developing what they
called Gender-Related Variables for Health Research. They developed a gender
assessment tool, to measure gender as a socio-cultural variable. Based on gender
theory, gender is conceived as multidimensional, comprising gender norms,
gender-related traits and gender relations. Exploratory and confirmatory factor
analyses yielded seven gender-related variables: caregiver strain, work strain, inde-
pendence, risk-taking, emotional intelligence, social support and discrimination,
variables that can be deployed quantitatively in diverse clinical research or large
health surveys (Nielsen et al. 2021).
An interesting contribution to the issue of measuring gender was made by a
consortium of six partners under the umbrella name of Gender Outcomes INterna-
tional Group: to Further Well-being Development (GOING-FWD. They emphasized
that there is no standard definition of gender and so there can be no standardized
measure thereof. They advocate that the selection of gender-related variables and
their relevance to the study should be inferred from the initial research question
(Tadiri et al. 2021).
6.3 Curriculum Development
In the first decade of the twenty-first century, a number of European universities had
started the dissemination of knowledge on sex and gender aspects in medicine by
organizing the so-called Ring Vorlesungen, which are weekly or monthly lectures on
sex and gender aspects by gender experts in the various medical disciplines intended
for the university or medical school as a whole. Examples of initiatives are the
University of Innsbruck (Austria), Berlin (Germany) and Göttingen (Germany). In
later years many universities have followed. Often these lecture series have turned
into publications or handbooks (Hurrelman and Kolip 2002).
Funded by the Erasmus Programme of the European Commission, the EUGiM
project organized Summerschools in 2010 in Berlin and in 2011 in Sassari, Italy.
Seven European universities (Berlin, Budapest, Innsbruck, Maastricht, Nijmegen,
Sassari, and Stockholm; and their associated partners) joined forces in EUGIM to
generate a flexible module Gender Medicine (GM) that teaches sex and gender
differences in widespread diseases, therapy and research throughout Europe. The
expertise of the partners in these disciplines was combined to assemble scientific
content and learning goals for a common GM module and to build a common pool of
teachers. The basic idea was that GM modules can be flexibly integrated into
bachelor or master programmes or vocational education at different universities
and should lead to an internationally recognized certificate. In this way innovation
in medical education is promoted and has contributed to harmonization of biomed-
ical study structures in Europe. The project has generated internationally recognized
experts for gender-sensitive medicine and has created an expandable European
network from university and non-university partners. Network members have sen-
sitized universities, medical professional organizations, health care politics, funding
organizations and insurance companies for sex and gender aspects (EUGiM
(European Curriculum in Gender Medicine) 2010).
Virginia Miller in the USA has been a pioneer in signalling the need for
integration of sex and gender in medical curricula (Miller et al. 2013).
6.4 Dissemination
Two projects funded by the European Commission can be considered best practices
in terms of dissemination.
EUGenMed (European Gender Medicine) (2013) aimed at developing an inno-
vative roadmap for implementation of sex and gender in biomedicine and health
research. Partners involved were: Institute of Gender in Medicine (GiM, Charité
40 I. Klinge
Universitätsmedizin) Berlin; European Institute of Women’s Health Dublin, Ireland;

Maastricht University, Department of Health, Ethics and Society, NL. Target audi-
ences were doctors, medical associations, teachers, students, researchers, industry,
health policy makers, funding agencies and politicians. 503 stakeholders were
identified from 25 EU and 10 non-EU countries. They were medical doctors and
(bio) medical scientists, researchers, teachers and students in academia, industry,
pharmaceutical companies, science funding organizations, regulatory bodies, health
policy makers, patient organizations, representatives of civil society and lay people.
Among them, there were 28 journalists and 28 partners working at different levels
for the EU commission and related agencies. Of these about one third participated in
EUGenMed’s conferences and workshops, others followed via the Internet. The
project covered four working fields; clinical medicine and pharmacology, public
health and prevention, basic biomedical research, medicines regulations and medical
education (EUGenMED (European Gender Medicine) n.d.).
EUGenMed produced information (publications) and recommendations (policy
briefs) for the implementation of sex and gender in biomedicine and health research
for different target audiences (Oertelt-Prigione et al. 2017).
GenCAD (Gender-specific mechanisms in coronary artery disease in Europe)
Over its three-year lifespan (from February 2015), the GenCAD project aimed to:
(1) Analyse existing knowledge on gender differences in CAD risk factors, disease
mechanisms, clinical manifestations, treatment options, access to health care, as well
as management and outcomes; (2) Assess the awareness of health professionals and
the general population to identify the most effective practices to raise awareness
about sex and gender manifestations of CAD; (3) Develop and disseminate infor-
mation materials based on the outcomes of the studies, surveys, and comprehensive
needs assessment. Important materials, made available in all 24 European
languages are: (a) Factsheets for the General Public: How to protect your heart;
are women and men different? (b) Factsheets for health professionals on gender
differences in coronary artery disease in Europe (GenCAD n.d.).
6.5 (Online) Trainings
Both IGH and NIH/OWHR have developed online trainings in recent years. For the
field of health research, the IGH training materials are currently the most advanced:
“Every cell is sexed and every person is gendered” (Online Trainings CA n.d.).
NIH/OWHR recently launched on online training on SABV (Online Trainings
US n.d.).
The conditions for funding set by science funding bodies in Canada, the USA and
Europe have been accompanied by training courses especially for early researchers
at a national level. A good practice is the International Gender in Research Course
for early researchers organized by the Netherlands Organisation for Health Research
and Development (ZonMw). Fellowships were available for attending the course and
covering travel costs. 42 applicants from six continents were selected and attended
the course in Summer 2019. The second edition took place in May/June 2021.
Experts and guest speakers from a multitude of disciplines jointly teach and share
their knowledge and research expertise with the next generation of researchers from
all over the world. Participants have the opportunity to build a community with
fellow talented early-career researchers from around the world and work in diverse
teams on a prize-winning competition within the course (Fellowships Gender in
Research Course – ZonMw n.d.).
6.6 Editorial Policies of Journals
Journals are gatekeepers in advancing sex and gender analysis in research. The
SAGER guidelines developed over a 3-year period by a multidisciplinary group of
academics, scientists and journal editors by means of literature reviews, expert
feedback and public consultations at conferences provide researchers and authors
with a tool to standardize sex and gender reporting in scientific publications. The GI
website has listed the journals that have issued guidelines for reporting on sex and
gender analysis (Heidari et al. 2017; Heidari et al. 2016; stanford.edu n.d.).
7 Current Challenges
7.1 Diversity and Inclusion
Gendered Innovations 2 has had a big impact on strengthening the integration of the
gender dimension in Horizon Europe. The title of the second policy review has
intersectionality prominently in its title: GI2 How inclusive analysis contributes to
research and innovation.
Influential work by Hankivsky on intersectionality (2008) has now become more
fully acknowledged (Hankivsky and Christoffersen 2008; Schulz and Mullings
2005; Epstein 2007). It has resulted from evolving insights that sex and gender do
not exist in a vacuum but play out in a context of intersecting other characteristics of
social position/inequality such as age, ethnicity, socioeconomic position, sexual
orientation, geographic location, (dis) ability, etc. In GI2 an intersectional approach
is described as important to consider when setting research priorities, developing
hypotheses and formulating study designs. Taking an intersectional approach can
better predict variations in health outcomes and determine user needs, and ultimately
lead to more inclusive research and engineering solutions. For example: recent
research demonstrates how an intersectional approach can improve the accuracy of
AI-based facial recognition and energy efficiency measures (Term Intersectionality
n.d.). Facial recognition systems (FRSs) can identify people in crowds, analyse
emotion, and detect gender, age, race, sexual orientation, facial characteristics, etc.
These systems are often employed in recruitment, authorizing payments, security,
42 I. Klinge
surveillance or unlocking phones. Despite efforts by academic and industrial

researchers to improve reliability and robustness, recent studies demonstrate that
these systems can discriminate based on characteristics such as race and gender and
their intersections. Bias in machine learning (ML) is multifaceted and can result from
data collection, or from data preparation and model selection. Applying the field
specific method ‘Analysing gender and intersectionality in machine bias’ offers
opportunities to counter amplification of different types of discrimination on an
individual or group.
The energy debates often ignore the human factor. An intersectional approach is
needed, one that recognizes people’s multiple, interdependent and overlapping axes
of social identity, e.g. gender, socioeconomic status and age. A single factor, such as
gender, both shapes and is shaped by other social attributes such as age, ethnicity and
socioeconomic status. Together, these factors influence the life experiences of
citizens engaging with the complex sociotechnical networks that constitute the
energy system.
Another policy initiative on intersectionality is the EDI (equity, diversity and
inclusion) policy of the Canadian Institutes of Health Research (Best Practices in
EDI n.d.).
7.2 Evaluation Criteria and Monitoring of Policies
Monitoring the implementation of a policy is crucial. The Canadian IGH evaluated

the SGBA+ policy by examining the uptake of accounting for sex and gender
(Johnson et al. 2014) and the effectiveness of online learning (Tannenbaum and
van Hoof 2018). They demonstrated that the online modules improved knowledge
and self-efficacy for integration of sex and gender in health research. NIH/OWHR
evaluated 5-year progress on SABV policy and concluded with a call on various
stakeholders to “double their efforts” (Arnegard et al. 2020).
Implementation of a policy is a multilevel process and stretches from policy texts
to everyday activities of staff members and eventually to evaluation forms for
scoring a proposal. In this respect it is crucial that evaluators of applications that
require the integration of the gender dimension (sex and gender analysis) have the
skills to be able to do so. IGH has developed an online training for peer reviewers
which guides them to reach at decisions on what is a good or not so good integration
of sex and gender (Assessing Sex and Gender Integration in Peer Review n.d.).
Many science funding bodies are still in the process of developing context-specific
criteria.
In conclusion, it took 20 years of EU policy investments in integrating sex,
gender and intersectional analysis into research and innovation content to reach at
the current situation under Horizon Europe where addressing the gender dimension
is the default requirement.
In this research policy field, next to the EU, Canada, the USA and Korea are
among the most influential players. A current study aims to compare efforts of
science funding bodies in this respect on a global scale (Schiebinger and Hunt n.d.).
Perhaps the scientific field started as Gender Medicine now gradually needs
another label. The newly launched Canadian Organization for Gender and Sex
Research (COGS) has proposed a new name for the field: Intersectional Sex and
Gender Science (Canadian Organisation for Gender and Sex Research n.d.). This
name conveys the relevance of sex and gender analysis for all fields of science and
secondly fully embraces an intersectional approach. A recent perspective in Nature
provides a roadmap for sex and gender analysis across scientific disciplines and calls
on researchers, funding agencies, peer-reviewed journals and universities to coordi-
nate efforts to implement robust methods of sex and gender analysis in order to foster
scientific discovery, improve experimental efficiency and enable social equality
(Tannenbaum et al. 2019).
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The Curious Case of the Naked Mole-Rat:
How Extreme Social and Reproductive
Adaptations Might Influence Sex
Differences in the Brain
Phoebe D. Edwards, Ilapreet Toor, and Melissa M. Holmes
Contents
1 Naked Mole-Rats: A Case for Social Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2 Brain Morphology: Status Differences, Not Sex Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3 Brain Gene and Receptor Expression: Status Differences and Sex Differences . . . . . . . . . . . . 58
4 Damaraland Mole-Rats as a Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5 Reduced Sex Differences: Why and How? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Abstract Research in the neurobiology of sex differences is inherently influenced

by the study species that are used. Some traditional animal research models, such as
rats and mice, show certain sex differences in the brain that have been foundational
to neurobiological research. However, subsequent work has demonstrated that these
differences are not always generalizable, especially to species with different social
structures and sex-associated roles or behaviors. One such example is the naked
mole-rat (Heterocephalus glaber), which has an unusual social structure among
mammals. Naked mole-rats live in large groups where reproduction is restricted to
a dominant female, called the “queen,” and often only one breeding male. All other
animals in the group, the “subordinates,” are socially suppressed from reproduction
and remain in a prepubescent state as adults, unless they are removed from the
presence of the queen. These subordinates show little to no sex differences in
external morphology, neural morphology, or behavior. However, there are a suite
of neurobiological differences between subordinate and breeding naked mole-rats.
After naked mole-rats attain breeding status, many of the classically sexually
differentiated brain regions increase in volume (paraventricular nucleus, medial
amygdala, bed nucleus of the stria terminalis). There are additionally social status
differences in sex hormone receptor expression in the brain, as well as other changes
in gene expression, some of which also show sex differences – though not always in
P. D. Edwards, I. Toor, and M. M. Holmes (*)

Department of Psychology, University of Toronto Mississauga, Mississauga, ON, Canada
e-mail: melissa.holmes@utoronto.ca

https://doi.org/10.1007/7854_2022_310
48 P. D. Edwards et al.
the predicted direction based on other rodent studies. Data from naked mole-rats
show that it is critical to consider the evolved social structure of a species when
studying sex differences in the brain.
Keywords Cooperative breeding · Eusocial · Mole-rat · Plasticity · Sex difference ·

Social status
Sex differences in the brain are traditionally interpreted through the lens of sex
differences in behavior. In many species, males and females differ in levels of
competition for access to mating opportunities, their particular roles in reproduction,
and the specific behaviors associated with these processes. Thus, it is not unreason-
able to postulate that sex differences in the brain would correspond to these sex
differences in behavior. Foundational work in traditional laboratory rodent models
(rats, mice, guinea pigs) has provided support for this framework. For example, in
rats and several other species, the sexually dimorphic nucleus of the medial preoptic
area (SDN-POA) is significantly larger in males than in females (Greenberg and
Trainor 2016). Accordingly, lesioning of this area in rats impairs male-typical sexual
behaviors, including mounting, intromission, and ejaculation (De Jonge et al. 1989).
There are also female-biased examples: in mice and rats, females have more cells
involved in dopamine synthesis in the anteroventral periventricular nucleus (AVPV)
of the brain, and this population of cells is functionally associated with levels of
maternal behavior (Simerly 1989; Forger et al. 2004; Scott et al. 2015).
Such sex differences in the brain are predominantly shaped during critical
developmental periods. Neural sex differences are often derived through an
organizational-activational process, where the brain is organized by gonadal hor-
mones early in development (prenatal and early postnatal life) and is then modulated
by gonadal hormones in adulthood (Phoenix et al. 1959; Arnold 2009). Increasingly,
puberty has been considered a second or extended critical period of brain organiza-
tion, where circulating sex hormones alter structure and circuits in numerous areas of
the brain (Sisk and Zehr 2005; Schulz et al. 2009). Puberty is of exceptional
importance because of the confluence of gonadal activation, neural rewiring, and
restructuring of the social environment (Sisk and Zehr 2005; Bell et al. 2013).
Organizational and activational events are not entirely distinct from one another
during this period, but rather part of a spectrum where organizational events can
transition into activational events during puberty (Juraska et al. 2013). The influx of
gonadal steroid hormones during puberty activates neural circuitry in a sex-specific
manner, and also further contributes to the sculpting of sex differences in various
brain regions (Sisk and Zehr 2005). For example, in the rat brain, sex differences in
region volume in the AVPV, SDN-POA, and medial amygdala (MeA) are
established through new cell birth during puberty, but these sex-specific changes
in cell number can be attenuated by prepubertal gonadectomy (Ahmed et al. 2008).
However, the same neurobiological sex differences are not consistently present
across species and may even be inconsistent among different strains of mice (Brown
et al. 1999; Holmes et al. 2009). Some species display seasonal sex differences in the
brain: common shrew (Sorex araneus) males have a larger hippocampal CA1 than
The Curious Case of the Naked Mole-Rat: How Extreme Social and. . . 49
females in the summer, when they have larger home ranges than females, but not in
the winter, when both sexes are less active (Lázaro et al. 2018). Similarly, there are
seasonal sex differences in the hippocampal dentate gyrus in meadow voles
(Microtus pennsylvanicus), where females have higher rates of cell proliferation
and death than males in the non-breeding season, but not in the breeding season
(Galea and McEwen 1999). Conditions in the social environment may also result in
sex-specific changes in the brain. For example, absence of social contact can have
sex-specific effects in Syrian hamsters (Mesocricetus auratus): isolated males have
higher V1a receptor binding in the anterior hypothalamus compared to grouped
males, whereas isolation does not affect V1a binding in females in this region (Ross
et al. 2019). Similar examples can be found in birds. In the zebra finch (Taeniopygia
guttata), mixed effects between sex, social context (exposure to novel or familiar
animals), and behavioral phenotype influence activation of several dopaminergic
populations (Kelly and Goodson 2015). Male zebra finches have more tyrosine
hydroxylase/Fos double-labeled cells in the medial hypothalamus/preoptic area
than females, though the interaction effects of social context and behavioral pheno-
type on this population are independent of sex (Kelly and Goodson 2015).
There are additionally striking interaction effects between social status and sex
differences in the brain. Social hierarchies exist in many species, and an individual’s
social status within the hierarchy (being dominant or subordinate) results in different
roles, behaviors, and often, physiological and reproductive changes (Sapolsky
2004). In some species, social dominance is simply associated with better chances
of successful reproduction (e.g., Holekamp et al. 1996), whereas in others, sub-
ordinates will fully suppress sexual maturation because their chances of accessing
reproductive opportunities are so low (e.g., Jarvis et al. 1994). Subordinates can also
display alternative phenotypes, for example, subordinate males of some species of
cichlid fish will appear and behave as females until they gain dominance, when they
will switch to the male-typical coloration and territorial behaviors (Maruska and
Fernald 2010).
Accordingly, social status can influence neurobiological sex differences in
species-specific ways. Acquisition of dominance in female Syrian hamsters
increases activation of serotonergic neurons in the anterior portion of the dorsal
raphe nucleus relative to subordinate females, but there is no detectable effect of
social status on this population in males (Terranova et al. 2016). In cooperatively
breeding white-browed sparrow weavers (Plocepasser mahali), dominant males
have larger HVC and RA regions in the forebrain (involved in song production)
than do subordinate males. This is thought to be because the dominant males have an
additional type of song that subordinate males and females do not sing (Voight and
Gahr 2011). Though subordinate males still have larger HVC and RA regions than
females, their mRNA expression of candidate genes in the HVC is more similar to
females than to dominant males (Voight and Gahr 2011).
Thus, ample evidence indicates we must acknowledge that the neural sex differ-
ences seen across vertebrates are not rigid phenomena. Rather, they can be dynamic
in response to social organization, reproductive strategy, and factors in the social
environment. The variability in regard to neural sex differences demonstrates it is
essential to consider species-specific social organization and breeding strategy in

order to understand both how and why sex differences in the brain evolve and
persist. Studying species that differ in social organization captures a more
multidimensional view of sex differences, facilitating interpretation of the signifi-
cance and function of sex differences in the brain. Here, we focus on the naked mole-
rat, a strange and remarkable eusocial rodent that shows minimal evidence of sexual
differentiation. Naked mole-rats have evolved a social system so extreme in its taxon
that it provides a powerful opportunity for investigation into the factors influencing
sex differences.
1 Naked Mole-Rats: A Case for Social Status
Naked mole-rats are subterranean rodents native to sub-Saharan Africa. They reside
in colonies typically containing 70–80 individuals, but can be upwards of 300, in an
expansive burrow system (Jarvis 1981; Brett 1991; Jarvis et al. 1994). They are
considered eusocial, with a reproductively active breeding caste consisting of a
breeding female and 1–3 breeding males, and a reproductively suppressed subordi-
nate caste (Jarvis 1981; Brett 1991). Social hierarchies seem to be maintained via
aggressive interactions between queen and subordinates (biting, shoving, etc.), as the
physical presence of the breeding female is required to maintain subordinate repro-
ductive suppression (Faulkes et al. 1990, 1991; Reeve and Sherman 1991).
While queens have an ovarian cycle length of approximately 34 days, subordinate
females have quiescent ovaries, low levels of luteinizing hormone, and consistently
low progesterone levels, signalling a lack of ovulation (Faulkes et al. 1990). Once
the suppression from the breeding female is removed, either by her death or by
removal of the subordinate from the colony, ovulation activates in female subordi-
nates (Faulkes and Abbott 1993; Smith et al. 1997). Male subordinates are also
reproductively suppressed and show smaller testis size and low sperm quality in
comparison with breeding males (Faulkes et al. 1991, 1994). However, there is
continued spermatogenesis in subordinate males, making reproductive suppression
less absolute than in females (Jarvis 1981). Subordinate animals are chronologically
adults but rarely sexually mature. Given the importance of puberty in wiring and
rewiring neural circuits involved in social cognition and reproductive behaviors,
naked mole-rats provide an opportunity to study what would occur in the adult brain
if puberty was never achieved.
Breeder naked mole-rats exhibit the same copulatory behaviors as other mam-
mals, where males will mount and intromit, and females will perform lordosis during
the peri-ovulatory phase of their ovulatory cycle (Jarvis 1991; Holmes and Goldman
2021). The breeders also exhibit a mutual genital nuzzling behavior that occurs
throughout the female’s ovulatory cycle, and also continues through pregnancy
(Lacey et al. 1991). Mounting behavior in males can be eliminated via castration
and rescued through testosterone treatment, but genital nuzzling is not completely
eliminated by castration (Goldman et al. 2006). Furthermore, aggressive interactions
Fig. 1 Differences in naked mole-rat external morphology by status and sex. (a) Breeding (BRE)
female and male on the left tend to be larger than subordinates (SUB). Subordinates have no sex
differences in overall body size and appearance. (b) Genitalia of breeding female on the left
compared to subordinate female on the right. The breeding females have enlarged genitals and a
more obvious vaginal fold or vaginal perforation relative to the subordinate females. (c) Genitalia of
breeding male on the left compared to subordinate male on the right. There are no status differences
between breeding and subordinate males, and minimal sex differences between subordinate females
and subordinate males. Modified from Faykoo-Martinez and Holmes (2021)
are also often performed by breeders, especially the breeding female. The breeding
female initiates more shoving interactions than any other individual within the
colony, including the breeding male, but is never the recipient of shoves (Clarke
and Faulkes 2001). The onset of increased aggressive interactions is closely associ-
ated with caste transition, increases in urinary progesterone in females, and testos-
terone in males, following removal of previous breeders (Clarke and Faulkes 2001).
Subordinates rarely, if ever, perform copulatory or aggressive behaviors within
the colony (Clarke and Faulkes 2001; Goldman et al. 2006). In fact, naked mole-rat
subordinates show very little sex differences both behaviorally and anatomically. All
subordinate individuals, regardless of sex, contribute to colony and litter care
(Sherman et al. 1992). Task specialization is more closely associated with the
body size of the individual rather than their sex (Sherman et al. 1992; Mooney
et al. 2015a). There is also evidence that dispersal behavior is not sex biased in either
wild or laboratory colonies, with males and females being equally likely to leave the
colony (Braude 2000; Toor et al. 2020; c.f. O’Riain et al. 1996 – male-biased
dispersal). In terms of external anatomy, there are no sex differences in size or
weight between male and female naked mole-rats (Fig. 1). External genitalia is
remarkably similar and can only be differentiated by an inconspicuous vaginal
fold in females, and there is no difference in anogenital distance or phallus size/
length (Fig. 1; Peroulakis et al. 2002).
The lack of sex differences in the naked mole-rat seems to be mediated by its
unique social system. When comparing the eusocial mole-rat to its solitary cousin
the silvery mole-rat, and its less-extreme eusocial cousin the Damaraland mole-rat,
social structure was found to predict genital morphology within the Bathyergidae
family with naked mole-rats having no sex differences in genitalia and associated
muscular, silvery mole-rats having sex differences, and Damaraland mole-rats being
intermediate (Seney et al. 2009).
2 Brain Morphology: Status Differences, Not Sex

Differences
The lack of sex differences in naked mole-rats (especially subordinates) is also

reflected in their neural morphology (Table 1). Regions of the brain that are typically
sexually differentiated in laboratory rats and mice have been examined in both
breeding and subordinate naked mole-rats. These regions include the bed nucleus
of the stria terminalis (BST), the paraventricular nucleus of the hypothalamus
(PVN), the ventromedial nucleus of the hypothalamus (VMH), and the medial
amygdala (MeA). In laboratory rats and guinea pigs, males have more neurons in
the BST, and this region is implicated in sexual behavior, aggressive behavior, and
response to social challenges (Forger et al. 2004; Greenberg and Trainor 2016). The
PVN is critically involved in stress axis reactivity, as it secretes corticotropin-
releasing factor (CRF), and has additional projections to the pituitary. In rats,
males show post-puberty differences in PVN cellular activation in response to stress,
whereas females show post-puberty differences in CRF expression (Viau et al.
2005). The VMH is involved in copulatory behavior and is larger in male rats,
with a greater density of cells (De Vries and Simerly 2002). Male rats also have a
larger MeA, which is functionally associated with olfactory investigation and sexual
arousal (Cooke et al. 1999).
In naked mole-rats, none of these regions significantly differ in volume, cell
number, or cell size between males and females (Holmes et al. 2007). This is true in
both subordinates and breeding animals. However, all of these regions have status
differences: breeders, regardless of sex, differ from subordinates. The BST, PVN,
and MeA are larger in volume in breeders than in subordinates. The VMH shows no
status differences in regional volume, but contains more cells within breeders, with a
marginally larger cell size (Holmes et al. 2007). The increase in volume in the PVN
occurs when subordinates are removed from reproductive suppression and paired
with an opposite sex animal (Fig. 2; Holmes et al. 2011), essentially when the animal
undergoes reproductive maturation. In contrast, the increase in volume in the BST
does not happen at initial pairing and reproductive maturation, but rather once the
animal successfully reproduces (Holmes et al. 2011). Further, these status differ-
ences are not due to an overall change in brain morphology in breeders, as other
regions unrelated to sexual behavior remain unchanged. The suprachiasmatic
nucleus, which controls circadian rhythms, and the anterior cortical amygdaloid
nucleus, which receives olfactory input, do not differ in volume, cell number, or
cell size between subordinates and breeders, nor is there a difference in cortical
thickness (Holmes et al. 2007).
Table 1 Naked mole-rat brain status differences and sex differences discovered to date
Region Measure Method Sex differences Status differences Reference
Brain Anterior cortical Cell size and Thionin staining No difference No difference Holmes
morphology amygdaloid number et al.
nucleus Region volume No difference No difference (2007)
Bed nucleus of Cell size and No difference No difference
the stria number
terminalis (BST) Region volume No difference Larger in breeders
Medial amygdala Cell size and No difference No difference
(MeA) number
Region volume No difference Larger in breeders
Onuf’s nucleus Cell size and Kluver-Barrera No difference More cells in breeders, fewer Seney
motoneurons number staining small cells in breeders et al.
(2006)
Paraventricular Cell size and Thionin staining No difference No difference Holmes
nucleus (PVN) number et al.
Region volume No difference Larger in breeders (2007)
Suprachiasmatic Cell size and No difference No difference
nucleus of the number
hypothalamus Region volume No difference No difference
(SCN)
The Curious Case of the Naked Mole-Rat: How Extreme Social and. . .
Ventromedial Cell size and No difference More cells in breeders, margin-

nucleus of the number ally larger cell size
hypothalamus Region volume No difference No difference
(VMH)
Oxytocin BST Oxytocin Autoradiography No difference No difference Mooney
receptors Hippocampus receptors No difference No difference et al.
Major islands of No difference No difference (2015b)
Calleja
(continued)
53
Table 1 (continued)
54

MeA More OTR in males No difference
NAcc No difference in subordinates, No difference
marginally lower OTR in
breeding females relative to
breeding males
VMH No difference No difference
Reproductive BST Androgen Immuno- No difference Less AR in breeders Holmes
axis MeA receptors histochemistry More AR in males Less AR in breeders et al.
PVN No difference Less AR in breeders (2008)
SCN No AR present in this region No AR present in this region
Ventral portion of More AR in males Less AR in breeders
the
premammillary
nucleus (PMv)
VMH More AR in males in VMHdm Less AR in breeders
and VMHdl but no difference in
VMHc
Whole right Androgen qPCR Higher levels in males Higher levels in breeding males Swift-
hemisphere of the receptors (Ar) than subordinate males Gallant
diencephalon Aromatase Higher levels in females Higher levels in breeding et al.
(Cyp19a1) females than subordinate (2015)
females
Estrogen recep- Higher levels in females Higher levels in breeding
tors (Esr1) females than subordinate
females
Progesterone – Higher levels in breeding
receptors (Pgr) females than subordinate
females; lower levels in breed-
P. D. Edwards et al.
ing males than subordinate

males
Reproductive NAcc Kisspeptin qPCR Marginally higher levels in No difference Faykoo-
onset related (Kiss1) males than in females Martinez
Neuropeptide Higher levels in males than in No difference et al.
VF precursor females (2018)
(Npvf)
Tachykinin Higher levels in males than in No difference
3 receptor females
(Tac3r)
PVN Kappa opioid Marginally higher levels in No difference
receptor (Kor) males than in females
RFRP receptor Higher levels in males than in No difference
(Gpr147) females
Stress axis Arcuate nucleus Corticotropin- qPCR No difference Higher levels in subordinates Faykoo-
releasing factor than in breeders Martinez
receptor et al.
2 (Crfr2) (2018)
Hippocampus Glucocorticoid No difference No difference between subordi-
receptor nates or breeders, but higher
(Nr3c1) levels in pubescent animals.
MeA Corticotropin- No difference Higher levels in subordinates
releasing factor than in breeders
receptor
The Curious Case of the Naked Mole-Rat: How Extreme Social and. . .
2 (Crfr2)
NAcc Corticotropin- No difference No difference between subordi-
releasing factor nates or breeders, but higher
receptor levels in pubescent animals
2 (Crfr2)
PVN Corticotropin- Marginally higher levels in Higher levels in breeding males
releasing factor breeding males relative to than in subordinates, no status
receptor breeding females, no sex differ- difference in females
2 (Crfr2) ence in subordinates
55
(continued)
Table 1 (continued)
56

Glucocorticoid No difference No difference between subordi-
receptor nates or breeders, but higher
(Nr3c1) levels in pubescent females
Whole brain, Corticotropin- Autoradiography Higher levels in females No difference Beery
amygdala releasing factor et al.
receptor 2 (2016)
Whole brain, Corticotropin- No difference Higher levels in subordinates
amygdala, releasing factor
piriform cortex receptor 1
P. D. Edwards et al.
Fig. 2 Examples of neurobiological status differences and lack of sex differences in naked mole-
rats. (a) Photomicrograph of a Kluver-Barrera-stained portion of Onuf’s nucleus. Motoneurons are
marked by the white arrow, and “small cells” are marked by the black arrow. Scale bar ¼ 50 μm. (b)
Mean ( SEM) counts of motoneurons in Onuf’s nucleus by sex and status. Breeders and sub-
ordinates differed in cell number but had no detectable sex differences. (c) Photomicrograph of a
thionin-stained coronal section of the PVN (d) Mean ( SEM) regional volume of the PVN by sex
and status, with males as black bars and females as white bars. Breeders and subordinates differed in
volume while no significant sex differences or sex and status interactions were detected. Modified
from Seney et al. (2006) and Holmes et al. (2007)
Lack of sex differences in volume and cell number also extend to other parts of
the nervous system. In the mammalian spinal cord, the population of motoneurons
that innervate the muscles of the phallus is called Onuf’s nucleus (ON motoneurons).
The homologous regions in rats and mice are referred to as the spinal nucleus of the
bulbocavernosus and the dorsolateral nucleus. Because this area is associated with
the muscles of the phallus, it displays sex differences in a wide variety of mammals
including rats (Breedlove and Arnold 1980), humans (Forger and Breedlove 1986),
dogs (Forger and Breedlove 1986), Mongolian gerbils (Meriones unguiculatus;
Ulibarri et al. 1995), and Asian musk shrews (Suncus murinus; Polak and Freeman
2010). Yet, in naked mole-rats, there is no sex difference in number or size of ON
motoneurons (Fig. 2; Peroulakis et al. 2002; Seney et al. 2006). This is true for both
subordinates and breeders, however breeders have more large ON motoneurons than
subordinates, regardless of sex (Seney et al. 2006). This again emphasizes the
predominance of status differences, rather than sex differences, in this species.
3 Brain Gene and Receptor Expression: Status Differences

and Sex Differences
Though naked mole-rats, as of yet, have no identified sex differences in brain

morphology, they display some sex differences, as well as status differences, in
gene and receptor expression in the brain (Table 1; Fig. 3). Brain steroid hormone
receptor expression and circulating steroid hormone levels do show some sex
differences in this species. Although subordinates do not have sex differences in
progesterone or testosterone, breeding females have elevated progesterone levels
relative to males and subordinate females, and breeding males have elevated testos-
terone levels relative to females and subordinate males (Clarke and Faulkes 1997,
1998; Zhou et al. 2013; Swift-Gallant et al. 2015; Faykoo-Martinez et al. 2018).
Thus, sex differences in progesterone and testosterone levels are latent until adult
subordinates undergo reproductive maturation, whereafter sex differences in gonadal
steroid hormones become pronounced. Estradiol is more variable as subordinate
females can have high estradiol levels, comparable to non-pregnant breeding
females (Zhou et al. 2013). However, estradiol levels are elevated in breeding
females when they initially become reproductively active (4 weeks after separation
from the natal colony) and during pregnancy (Swift-Gallant et al. 2015; Edwards
et al. 2021). The estradiol levels in subordinates are positively associated with queen
levels and increase during her pregnancy. This is possibly by social transmission of
queen hormones or due to reduced queen aggression during pregnancy which may
allow subordinates to begin to sexually mature during that time (Watarai et al. 2018;
Edwards et al. 2021). Breeding males and gonadectomized animals of both sexes can
also have high and variable estradiol levels, potentially adrenal in origin (Zhou et al.
2013; Swift-Gallant et al. 2015).
The sex differences in neural androgen receptor levels in naked mole-rats are
generally consistent with expectations based on other species, with males having
higher levels than females. Quantification of brain androgen receptors with immu-
nohistochemistry has demonstrated that male naked mole-rats have more androgen
receptors than females in three particular regions of the brain: the MeA, VMH, and
premammillary nucleus (Holmes et al. 2008). Though this sex effect appeared more
pronounced in breeders, no significant sex by reproductive status interaction effect
was detected (Holmes et al. 2008). When androgen receptor (Ar) mRNA expression
is quantified from the right hemisphere of the diencephalon (pooled tissue), again
male naked mole-rats are found to have higher androgen receptor expression than
females (Swift-Gallant et al. 2015). Status differences in androgen receptors are also
abundant. Regardless of sex, subordinates surprisingly have higher levels of andro-
gen receptor protein expression in the BST, PVN, VMH, and MeA relative to
Fig. 3 Heatmap of mean gene expression (mRNA) in naked mole-rat brain regions, arranged using
hierarchical clustering separated by sex. Each row represents a single gene in a specific brain region.
Details can be found in source publication (Faykoo-Martinez et al. 2018). Subordinates (SUB) of
each sex are on the left and breeders (BRE) of each sex are on the right. Means were adjusted to
z-scores with blue indicating low relative expression and red indicating high relative expression.
Modified from Faykoo-Martinez et al. (2018)
breeders (Holmes et al. 2008). However, subsequent gene expression results from
the whole right hemisphere of the diencephalon displayed the opposite effect, with
higher androgen receptor mRNA expression in breeding males than subordinate
males, and no difference in androgen receptor expression between female breeders
and subordinates (Swift-Gallant et al. 2015). This indicates that the status differences
in androgen receptor expression may be region-specific and/or depend on
methodology.
As for receptors and genes related to the female-biased sex hormones, estrogen
receptor alpha (Esr1) and aromatase (Cyp19a1) are expressed at higher levels in the
diencephalon in female naked mole-rats relative to males. There is a status difference
in females for both genes, with higher levels of expression in breeding females
relative to subordinate females (Swift-Gallant et al. 2015). Hence, even though
subordinate females and males can have relatively high estradiol levels, they may
not be as responsive to circulating estrogens as are the breeding females. Female and
male naked mole-rats both also have status differences in progesterone receptor
(Pgr) expression, with breeding females having higher progesterone receptor mRNA
levels than subordinate females, and breeding males having lower progesterone
receptor mRNA levels than subordinate males (Swift-Gallant et al. 2015).
Additionally, several genes related to reproductive axis function and the onset of
reproductive competence have higher levels of expression in male naked mole-rats
relative to females. In the nucleus accumbens, males have higher expression of
kisspeptin (Kiss1), neuropeptide VF precursor (Npvf), and tachykinin 3 receptor
(Tac3r), and in the PVN, males have higher expression of RFRP receptor (Gpr147)
and kappa opioid receptor (Kor) (Faykoo-Martinez et al. 2018). Though no main
effect of status was detected on any of these reproductive genes, subordinate males
in particular had the highest levels of Npvf, Tac3r, and Gpr147 in the nucleus
accumbens. These sex differences in neural gene expression may be due to sex
differences in mechanisms and degrees of reproductive suppression in naked
mole-rats. Reproductive suppression is less pronounced in subordinate males than
subordinate females, as the majority of subordinate male naked mole-rats produce
spermatozoa, whereas subordinate females lack mature gametes and do not ovulate
(Faulkes et al. 1991; Coen et al. 2021; Faykoo-Martinez et al. 2021).
Other sex and status differences in naked mole-rat neural gene expression are in
hypothalamic-pituitary-adrenal axis (HPA axis; “stress axis”) related genes and
receptors. The stress axis has pronounced sex differences in other rodent species,
at multiple levels of regulation. In laboratory rats, females have higher circulating
glucocorticoid levels than males and it takes longer for their glucocorticoid levels to
return to baseline following an acute stressor (Oyola and Handa 2017; Heck and
Handa 2019). This difference in stress axis regulation may be partially driven by sex
differences in glucocorticoid receptors in the brain, which are involved in negative
feedback of the stress axis. Female rats have less glucocorticoid receptors in the
hypothalamus and pituitary than males, though there are likely additional differences
in stress axis regulation beyond glucocorticoid receptor expression (Heck and Handa
2019). Further, corticotropin-releasing factor (CRF), which is synthesized in the
PVN and initiates stress axis activity, is expressed at higher levels in female rats
relative to male rats, and CRF expression is stimulated by estrogens and suppressed
by testosterone (Heck and Handa 2019). CRF and other CRF family ligands bind to
two receptor types in the brain: CRFR1 and CRFR2. In general, CRFR1 activation is
thought to stimulate the stress response, whereas CRFR2 activation dampens it (Bale
and Vale 2004). In several species of voles in genus Microtus, CRFR2 density is
higher in the BST of male voles than females voles, and estradiol treatment is known
to alter CRFR2 density in meadow voles (Beery et al. 2014, 2016). Male meadow
voles additionally tend to have lower corticosterone levels than females, particularly
in breeding individuals (Galea and McEwen 1999; Edwards et al. 2019).
In naked mole-rats, however, these sex differences in the stress axis are largely
absent or reversed. Work comparing blood cortisol levels (the primary
glucocorticoid in naked mole-rats; Ganem and Bennett 2004) in male and female
naked mole-rats, collected during sacrifice, has shown that males have marginally
higher circulating cortisol levels than females, regardless of status, though this
difference appeared to be primarily driven by higher levels in reproductive males
(Faykoo-Martinez et al. 2018). Additional research examining urinary or fecal
cortisol metabolites, as a measure of integrated baseline cortisol levels, has generally

demonstrated no sex or status differences in cortisol levels in naked mole-rats
(Clarke and Faulkes 1998, 2001; Edwards et al. 2020; c.f. Faulkes and Abbott
1997 – lower urinary cortisol in non-pregnant breeding females; Clarke and Faulkes
1997 – higher urinary cortisol levels in high ranking individuals in some, but not all,
colonies). Although sex differences in CRF have not been quantified in naked mole-
rats, this species has no sex differences in CRFR1 levels, neither globally nor in
specific brain regions (Beery et al. 2016; Faykoo-Martinez et al. 2018). Female
naked mole-rats have higher CRFR2 density than males globally and in the amygdala
in particular, regardless of status, as demonstrated by receptor autoradiography
(Beery et al. 2016). Subsequent work quantifying mRNA levels found no detectable
sex difference in CRFR2 expression, aside from a marginal increase in levels
between breeding males relative to breeding females (but not subordinate males
relative to subordinate females) in the PVN.
A few status differences in stress axis related genes in the brain have been found
in naked mole-rats. Subordinates have higher CRFR1 density than breeders, globally
and in the piriform cortex and amygdala in particular (Beery et al. 2016), though this
status difference was not found in subsequent CRFR1 mRNA expression data from
distinct brain regions. The mRNA expression data additionally indicates that
breeders have higher CRFR2 expression levels than subordinates in the MeA and
arcuate nucleus, regardless of sex. No glucocorticoid receptor (Nr3c1) expression
differences between breeders and subordinates were found for any region (Faykoo-
Martinez et al. 2018).
Finally, status but not sex is associated with number of oxytocinergic neurons in
the PVN with subordinates having more than breeders (Mooney and Holmes 2013).
There is a sex difference in oxytocin receptor density in naked mole-rats, with males
having higher levels in the MeA than females. This difference appears to be driven
primarily by breeders, particularly by low oxytocin receptor levels in breeding
females, though no significant main effect of status was detected in the analysis
(Mooney et al. 2015b). No other sex differences in oxytocin receptor density were
detected in any other region (Mooney et al. 2015b). Male-biased oxytocin receptor
expression in naked mole-rats is consistent with sex differences found in some other
rodent species, though less pronounced. Male singing mice (Scotinomys
xerampelinus) and mandarin voles (Lasiopodomys mandarinus) both have higher
levels of oxytocin receptor in the MeA than females of their respective species
(Campbell et al. 2009; Smorkatcheva 2011). Male rats have higher densities of
oxytocin receptors than females in several brain regions, including the MeA
(Dumais et al. 2013). This male bias in rats was hypothesized to be due to a sex
difference in social interest, with virgin males having higher levels of social inves-
tigation of conspecifics than virgin females (Dumais et al. 2013). This explanation
perhaps does not apply to naked mole-rats, as there are minimal sex differences in
social behavior, aside from the queen being more aggressive than other colony
members. As such, sex differences in oxytocin receptor levels in the naked mole-
rat may be related to a compensation effect (De Vries 2004) where increased
oxytocin receptor levels are needed to promote baseline prosocial behavior in males.
4 Damaraland Mole-Rats as a Comparison
Naked mole-rats can be compared with another cooperatively breeding mole-rat, the
Damaraland mole-rat (Fukomys damarensis). Damaraland mole-rats are also in
family Bathyergidae, and similarly have a single breeding pair per colony supported
by reproductively suppressed subordinates. Evidence suggests that eusociality
evolved independently in naked mole-rats and Damaraland mole-rats, making
them an interesting contrast to examine convergent characteristics of social structure
(Allard and Honeycutt 1992; Jarvis and Bennett 1993; Faulkes and Bennet 2021).
Damaraland mole-rats appear to be an intermediate between the extreme cooperative
lifestyle of naked mole-rats and the solitary lifestyle of some other mole-rat species.
The reproductive skew is lower in Damaraland than in naked mole-rats; Damaraland
mole-rat colonies contain an average of 16 animals (maximum 41), as opposed to
naked mole-rat colonies, which can have up to 300 individuals (Jarvis and Bennett
1993). It is estimated that 90% of Damaraland mole-rats will remain
non-reproductive throughout their lives, whereas over 99% of naked mole-rats will
remain non-reproductive (Jarvis and Bennett 1993; Jarvis et al. 1994).
The extent of the physiological reproductive suppression in Damaraland mole-
rats is not quite as extreme as in naked mole-rats. In females, subordinate
Damaraland mole-rats have greater follicular development in their ovaries than
naked mole-rats. In males, subordinate Damaraland mole-rats have circulating
testosterone levels comparable to breeding Damaraland mole-rats, whereas subordi-
nate naked mole-rats tend to have much lower testosterone levels than breeding male
naked mole-rats (Jarvis and Bennett 1993). Damaraland mole-rats additionally have
more visible sexual dimorphism than naked mole-rats. Whereas subordinate male
and female naked mole-rats have virtually no differences in their external genitalia,
both subordinate and breeding Damaraland mole-rats do have sex differences
(longer anogenital distance and phallus length in males), though these differences
are less pronounced than in solitary silvery mole-rats (Heliophobius
argenteocinereus) (Seney et al. 2009).
Like naked mole-rats, Damaraland mole-rats do not have sex differences in
volume in the BST and PVN, but do have status differences in these regions, with
breeders having larger regional volumes. Further, breeders have larger cells in the
PVN and MeA, an additional status difference that has not been detected in naked
mole-rats (Anyan et al. 2011). These differences reinforce the importance of repro-
ductive status over sex in cooperative breeders. However, Damaraland mole-rats
also have sex differences that were not detected in naked mole-rats: males have a
larger MeA volume and more ON motoneurons than females. While the sex differ-
ences in MeA volume occurred regardless of breeding status, the difference in
number of ON motoneurons appeared to be primarily driven by breeding males,
with a marginal sex and status interaction effect (Anyan et al. 2011). This sex
difference in ON motoneurons in Damaraland mole-rats is in contrast with naked
mole-rats that have no sex differences in this measure, but is consistent with a variety
of other mammals where males have more ON motoneurons (Peroulakis et al. 2002;
Seney et al. 2006).
5 Reduced Sex Differences: Why and How?
The reduction of sex differences in naked mole-rats, relative to other rodent species,
raises two major questions. The first is why naked mole-rats mostly lack sexual
dimorphism in the brain. If we assume that sex differences in the brain are selected
for to drive sex differences in reproduction, then the reduction in naked mole-rats
would be associated with a lack of differences in reproductive behavior in sub-
ordinates of this species. Only after sexual maturation into a breeder do the female
and male reproductive and social roles diverge. Supporting this are the observations
of more pronounced reproductive status differences, not sex differences, in this
species. The only major sex differences in subordinate naked mole-rats brains,
discovered to date, are related to genes and receptors involved with puberty and
the sex hormones, though there may be other sex differences in gene expression as of
yet unstudied.
The second question is how, mechanistically, this lack of sexual dimorphism
occurs in naked mole-rats. Many sex differences in mammals are known to be driven
by androgen action during development, as opposed to genetic sex alone (Cooke
et al. 1999; Forger et al. 2004). Androgens can act on tissues by interaction with
androgen receptors, and by aromatization to estradiol and interaction with estrogen
receptors, with the evidence of the latter being more important in inducing sex
differences in the brain during development (McCarthy 2008). Sex differences in
the BST and PVN in laboratory rodents can be reversed by manipulating androgen
levels in the perinatal period (Forger et al. 2004). The same principle applies to the
MeA in rats, where castrated males had posterodorsal regions of the MeA that were
similar to females in volume (Cooke et al. 1999). This is also true of ON motoneu-
rons: male rats with androgen insensitivity lack a developed spinal nucleus of the
bulbocavernosus (ON homologous region) and in castrated Mongolian gerbils, size
of the motoneurons was decreased (Breedlove and Arnold 1980; Ulibarri et al.
1995). Similarly, prenatally treating female dogs with androgens increased the
number of ON motoneurons to the same amount as in males (Forger and Breedlove
1986).
Thus, Seney et al. (2009) proposed that naked mole-rats either have minimal sex
differences in androgen exposure during development, or certain tissues are
androgen-insensitive during development. Female naked mole-rats are not “mascu-
linized” like female hyenas, which have masculinized external genitalia
(pseudopenis); rather, Seney et al. (2009) suggested that it may be more appropriate
to consider male naked mole-rats as “feminized.” Similarly, Peroulakis et al. (2002)
noted that while naked mole-rats have sexually differentiated internal sex organs,
they do not have sexually differentiated external genitalia, perineal muscles, and
motoneurons, which is consistent with differential androgen exposure earlier in
gestation, when internal genitalia differentiate, but not later in gestation or early
postnatal life when these other sex differences occur in rodents. Hence, timing of
differential androgen exposure during sensitive periods could drive these lack of
differences in male and female naked mole-rats.
While prenatal and early life androgens have been heavily studied in traditional
laboratory rodents for their role in sexual differentiation, subsequent work has
revealed other mechanisms that are important agents driving sex differences in the
brain. For example, estrogen receptor activation in the brain, by testicular androgens
aromatized into estradiol, triggers expression of the cyclooxygenase enzymes, which
markedly elevate levels of prostaglandin E2. Prostaglandin E2, in turn, induces
maturation of dendritic spines, thus altering brain morphology (McCarthy et al.
2009). Disrupting any step in this chain can alter sexual differentiation of the
brain: blocking cyclooxygenase enzyme action leads to a female-like level of
dendritic spines, and treatment with prostaglandin E2 leads to a male-like level of
dendritic spines (McCarthy et al. 2009). Hence, there are potentially many places
where natural selection could act to disrupt the development of neural sex differ-
ences in the brain of naked mole-rats.
Although naked mole-rats show few sex differences as subordinates, a number of
status differences in sexually-relevant brain regions emerge following reproductive
activation. These differences are likely induced by hormone and gene expression
changes occurring during naked mole-rat “puberty,” as individuals ascend to breed-
ing status. Indeed, naked mole-rats that are removed from the colony and paired with
an opposite sex conspecific – thus becoming pubescent but not yet established
breeders – display a suite of hormone and gene expression changes. After 4 weeks
of separation from the colony and pairing with an opposite sex conspecific, females
and males have elevated estradiol and testosterone levels, respectively, relative to
subordinates (Swift-Gallant et al. 2015; Faykoo-Martinez et al. 2018). The pubes-
cent individuals additionally have altered expression of reproductive and stress
related genes in the brain, in a unique signature that is distinct from both sub-
ordinates and breeders (Faykoo-Martinez et al. 2018). These changes during puberty
likely drive many of the neurobiological differences observed between subordinate
and breeder naked mole-rats. Additionally, gonadectomy of breeders does not
reverse the differences in volume that develop in the PVN and BST in reproductively
mature animals (Holmes et al. 2011), supporting the idea that irreversible changes in
brain organization occur as naked mole-rats transition from subordinate to breeding
status.
6 Conclusion
Our understanding of sex differences is inherently based on the species that have
been studied in this respect. The majority of work to date focuses on laboratory rats
and mice, which have different social and reproductive strategies between the sexes.
This, in turn, presumably relates to the many observed neurobiological sex differ-
ences. However, investigation of diverse species indicates that the sex differences
originally found in rats and mice cannot necessarily be generalized across species,
especially to species lacking an analogous social structure. Naked mole-rats dem-
onstrate that when considering the evolution of neurobiological sex differences, the
social organization of the species is critical in predicting and understanding the sex
differences or lack thereof that may be observed.
Beyond research in social neurobiology, naked mole-rats are an exciting study
system based on other aspects of their unique biology. They are very long-lived for a
small mammal, and can live for over 30 years in captivity, with the oldest known
naked mole-rat being 37 (Buffenstein and Craft 2021). They show resistance to
many aspects of senescence, and there are no age effects on death rate, incidences of
disease (including cancer), metabolic rate, bone integrity, or vascular function, until
they approach their maximum lifespan where their condition finally deteriorates
(Buffenstein 2008). Breeding females do not decrease reproduction with age and can
breed until they die, and old males in their late 20s have been documented siring
litters (Buffenstein 2005, 2008). The lack of age-related mortality risk, or “non-
increasing mortality hazard,” is present regardless of sex or status, though breeders
have better survival rates than subordinates generally speaking (Ruby et al. 2018).
Thus, in addition to foundational questions about sexual differentiation of the
nervous system and behavior, naked mole-rats will also be an informative model
for understanding aspects of aging and pathologies that show sex biases in humans
and other species.
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Sex/Gender Differences in Brain
Lateralisation and Connectivity
Sophie Hodgetts and Markus Hausmann
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
2 Sex/Gender Differences in Lateralisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
2.1 Sex/Gender Differences in Structural Cerebral Asymmetries . . . . . . . . . . . . . . . . . . . . . . . . . 73
3 Sex/Gender Differences in Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.1 Structural Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.2 Functional Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.3 Sex/Gender Differences in Functional Cerebral Asymmetries . . . . . . . . . . . . . . . . . . . . . . . . 80
3.4 Hormonal Influences on Lateralisation and Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.5 The Case for a Biopsychosocial Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Abstract There is now a significant body of literature concerning sex/gender

differences in the human brain. This chapter will critically review and synthesise
key findings from several studies that have investigated sex/gender differences in
structural and functional lateralisation and connectivity. We argue that while small,
relative sex/gender differences reliably exist in lateralisation and connectivity, there
is considerable overlap between the sexes. Some inconsistencies exist, however, and
this is likely due to considerable variability in the methodologies, tasks, measures,
and sample compositions between studies. Moreover, research to date is limited in
its consideration of sex/gender-related factors, such as sex hormones and gender
roles, that can explain inter-and inter-individual differences in brain and behaviour
better than sex/gender alone. We conclude that conceptualising the brain as ‘sexually
dimorphic’ is incorrect, and the terms ‘male brain’ and ‘female brain’ should be
avoided in the neuroscientific literature. However, this does not necessarily mean
S. Hodgetts
School of Psychology, University of Sunderland, Sunderland, UK
e-mail: sophie.hodgetts@sunderland.ac.uk
M. Hausmann (*)
Department of Psychology, Durham University, Durham, UK
e-mail: markus.hausmann@durham.ac.uk

https://doi.org/10.1007/7854_2022_303
72 S. Hodgetts and M. Hausmann
that sex/gender differences in the brain are trivial. Future research involving
sex/gender should adopt a biopsychosocial approach whenever possible, to ensure
that non-binary psychological, biological, and environmental/social factors related
to sex/gender, and their interactions, are routinely accounted for.
Keywords Biopsychosocial approach · Brain connectivity · Commissures ·

Default-mode network · Diffusion tensor imaging · Functional cerebral
asymmetries · Interhemispheric interaction · Resting-state network · Sex hormones
1 Introduction
Recent decades have seen a substantial increase in the number of studies investigat-
ing sex/gender differences in the brain. These studies have generally supported the
notion that sex/gender differences in the brain exist at multiple levels, including
structural and functional lateralisation and connectivity (e.g., Cahill 2017; Jäncke
2018; Hodgetts and Hausmann 2020b). Historically, many cognitive neuroscientists
have conceptualised sex/gender differences in brain structure and function as large
and ‘hard-wired’. Consequently, explanations for such differences typically
emphasised early genetic factors and prenatal hormonal influences (Jäncke 2018).
Recent evidence has prompted a shift away from such ideas, and a growing number
of neuroscientists now agree that while sex/gender differences in the brain do exist
they are small in size, and thus it cannot be argued that the brain is ‘sexually
dimorphic’ (Joel et al. 2015; Hirnstein and Hausmann 2021; Hodgetts and
Hausmann 2020b; Eliot et al. 2021). Moreover, findings from several recent studies
support taking a biopsychosocial approach to the study of sex/gender differences in
the brain, such that both the influence of biological and environmental factors and
their complex interaction(s) are investigated (Halpern 2013; Cahill 2017; Jäncke
2018; Hausmann 2020; Hodgetts and Hausmann 2020b; Joel 2021).
This chapter will review the findings from current research into sex/gender
differences in brain lateralisation and connectivity. In this chapter, we will argue
that some sex/gender differences reliably exist in both lateralisation and connectivity
and that they are not trivial, while at the same time they are not ‘sexual dimorphisms’
(see also Galea 2021; Hirnstein and Hausmann 2021). We will argue that some
sex/gender differences exist regarding both brain lateralisation and connectivity, but
that there is a high level of inter-and intra-individual variability resulting in a
considerable amount of overlap between the sexes at population level. We will
also argue that future research should adopt a biopsychosocial approach (Hausmann
2020), to ensure that environmental factors and intra-individual variation related to
sex/gender are considered more routinely.
The term ‘sex’ is typically used when referring to the biological classification of
an individual as male or female, based on their reproductive organs (e.g., genitalia)
while ‘gender’ refers to the psychosocial constructs typically associated with sex
Sex/Gender Differences in Brain Lateralisation and Connectivity 73
(e.g., masculinity, femininity). However, the two terms are often misleadingly
conflated in previous research, and it is not always clear whether a given study is
investigating sex, gender, or both. Therefore, to account for this inconsistency, this
chapter will use sex/gender, rather than ‘sex’ or ‘gender’ separately. It should also be
noted that we are discussing data from averages of groups, not individuals. This
chapter will also focus primarily on large-scale studies and meta-analyses, meaning
several, high-qualities studies will not be reviewed in detail here. Finally, we
explicitly avoid the term ‘sexual dimorphism’ as this term implies two distinct brains
in a binary sense: one male and one female, as all empirical evidence suggests that
such dual categorisation is incorrect in the context of the brain.
2 Sex/Gender Differences in Lateralisation
Sex/gender has been extensively investigated concerning cerebral lateralisation, a

fundamental principle of functional brain organisation referring to the asymmetrical
representation of a specific cognitive process to either the left or right cerebral
hemisphere. For example, in the healthy adult human brain, the left hemisphere is
typically dominant for some language processes (Broca 1861; Kimura 1967), while
the right hemisphere is dominant for some visuospatial processes (Hellige 1993;
Hugdahl and Westerhausen 2013). Cerebral lateralisation can be assessed at both the
structural level (i.e. asymmetry with regard to size/volume/shape of specific brain
areas) and at the functional level (see Sect. 3.3).
2.1 Sex/Gender Differences in Structural Cerebral

Asymmetries
Given that a body of literature has supported the existence of sex/gender differences
in specific language processes, research into sex/gender differences in structural
cerebral asymmetries has focused on cortical areas known to be involved in language
processes such as the planum temporale, a region that overlaps with Wernicke’s area
and is involved in language comprehension (Shapleske et al. 1999).
An early study with only 24 healthy participants revealed that the left planum
temporale is bigger in men compared to women (Kulynych et al. 1994). Moreover,
men showed an asymmetry (i.e., larger left PT compared to right), but women did
not. A recent study of three large magnetic resonance imaging (MRI) datasets
(n ¼ 2,337, 935, and 888) found a stronger leftward asymmetry in the planum
temporale for men compared to women (Guadalupe et al. 2015). However, this
finding is inconsistent. For example, a meta-analysis of 13 studies (n ¼ 807) inves-
tigating structural asymmetry of the PT revealed no sex/gender difference (Sommer
et al. 2008). In addition, two reviews of clinical data suggest that aphasia following
left-hemispheric damage is not associated with sex/gender (Plowman et al. 2012;

Watila and Balarabe 2015). Taken together, these findings suggest that (1) a
sex/gender difference in the structure of the planum temporale might exist, (2) the
effect size is small, and (3) a significant difference will only be detected with larger
samples.
3 Sex/Gender Differences in Connectivity
3.1 Structural Connectivity
One of the most consistently reported sex/gender differences in brain macrostructure

is brain size and volume. These studies suggest that the average male brain is larger
and heavier than the average female brain (Peters 1991), even if sex/gender differ-
ences in body size are controlled for (Ruigrok et al. 2014). However, several authors
have argued that this finding is a false positive that results from the specific analysis
method and the specific method by which sex/gender differences in body size are
controlled (Schluter 1992; Forstmeier 2011). For example, a recent study Sanchis-
Segura et al. (2019) demonstrated that sex/gender differences in regional grey matter
volumes were significantly reduced when the total intracranial volume was con-
trolled for. As such, the authors argued that male–female differences in brain size are
better conceptualised as size differences resulting from variation in total intracranial
volume as opposed to sex/gender effects per se. The authors further argued that not
all methods of controlling for total intracranial volume are equally valid/reliable, and
some methods may produce misleading results.
Further MRI studies have demonstrated sex/gender differences in measures that
are associated with within- and between-hemisphere structural connectivity, such as
the relative composition of grey and white matter (GM, WM) volumes. Such studies
have shown that WM volume is generally larger across the cerebrum in men
compared to women (Allen et al. 2003; Filipek et al. 1994; Gur et al. 1999; Lentini
et al. 2013; Passe et al. 1997). However, inconsistencies exist in the literature when
several potentially confounding factors are considered. For example, when
sex/gender differences in cranial size are controlled for, women typically yield a
higher volume of GM compared to men (Gur et al. 1999; Goldstein 2001), while
sex/gender differences in the relative proportions of GM and WM were considerably
reduced when differences in brain size are controlled (Lüders et al. 2002; Leonard
et al. 2008; Jäncke et al. 2015). Furthermore, Allen et al. (2003) reported that women
had a higher GM:WM ratio consistently across all brain structures and lobes
analysed. This study with a relatively small sample size (n ¼ 36) also showed that
the effect of sex/gender was greater on WM than GM; that is, the higher GM:WM
ratios in women were a result of reduced WM in women compared to men. More
recently, a large MRI study (n ¼ 900, van der Linden et al. 2017) also reported that
women yielded a higher GM:WM ratio throughout the cortex compared to men,
even after controlling for sex/gender differences in body size. However, it should be
noted that van der Linden et al. (2017) used body height to control for body size
differences, a measure previously suggested to lead to an overestimation of
sex/gender differences in brain size (Jäncke et al. 2015). It is also likely that age is
a relevant factor to consider regarding sex/gender differences in GM:WM, as several
studies have shown that WM volume typically decreases during the perimenopause
phase and throughout menopause itself (Brinton 2016; Rahman et al. 2020). Never-
theless, sex/gender differences in GM:WM proportions have been reported in two
recent studies with large sample sizes (n ¼ 1,400, Joel et al. 2015; n ¼ 2,838, Lotze
et al. 2019). Together, these findings suggest that while brain/body size may explain
some of the variance in GM:WM ratio, it is likely that sex/gender effects are also
present.
The size and/or shape of the corpus callosum is one of the most frequently cited
examples of sex/gender differences in structural connectivity (Eliot et al. 2021).
Several early studies reported that the posterior subsections of the corpus callosum
were larger in women compared to men, including the splenium (e.g., Allen et al.
1991; DeLacoste-Utamsing and Holloway 1982), posterior midbody (Habib et al.
1991), and the isthmus (Witelson 1989). Such findings are inconsistent, with some
studies demonstrating larger corpus callosum sections in men compared to women
(e.g., Allen et al. 2002; Westerhausen et al. 2011), or no sex/gender differences
(Oppenheim et al. 1987). Moreover, evidence from meta-analyses of such studies
has demonstrated that larger callosal indices in women become evident only when
sex/gender differences in brain size are controlled for (Bishop and Wahlsten 1997;
Smith 2005; see also Cahill 2017). Consequently, more recent studies have inves-
tigated sex/gender differences in the corpus callosum while statistically controlling
for variation in brain size and/or by matching male and female samples for brain size.
Using both methods, a larger study (n ¼ 316) by Ardekani et al. (2013) found that
the whole corpus callosum was larger in women compared to men (see also, Shiino
et al. 2017). In contrast, Luders et al. (2014) found in a smaller sample (n ¼ 96) that
sex/gender differences in callosal thickness were reduced when male/female samples
were matched for intracranial volume. Another possible explanation for these
inconsistencies is the small sample sizes used in most individual studies. Indeed,
in a recent review, Eliot et al. (2021) argued that since most studies on sex/gender
differences in the corpus callosum included <100 participants they are likely
underpowered and not able to detect the estimated effect size (d ¼ 0.22, as reported
in an earlier meta-analysis by Smith 2005).
Aside from the corpus callosum, two other interhemispheric connections have
been shown to differ in size between men and women: the anterior commissure and
interthalamic adhesion, also known as massa intermedia (Eliot et al. 2021). To date,
findings regarding the anterior commissure are inconsistent with some evidence
suggesting it is bigger in men (Demeter et al. 1988), some reporting a larger anterior
commissure in women (Allen and Gorski 1991), while others showed no sex/gender
difference (Lasco et al. 2002). More recently, an MRI study by Choi et al. (2011)
included correction for sex/gender differences in intracranial volume (ICV) and
reported larger anterior commissure volumes in middle-aged (but not young adult)
women. Findings regarding a sex/gender difference in the size of the interthalamic
adhesion are more reliable with studies typically showing it is larger in women
compared to men (Allen and Gorski 1991; Damle et al. 2017). However, it should be
noted that the number of studies of this structure is limited. For example, it is unclear
to what extent the size of the interthalamic adhesion is sensitive to brain size, and
more importantly, its functional relevance is largely unknown (Damle et al. 2017).
More recent studies have used diffusion tensor imaging (DTI) to investigate
sex/gender differences in microstructural connectivity. Studies of microstructure
typically report measures of fractional anisotropy (FA) and mean diffusivity
(MD) as indices of white matter (WM) fibre organisation and structural integrity,
respectively. Several studies investigated sex/gender differences in WM microstruc-
ture of the corpus callosum, which allows for inferences to be made regarding
structural interhemispheric connectivity (Westerhausen et al. 2003, 2011; Hsu
et al. 2008; Schmithorst et al. 2008; Kanaan et al. 2012). For example, Westerhausen
et al. (2003) found higher FA throughout the corpus callosum in men compared to
women, possibly reflective of thicker myelination and/or less inter-fibre space in the
average male corpus callosum. In a further study, Westerhausen et al. (2011)
reported greater FA and lower MD in the anterior genu subregion of the corpus
callosum in men. Given that this subregion facilitates interhemispheric connectivity
between the two frontal lobes, these results were interpreted as evidence for stronger,
more efficient callosal-frontal connectivity in men. While both studies are limited by
relatively small samples, Westerhausen et al. (2011) concluded that this may be
related to sex/gender differences in lateralisation (see Sect. 3.3 of this chapter). An
alternative interpretation of these findings is that the stronger callosal connectivity in
males may be due to the greater distance between the hemispheres, resulting from
their larger overall brain size. Other studies reported inconsistent results, with some
demonstrating higher FA in the corpus callosum in women compared to men
(Schmithorst et al. 2008; Kanaan et al. 2012), while others suggested there are no
sex/gender differences in global FA and MD (Eluvathingal et al. 2007; Hsu et al.
2008; Clayden et al. 2012). Moreover, evidence suggests that sex/gender differences
in WM microstructure become non-significant after controlling for differences in
intracranial volume (Takao et al. 2014). Overall, there is an on-going debate
regarding whether sex/gender differences in the macro-and microanatomy of the
corpus callosum truly exist, as well as whether sex/gender differences in WM exist in
the brain globally.
Further DTI studies have investigated sex/gender differences in the structural
connectivity via whole-brain analysis (Iturria-Medina et al. 2007, 2008; Tian et al.
2011; Ingalhalikar et al. 2014). For example, across two smaller sample studies
(n ¼ 5 and 20, respectively), Iturria-Medina et al. (2007, 2008) reported no
sex/gender differences in the small-world attributes (high clustering of network
nodes, short paths between nodes) of the whole brain. In contrast, Yan et al.
(2011) found that women exhibited greater clustering within several specific brain
regions, such as the precuneus, precentral gyrus, lingual gyrus, and the calcarine
fissure, suggesting sex/gender differences in local network efficiency. Ingalhalikar
et al. (2014) investigated sex/gender differences in structural connectivity across the
whole brain in a large sample of healthy participants (n ¼ 949), including children
and young adults (8–22 years of age). Results showed that men exhibited greater
intrahemispheric structural connectivity compared to women, particularly between
the frontal, temporal, and parietal lobes. In contrast, women exhibited greater
interhemispheric structural connectivity. Such differences in structural connectivity
were not seen in similar studies (Duarte-Carvajalino et al. 2012; Dennis et al. 2013).
Ingalhalikar et al. (2014) argued that the inconsistency between their results and
previous studies may be due to small effect sizes, meaning the effects will only be
detected by larger samples. However, Hänggi et al. (2014) revealed similar differ-
ences in inter-and intra-hemispheric connectivity using a smaller sample (n ¼ 138),
suggesting that the inconsistent results are not entirely due to small effect sizes and
underpowered studies. Critically, Hänggi et al. (2014) demonstrated that differences
in inter-and intra-hemispheric connectivity were driven by differences in brain size,
not sex/gender per se. That is, larger brains were more often associated with more
intrahemispheric connectivity and smaller brains were more often associated with
more interhemispheric connectivity, even when the sample was pooled according to
sex/gender. It should also be noted that the study reported by Ingalhalikar et al.
(2014) has been subject to multiple criticisms concerning the authors’ methodology
and interpretations of their data (Joel and Tarrasch 2014). Regarding methodology,
this study did not control for brain size, a factor known to both vary according to
sex/gender and to influence structural connectivity. Moreover, as noted by Joel and
Tarrasch (2014), it is important to place the significant results in context as
Ingalhalikar et al. (2014) only found sex/gender differences in a small subsample
of the 9,000 connections assessed in their study. Moreover, Ingalhalikar et al. (2014)
argued that such sex/gender differences in structural connectivity suggested that
‘male brains are structured to facilitate connectivity between perception and coordi-
nated action, whereas female brains are designed to facilitate communication
between analytical and intuitive processing modes’. (p. 1), despite the study lacking
behavioural measures. In contrast, an earlier, larger study (from which Ingalhalikar
et al.’s participants were sampled) demonstrated that although sex/gender differ-
ences in social cognition and spatial processing were present, the effect sizes were
small (Gur et al. 2012), suggesting that the conclusions drawn in 2014 are highly
speculative at best.
A further study aimed to investigate whether structural sex/gender differences in
functionally defined cortical networks such as auditory, visual, and motor networks
are related to behavioural sex/gender differences (Tunç et al. 2016). In this study, a
large sample of healthy participants (n ¼ 900) underwent both DTI and
neurocognitive testing. The results demonstrated more structural connectivity in
men within the motor, sensory, and executive function networks, while women
exhibited greater connectivity within networks associated with memory, attention,
and social cognition. The results suggest that sex/gender differences in structural
connectivity can predict some sex/gender differences in cognition. A further recent
structural connectivity study with a sample of 312 males and 362 females, aged
9–22 years, suggested that ‘the degree to which a given participant’s cognitive
profile was “male” or “female” was significantly related to the masculinity or
femininity of their pattern of brain connectivity’ (Satterthwaite et al. 2015,
p. 2383). Although these studies indicated sex/gender differences in structural

connectivity, the overall picture is inconsistent because other studies with large
samples revealed no sex/gender differences (e.g., Nielsen et al. 2013) as well as
substantial variability and overlap both within and between men and women.
3.2 Functional Connectivity
Several studies have investigated sex/gender differences in functional connectivity

using functional MRI paradigms (fMRI), in which participants are scanned while
completing a specific cognitive task. Most studies using such paradigms to investi-
gate sex/gender differences have focused on differences in task-related activity in
specific cortical regions (for a review, see Eliot et al. 2021), while other studies
investigated sex/gender differences in task-related functional connectivity by exam-
ining the temporal correlation of activity between brain areas during a given task.
Other studies have focused on sex/gender differences in resting-state functional
connectivity, defined as the temporal correlation of activity between brain regions
generally in the absence of a specific cognitive task (Tomasi and Volkow 2012a;
Mao et al. 2017; Ritchie et al. 2018; Zhang et al. 2018; Weis et al. 2019; Wheelock
et al. 2019). Further resting-state fMRI studies have investigated sex/gender differ-
ences in the resting functional connectivity of several task-related cortical networks
in the absence of a specific task.
3.2.1 Task-Related Connectivity
Sex/gender differences in task-related functional connectivity have been investi-

gated using fMRI during sex/gender-sensitive visuospatial tasks, such as mental
rotation. These studies typically showed that sex/gender differences exist in the
networks that underpin mental rotation. For example, it was shown that accurate
mental rotation performance was associated with deactivation of the parieto-insular
vestibular cortex in men only (Butler et al. 2006), whereas accurate performance in
women was underpinned by functional connectivity between frontal and parietal
cortical regions (Thomsen et al. 2000; Weiss et al. 2003; Hugdahl et al. 2006),
though contradictions have also been reported (see Eliot et al. 2021, for a tabulated
review). In an earlier review, Cahill (2006) noted a common misconception regard-
ing sex/gender differences in brain and behaviour which assumes that no sex/gender
differences in (cognitive) behaviour imply no sex/gender differences in the under-
lying neural network. Several studies have shown that this is incorrect. For example,
Jordan et al. (2002) demonstrated significant sex/gender differences in mental
rotation-related brain activity, despite similar task performance between men and
women in the same study. Together, these findings indicate that the lack of a
sex/gender effect in task performance cannot necessarily be used to infer a lack of
sex/gender effects at the neural level (Cahill 2006).
Additional studies have investigated sex/gender differences in task-related func-

tional connectivity associated with the integration of cognitive and emotional pro-
cesses (Weissman-Fogel et al. 2010). These studies revealed sex/gender differences
in the underlying mechanisms associated with cognitive control and emotion (Butler
et al. 2007; Cahill 2003; Hamann and Canli 2004; Koch et al. 2007 for a review see
Cahill 2017). For example, in an fMRI study, Butler et al. (2007) demonstrated in a
small sample (n ¼ 32) that performing a cognitively demanding task was associated
with a suppression of activity in the ventral anterior cingulate cortex, a region
associated with affect regulation (Stevens et al. 2011) and emotion recognition
(Etkin et al. 2011) among other processes related to social cognition (Rigney et al.
2018), in women only. Butler et al. (2007) also reported negatively correlated
functional connectivity between the ventral and dorsal anterior cingulate cortices
in women only. Consequently, and similar to Cahill (2006), Butler et al. (2007)
suggested that sex/gender differences in task-related functional connectivity that
may partly reflect sex/gender differences in the neurocognitive strategies used to
complete the task do not necessarily result in sex/gender differences in task perfor-
mance. Specifically, Butler et al. argued that suppression of the ventral anterior
cingulate likely reflects the greater cognitive effort, consistent with a ‘top-down’
approach to mental rotation in women. Taken together, findings from studies of task-
related connectivity support the notion that sex/gender differences reflect different
neural correlates of the behaviour – i.e., comparable task performance underpinned
by different patterns of neural activity (De Vries 2004; Becker and Koob 2016;
Hirnstein and Hausmann 2021).
3.2.2 Resting-State Connectivity
Several recent studies have investigated sex/gender differences in functional con-

nectivity using resting-state fMRI (rs-fMRI) paradigms, in which awake participants
are scanned without a specific cognitive task to complete. Smaller early studies
yielded no (n ¼ 49, Weissman-Fogel et al. 2010) or small sex/gender differences
(n ¼ 40, Bluhm et al. 2008), with the latter finding demonstrating greater connec-
tivity in the default-mode network (DMN) of women. The DMN is comprised of the
dorsal and ventral medial prefrontal cortex, the posterior cingulate cortex, precuneus,
and lateral parietal cortex (Laird et al. 2011). The function of the DMN was initially
thought to be ‘spontaneous cognition’, such as daydreaming or mind-wandering, but
more recent findings suggest that it is involved in fundamental processes such as the
functional integration of multiple cortical regions (for a review, see Raichle 2015a).
Unlike other resting-state networks, which become more active during cognitive
processing, activity in the DMN typically reduces (Raichle 2015b). In line with this
suggestion, hyperconnectivity and hyperactivity in the DMN has been demonstrated
in psychiatric disorders such as schizophrenia (Whitfield-Gabrieli et al. 2009).
Several large rs-fMRI studies have revealed stronger functional connectivity in
the DMN of women compared to men (Biswal et al. 2010; Allen et al. 2011; Tomasi
and Volkow 2012b; Ritchie et al. 2018; Zhang et al. 2018; De Lacy et al. 2019; Weis
et al. 2019), though contradictions also exist (Weissman-Fogel et al. 2010). This
finding is important, as atypical organisation and function of the DMN has been
demonstrated in clinical populations where men are overrepresented, such as schizo-
phrenia and bipolar disorder (Garrity et al. 2007; Whitfield-Gabrieli et al. 2009;
Öngür et al. 2010). It should be noted, however, that if schizophrenia is associated
with higher DMN activity, and women have higher resting DMN activity than men,
then it seems contradictory that schizophrenia is more prevalent in men. This might
suggest that the relationship between connectivity and psychopathology differs
between men and women. Similar contradictions apply to sex/gender differences
in lateralisation as reduced cerebral asymmetries have been frequently reported in
schizophrenic samples compared to healthy controls, and in women compared to
men (see below).
In a recent review, Eliot et al. (2021) noted that several inconsistent findings exist
when the whole connectome is considered (see Table 1). For example, the specific
regions of difference, as well as the direction of the sex/gender difference, vary
depending on the chosen analysis method. Evidence supporting this claim can be
found in studies that applied different analysis methods to the same data sets, such as
those reported by Tomasi and Volkow (2012a) and Zuo et al. (2012). Although both
studies used data from the ‘1000 Functional Connectomes Project’ (Biswal et al.
2010), differences in analysis methods resulted in differing conclusions; Tomasi and
Volkow (2012a) showed that women had higher local functional-connectivity den-
sity across multiple cortical regions compared to men, while Zuo et al. (2012) found
that women had higher functional-connectivity density in some areas while men had
higher functional density connectivity in other areas. Still further studies suggest that
sex/gender differences in functional connectivity exist, but they are small and there
is considerable overlap between men and women (e.g., Weis et al. 2020). Taken
together, these findings suggest that sex/gender differences exist, but studies are
quite inconsistent in terms of sample size, specific resting-state networks investi-
gated, and findings. Comparisons between studies are further complicated as only a
very few studies (e.g., Hjelmervik et al. 2014; Weis et al. 2019) included sex
hormonal factors in their analyses. Further caution is warranted as many fMRI
studies are underpowered and significant results may be subject to publication bias
(David et al. 2018).
3.3 Sex/Gender Differences in Functional Cerebral

Asymmetries
In addition to structural cerebral asymmetries and structural/functional connectivity,

sex/gender differences have been extensively investigated with respect to functional
cerebral asymmetries (FCAs, Hodgetts and Hausmann 2020a). Although it has been
shown that FCAs are relatively stable over time (e.g., Vingerhoets 2019), several
factors have been shown to contribute to variations and dynamic changes in FCAs
Table 1 Studies investigating the effect of sex/gender on connectivity of different resting-state networks
Mean
Number of age ( Sex/gender-related factors Resting-state network
Study participants (M:F) SD) considered (s) investigated Main results
Allen et al. 603 (298:305) 23.4 None Attentional, auditory, basil Increased within-network con-
(2011) (9.20) ganglia, default mode, frontal, nectivity in women, particularly
sensorimotor, visual in default-mode network and
some nodes of the frontal net-
works (including Broca’s area)
Increased between-network con-
nectivity in men, particularly
between motor and sensory
networks
Biswal 1,093 (not reported) 30.18 None Default mode (and whole-brain Increased functional connectivity
et al. (6.40) connectome) in the default-mode network in
(2010)a women compared to men
De Lacy 670 (335:335) Not None Cerebellar, control, default mode, Sex/gender differences present in
et al. (2019) reported language, sensorimotor, visual majority of resting-state net-
works, most robust differences
were increased connectivity in
the default-mode network of
Sex/Gender Differences in Brain Lateralisation and Connectivity
women compared to men

Filippi et al. 104 (48:56) Not None Attention, auditory, control, Men showed increased connec-
(2013) reported default mode, fronto-parietal tivity in parietal and occipital
working memory, salience, sen- regions across most networks
sorimotor, visual compared to women
Women showed increased con-
nectivity in frontal and temporal
regions across networks com-
pared men
Women also showed increased
81
(continued)
Table 1 (continued)
82
Mean
connectivity between attention
and fronto-parietal working
memory networks compared to
men
Hjelmervik 31 (15:16) 23.19 Menstrual cycle (women tested Fronto-parietal Women showed increased con-
et al. (2014) (3.72) three times, cycle phase verified nectivity in two of four fronto-
via saliva assays of estradiol and parietal networks investigated
progesterone)
Ritchie 5,216 (2,466:2,750) 61.72 None Default mode, frontal, sensorimo- Men showed increased connec-
et al. (2018) (7.51) tor, visual tivity between the sensorimotor,
visual, and rostral lateral pre-
frontal areas compared to women
nectivity within the default-mode
network compared to men
Scheinost 103 (52:51) 34.1 None Auditory, default mode, fronto- Men showed increased connec-
et al. (2015) (11.20) parietal, sensorimotor, tivity in sensorimotor network
subcortical-limbic, visual compared to women
nectivity in subcortical and lim-
bic networks compared to men
Smith et al. 188 (85:103) 21.85 Testosterone levels included as a Auditory, frontal-parietal, visual Compared with women, men
(2014) (SD not covariate; type of sample (i.e., showed increased connectivity
reported) blood or saliva) not reported between the visual network and
the intracalcerine cortex, cuneus,
supracalcerine and lingual gyrus
Men also showed increased
S. Hodgetts and M. Hausmann
connectivity between the audi-
tory network and Heschl’s gyrus,
planum temporale, insula, and
temporal pole
Finally, men also showed
increased connectivity between
the frontal-parietal network and
the middle, superior, and inferior
frontal gyrus
Testosterone levels did not cor-
relate with any of the above
measures
Tomasi and 561 (225:336) Not None Whole brain Women had 14% higher connec-
Volkow reported tivity compared to men, even
(2012b)a after differences in total brain
volume, grey matter, white mat-
ter, and age were controlled for.
Largest differences were seen in
the anterior thalamus
Weis et al. 38 (19:19) 24.73 Menstrual cycle (women tested Auditory, default mode No sex/gender differences pre-
(2019) (3.58) three times, cycle phase verified sent in connectivity of the
Sex/Gender Differences in Brain Lateralisation and Connectivity
via blood assays of estradiol and default-mode network.

progesterone) For the auditory network, men
showed increased connectivity
between the superior temporal
gyrus and the postcentral gyrus
compared to women
Weissman- 49 (23:26) 30.00 None Control, default mode, salience No sex/gender differences pre-
Fogel et al. (7.00) sent in functional connectivity in
(2010) any of the networks investigated
(continued)
83
Table 1 (continued)
84
Mean
Zhang et al. 820 (366:454) Not None Whole brain Functional connectivity of the
(2018) reported default mode, fronto-parietal, and
sensorimotor networks contrib-
uted most to predictions of
sex/gender
Zuo et al. 1,003 (434:569) 28.10 None Whole brain Women showed increased func-
(2012)a (12.70) tional connectivity in the hippo-
campus and medial occipital
regions compared to men
Men showed increased functional
connectivity in pre-and
postcentral lobules compared to
men. Findings were inconsistent
across analysis methods
Auditory: superior temporal gyrus, auditory cortices
Attentional: inferior parietal lobule, middle and superior frontal gyrus, precuneus, middle and superior temporal gyrus, angular gyrus, cingulate gyrus, insula
Basal ganglia: left and right putamen
Cerebellar: anterior and posterior lobe
Control: anterior cingulate, anterior prefrontal cortex, dorsolateral and ventrolateral prefrontal cortex, inferior parietal cortex, insula
Default mode: dorsal and ventral medial prefrontal cortex, posterior cingulate cortex, precuneus lateral parietal cortex
Frontal: inferior and middle frontal gyrus, supramarginal gyrus, middle temporal gyrus, caudate, pyramis, superior medial gyrus, superior parietal lobule
Fronto-parietal/frontro-parietal working memory: dorsolateral prefrontal cortex, intraparietal sulcus, posterior parietal sulcus
Salience: dorsal interior cingulate and bilateral fronto-insular cortices
Sensorimotor: precentral gyrus, cerebellum, postcentral gyrus, precuneus, inferior frontal gyrus, middle temporal gyrus, insula, supramarginal gyrus,
supplementary motor area, inferior parietal lobule
Subcortical-limbic: parahippocampal gyrus, hippocampus, thalamus, insular cortex, amygdala
Visual: lingual gyrus, calcarine gyrus, visual cortices, inferior parietal lobule, inferior temporal lobule
a
Indicates data sampled from the 1,000 Functional Connectomes Project (http://fcon_1000.projects.nitrc.org/)
S. Hodgetts and M. Hausmann
(e.g., Hausmann 2019; Hausmann et al. 2016), including biological sex and sex
hormones (e.g., Hausmann 2017).
In healthy adults, a large body of research showed sex/gender differences in
FCAs related to language (Hausmann et al. 1998; Shaywitz et al. 1995), spatial
ability (Chiarello et al. 1989; Hausmann and Güntürkün 2000), and face recognition
(Rizzolatti and Buchtel 1977; Borod et al. 2005). While contradictions exist (Boles
2005; Sommer et al. 2004), these studies suggest that women show reduced FCAs
(i.e. greater bilateral brain activity) relative to men. Several reviews, meta-analyses,
and large-scale behavioural studies have been conducted to quantify the size of
sex/gender effects on FCAs across a range of lateralised processes (e.g., Bless et al.
2015; Hausmann et al. 2019; Hiscock et al. 1994; Vogel et al. 2003; Voyer et al.
1995). A recent systematic review summarised these findings spanning 40 years
(Hirnstein et al. 2019) and concluded that a small but robust effect size regarding
greater FCAs in men (d ¼ 0.05–0.15). This suggests that such sex/gender differences
in FCAs do exist at population level but may only be detected in studies with larger
samples.
Several MRI studies also investigated the effect of sex/gender on FCAs. For
example, using rs-fMRI Liu et al. (2009) found small but significant sex/gender
differences for both left- and right-lateralised resting-state networks, with men
showing stronger FCAs than women. However, both sexes showed strong FCAs
in this study, and there was much overlap between them. In a larger rs-fMRI study
(n ¼ 913), Tomasi and Volkow (2012a) reported that men had stronger rightward
FCAs for short-range connectivity in specific regions of the superior temporal,
inferior frontal, and inferior occipital cortices, while women had stronger leftward
FCAs for long-range connectivity in the inferior frontal cortex. Furthermore, in a
large rs-fMRI study (n ¼ 1,011), Nielsen et al. (2013) identified several resting-state
networks characterised by lateralisation (e.g. the DMN and language network
included several left-lateralised regions), but the patterns of FCAs did not differ
between men and women. Finally, a recent structural MRI study used measures of
cortical thickness to create ‘hemispheric morphological networks’ in a sample of
285 participants (Choi et al. 2020). Results showed that the patterns of FCA differed
between men and women in several cortical regions, with men showing stronger
FCAs in the cingulate and superior parietal gyrus, and women showing stronger
FCAs in the temporal pole.
In sum, although inconsistencies exist regarding specific brain areas and meth-
odologies used, several meta-analyses and large-scale studies suggested that
sex/gender differences in FCAs do exist and that interactions with sex/gender should
be considered when investigating lateralisation and brain connectivity. Sex/gender
and FCAs have also been identified as potentially relevant factors with regard to
several clinical disorders (e.g., schizophrenia and other psychotic disorders, for a
review, see Hodgetts and Hausmann 2020a). Although the relationship between
sex/gender, lateralisation, and psychopathology is not well understood, and therefore
remains speculative, future research investigating these relationships might help to
better understand individual differences in clinical populations, which in turn will
support the development of stratified treatments for disorders characterised by

sex/gender differences and/or atypical FCAs.
3.4 Hormonal Influences on Lateralisation and Connectivity
Considering the evidence presented above, sex/gender differences exist concerning

both connectivity and lateralisation. However, it is incorrect to refer to brains as
‘sexually dimorphic’ as there is clear empirical evidence showing that prototypical
‘male brains’ or ‘female brains’ do not exist. Instead, there is overlap in brain
structure and function between the sexes, and there is substantial within-sex/gender
variation. As such, an increasing number of studies support the notion that
sex/gender differences are best conceptualised as a product of several non-binary
factors, including biological (e.g. sex hormones) and environmental influences.
Several studies have shown that an individual’s distinct hormonal profile can be
an important factor in the generation and maintenance of sex/gender differences in
lateralisation (i.e. FCAs) and connectivity (Weis and Hausmann 2010; Hodgetts and
Hausmann 2018, 2020a). However, sex hormone levels are dynamic, both across
short-time intervals (e.g., fluctuating during the menstrual cycle), across the lifespan
(e.g., reducing after menopause). Indeed, many studies investigating menstrual
cycle-related effects of sex hormones on lateralisation have revealed reduced
FCAs during the follicular phase (high levels of estradiol) and/or during the
postovulatory luteal phase and increased FCAs during menstruation (lowest levels
of estradiol and progesterone, (Hausmann 2017; Hodgetts and Hausmann 2018). In
contrast, other studies showed larger FCAs during the follicular and/or luteal phase
in combination with reduced FCAs during menstruation (e.g., Cowell et al. 2011;
Mead and Hampson 1996; Sanders and Wenmoth 1998; Wadnerkar et al. 2008). The
conflicting results, sometimes occurring even in the same study (e.g., Mead and
Hampson 1996; Sanders and Wenmoth 1998), may suggest that some studies are
reporting false positives, or they indicate that size and direction of the effects
partially depend on the specific task and test modality (Hausmann and Bayer
2010; Hjelmervik et al. 2012; Hodgetts et al. 2015, 2017).
Although different explanations on how sex hormones might influence
lateralisation have been proposed, one explanation takes the neuromodulatory prop-
erties of sex hormones into account and hypothesised that sex hormones affect
lateralisation via their effects on functional connectivity. The hypothesis of
progesterone-mediated interhemispheric decoupling (Hausmann and Güntürkün
2000) stated that high levels of progesterone lead to a reduction in interhemispheric
inhibition (i.e., activity in the nondominant hemisphere is no longer suppressed by
the dominant hemisphere). Specifically, it was hypothesised that higher levels of
progesterone can reduce interhemispheric inhibition by suppressing the excitatory
responses of neurons to glutamate (Smith et al. 1987) and by enhancing their
inhibitory responses to GABA (Smith 1991), leading to increased bilateral activation
and reduced FCAs (e.g., Cook 1984; Regard et al. 1994). This mechanism presumes
that FCAs arise because the hemisphere dominant in a particular task inhibits the
nondominant hemisphere via the corpus callosum (Cook 1984; Chiarello et al.
1989). Although cortico-cortical transmission is primarily excitatory, callosal pro-
jections terminate on pyramidal neurons, which subsequently activate GABAergic
interneurons (Toyama and Matsunami 1976), inhibiting the contralateral hemisphere
(Innocenti 1980). Moreover, it has been shown that callosal projections terminate
directly on GABAergic interneurons (Conti and Manzoni 1994). Both mechanisms
would result in widespread inhibition of homotopic regions of the nondominant
hemisphere by the dominant hemisphere. This hypothesis was supported empirically
by several behavioural studies of FCAs (e.g., (Hausmann and Güntürkün 2000;
Hausmann et al. 2002, 2013; Bayer et al. 2008), transcranial magnetic stimulation
experiments (Hausmann et al. 2006), and fMRI (Weis et al. 2008, 2011).
Further research revealed that high estradiol levels may also be capable of
reducing FCAs via an effect on interhemispheric inhibition (Hausmann 2005,
2017; Hausmann et al. 2006; Weis et al. 2008; Hausmann and Bayer 2010). For
example, Weis et al. (2008) used fMRI to scan a sample of naturally cycling women
while completing a word-matching task. Results revealed that high levels of estradiol
during the follicular phase were associated with reduced interhemispheric inhibition,
and in turn, reduced FCAs. Hausmann et al. (2013) used electroencephalography to
directly measure interhemispheric connectivity by using visual-evoked potentials to
estimate interhemispheric transfer time (IHTT). The results showed that IHTT from
right-to-left was longer during the luteal phase as compared to the menstrual phase
and that this effect was related to high levels of estradiol, as opposed to progesterone.
Further research has investigated the effect of sex hormones on intrahemispheric
activity. For example, Weis et al. (2011) used fMRI to scan natural cycling women
as they completed a figure-matching task. The results revealed cycle-phase related
reduced functional connectivity within right hemispheric networks during the luteal
phase, as compared to both the menstrual and the follicular phase. Consequently, the
authors suggested that sex hormones modulate not only interhemispheric inhibition
between homotopic areas (Weis et al. 2008) but can also influence intrahemispheric
integration, and interhemispheric connectivity between heterotopic brain regions
(Weis et al. 2011).
Considering the evidence presented in this chapter, it seems that sex/gender
differences in lateralisation and connectivity exist, but there is much overlap
between the sexes (Joel et al. 2015; Weis et al. 2020). Moreover, the detection of
sex/gender differences seems to partly be dependent on individuals’ sex hormonal
environment. Indeed, it has been argued that FCAs are double-coded by stable
characteristics (e.g., genetics) and temporary situational aspects (e.g., sex hormones,
environmental influences) (e.g., Hausmann 2019). It is noteworthy in this context
that sex hormones have been shown to have antipsychotic properties, for example in
schizophrenia (Kulkarni et al. 2015; McGregor et al. 2017; Riecher-Rossler et al.
2018). However, if sex hormonal effects on FCAs contribute to these clinical
observations is not known, as is the causality in the relationship between sex
hormones, FCAs, and psychiatric symptoms (see Fig. 1).
Genetic Environmental
factors factors
Estradiol
Psychotic
symptoms
(Atypical) FCAs
Fig. 1 The influence of estradiol on functional cerebral asymmetries (FCAs) and psychotic
symptoms in schizophrenia and other mental disorders such as major depression, bipolar disorder,
anxiety, and neurodevelopmental disorders. Estradiol can affect functional asymmetries and cog-
nitive control and can reduce psychotic symptoms. The causality of the relationship between
atypical asymmetries and psychotic symptoms is still unclear. There is empirical evidence for
both atypical asymmetries as a trait marker of the disorder and as a neuro-compensatory mecha-
nism. The relationships considered in this chapter are indicated by the solid arrows. Reprint from
Cortex, 127, S. Hodgetts & M. Hausmann, Antipsychotic effects of sex hormones and atypical
hemispheric asymmetries, p. 317, Copyright (2020), with permission from Elsevier
3.5 The Case for a Biopsychosocial Approach
Although the evidence presented above demonstrates that sex hormones can signif-
icantly influence both lateralisation and connectivity, it is incorrect to assume that
they underpin all of the demonstrated sex/gender differences in the brain (Cahill
2006; Hausmann 2020). Indeed, studies have revealed that environmental factors
associated with sex/gender, such as gender roles and stereotypes, can also influence
sex/gender differences such as those seen for lateralisation and connectivity (Jäncke
2018; Hodgetts and Hausmann 2020b).
It has been suggested that a relationship exists between socially derived gender
roles (i.e., masculinity, femininity) and brain structure (Belfi et al. 2014). In this
study, 108 children (56 boys and 52 girls), aged 7–17 years, completed a gender role
questionnaire and underwent structural MRI. The results showed that masculinity
positively correlated with WM volumes in the frontal lobe, while femininity posi-
tively correlated with GM volumes in the temporal lobe, even after biological sex
was controlled for. Although the causality of this relationship is not clear, the authors
suggested that this effect may represent an environmental influence on sex/gender
differences in the brain (see Bourne and Maxwell 2010). Similar results have been
found for sex/gender differences in cognition, with several studies suggesting that
gender roles (Hoffman et al. 2011; Reilly et al. 2016; Compère et al. 2018; Pletzer
et al. 2019) and gender stereotypes (Hausmann 2014; Hausmann et al. 2009,
Hirnstein et al. 2014) underlie differences in sex/gender-sensitive (some of them
lateralised) cognitive tasks such as mental rotation and verbal fluency.
In light of the evidence presented above, an increasing number of studies have
begun to consider the interactive effects of biological and environmental factors
concerning sex/gender differences in the brain and behaviour (Hausmann 2020;
Hodgetts and Hausmann 2020b). The biopsychosocial approach (Halpern and Tan
2001; Miller and Halpern 2014) suggests that environmental factors, including
gender roles and stereotypes, can interact with biological factors, including sex
hormones, to influence sex/gender differences in the brain and behaviour (Halpern
and Tan 2001; Wraga et al. 2007; Krendl et al. 2008; Haier et al. 2009; Hausmann
et al. 2009; Smith et al. 2013; Dunst et al. 2013; Pletzer et al. 2019). Using fMRI,
Wraga et al. (2007) investigated the effects of gender stereotyping on the neural
underpinnings of visuospatial performance in 54 women. As expected, significantly
poorer performance was found in women exposed to a negative gender stereotype,
when compared to women given a positive gender stereotype. Additionally, poorer
performance in the negative stereotype group was characterised by greater activation
in brain regions associated with emotional processing, including the orbital and
medial frontal gyri, and the anterior cingulate cortex. These results suggest that
gender stereotypes can influence the efficiency of neural activity in sex/gender-
sensitive tasks. Hausmann et al. (2009) investigated the effect of gender stereotypes
in both men and women using a battery of sex/gender-sensitive cognitive tasks (incl.
mental rotation and verbal fluency) and determined testosterone levels in all partic-
ipants. To prime gender, one-half of the entire sample completed a questionnaire
concerning gender stereotypes, while the other half completed a gender-neutral
version of the same questionnaire. Overall, the expected sex/gender differences
favouring men and women were found for mental rotation and verbal fluency,
respectively. However, the sex/gender difference in mental rotation was significantly
driven by the gender-primed group. Moreover, testosterone levels in the gender
stereotype group were 60% higher than those in the control group. These results
suggest that the effect of gender priming on cognitive performance can be mediated
by the accompanying changes in sex hormone levels, probably depending on
whether participants interpreted the testing situation after priming as challenging
or threatening. Similar results were recently reported by Pletzer et al. (2019), who
showed that high levels of testosterone in conjunction with high levels of self-
reported masculinity yielded highly accurate mental rotation scores regardless of
biological sex.
4 Conclusion
Research to date supports the notion that sex/gender differences in brain and
behaviour exist at many levels, including lateralisation and connectivity. However,
the notion that the brain is ‘sexually dimorphic’ is incorrect, and referring to an
oversimplified binary (e.g. ‘male brain’, ‘female brain’) is misleading and should be
avoided in future research. Although many of these sex/gender differences are
characterised by small effect sizes, some of these differences are reliably found
and should not be considered trivial. Indeed, there are various reasons for inconsis-
tencies in the literature. Some of them arise from (1) different methodological
approaches and reference measures, (2) small and/or heterogeneous sample compo-
sition, (3) the specific tasks/paradigm used, and (4) the limited consideration of sex/
gender-related factors that can explain inter-and inter-individual differences in brain
and behaviour better than sex/gender per se. Sex/gender differences in laterality and
connectivity are no exceptions to this. Sex/gender-related factors include biological
factors such as levels of sex hormones and sex-linked genes and environmental
factors such as gender roles and stereotypes. These and related factors should be
acknowledged routinely within a biopsychosocial approach when studying
sex/gender differences in brain and behaviour.
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Part II
Sex Differences in Cognitive and Mental
Health Across the Lifespan
Sex Differences in Depression and Anxiety
Pavlina Pavlidi, Nikolaos Kokras, and Christina Dalla
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
2 Sex Differences in Depression and Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
2.1 Sex Differences in Transcriptomic and Genetic Evidence of Depression and Anxiety 106
2.2 Sex Differences in the Immune System Response in Depression and Anxiety . . . . . 108
2.3 Sex Differences in Neuroendocrine Aspects of Depression and Anxiety . . . . . . . . . . . 109
2.4 Sex Differences in Cognition During Depression and Anxiety . . . . . . . . . . . . . . . . . . . . . . 110
3 Sex Differences in Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.1 Sex Differences in Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.2 Sex Differences in Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.3 Sex Differences in Side Effects of Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
3.4 Hormonal Milieu and Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.5 Neuroestrogens in Depression and Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.6 Effect of Antidepressants on Hormonal Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4 Conclusion: Sex as a Biological Variable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Abstract Depression and anxiety disorders carry a tremendous worldwide burden

and emerge as a significant cause of disability among western societies. Both
disorders are known to disproportionally affect women, as they are twice more
likely to be diagnosed and moreover, they are also prone to suffer from female-
specific mood disorders. Importantly, the prevalence of these affective disorders has
Nikolaos Kokras and Christina Dalla contributed equally to this work.
P. Pavlidi and C. Dalla (*)

Department of Pharmacology, Medical School, National and Kapodistrian University of Athens,
Athens, Greece
e-mail: cdalla@med.uoa.gr
N. Kokras
Department of Pharmacology, Medical School, National and Kapodistrian University of Athens,
Athens, Greece
First Department of Psychiatry, Eginition Hospital, Medical School, National and Kapodistrian
University of Athens, Athens, Greece
https://doi.org/10.1007/7854_2022_375
Published Online: 2 August 2022
104 P. Pavlidi et al.
notably risen after the COVID pandemic, especially in women. In this chapter, we
describe factors that are possibly contributing to the expression of such sex differ-
ences in depression and anxiety. For this, we overview the effect of transcriptomic
and genetic factors, the immune system, neuroendocrine aspects, and cognition.
Furthermore, we also provide evidence of sex differences in antidepressant response
and their causes. Finally, we emphasize the importance to consider sex as a biolog-
ical variable in preclinical and clinical research, which may facilitate the discovery
and development of new and more efficacious antidepressant and anxiolytic phar-
macotherapies for both women and men.
Keywords Antidepressants · Anxiety · Depression · Sex differences · Stress
1 Introduction
According to the World Health Organization, depression carries a tremendous

worldwide burden of disease and emerges as a significant cause of disability
among western societies (Ferrari et al. 2013). Indeed, an early study evaluating the
prevalence of DSM-III-R psychiatric disorders in the United States, reported that
depression is the most common mental illness, carrying devastating economic and
social consequences (Kessler et al. 1994). A cross-national epidemiological study
reported that the average lifetime and 12-month prevalence of DSM-IV major
depressive episodes are 14.6% and 5.5%, respectively, in ten high-income countries,
and 11.1% and 5.9%, respectively, in eight low- to middle-income countries
(Bromet et al. 2011). A more recent meta-analysis reported the one-year and lifetime
prevalence of depression at 7.2% and 10.8%, respectively (Lim et al. 2018). Fur-
thermore, a systematic review estimated the effects of COVID-19 pandemic in major
depressive disorder (MDD). For MDD in particular, due to COVID-19, an increase
of 27.6% was estimated in global prevalence for 2020 (COVID-19 Mental Disorders
Collaborators 2021). Interestingly, a recent study evaluating the influences of
sex/gender, age, and ethnicity on psychosocial factors during the COVID-19 pan-
demic, emphasized the prioritization of women, gender-diverse individuals, and
young people during public health measures in future pandemics (Brotto et al.
2021). Regardless of tremendous research, the pathophysiology of depression
remains only partially understood, whereas currently used pharmacotherapy has a
delayed onset of action, lacks absolute efficacy and has no effect in 35% of patients
(Berton and Nestler 2006; O'Leary et al. 2015). Moreover, a limited progress has
been made over the last decades toward faster and more efficacious treatments
(Wong and Licinio 2001). The only recent and novel addition to available treatments
is that of esketamine in 2019, which is a fast-acting antidepressant for treatment-
resistant depression (TRD) (Zarate and Niciu 2015; Kaur et al. 2021). A significant
contributor that hinders new drug discovery is still the knowledge gap regarding the
Sex Differences in Depression and Anxiety 105
precise neurobiological alterations that furnish the pathophysiology and treatment of

depression (Kuehner 2017).
Sex differences in the prevalence of major depressive disorder are known since at
least 1970s, noting that women are twice more likely to experience depression than
men in clinical and non-clinical samples (Weissman and Klerman 1977). In 2013,
the global 12-month prevalence of MDD was 5.8% in females and 3.5% in males,
and later studies reaffirm this finding (Ferrari et al. 2013; Gutierrez-Rojas et al.
2020). Besides the increased vulnerability of women to suffer from affective disor-
ders (Kokras and Dalla 2017), they are also prone to suffer from female-specific
mood disorders (Kornstein 1997; Balta et al. 2019), such as premenstrual dysphoric
disorder (PMDD) and postpartum depression (PPD) (Halbreich and Karkun 2006).
Premenstrual syndrome (PMS) is very commonly seen among women of reproduc-
tive age, with 20% of women experiencing severe symptoms that may warrant
treatment at some time. Furthermore, up to 8% of women experience PMDD, a
condition at the severe end of the PMS spectrum (Halbreich et al. 2003, 2007).
Along with the well-established sex ratio in the epidemiology of depressive disor-
ders, there are also notable differences regarding symptomatology and response to
pharmacological treatment. In fact, women with depression experience greater
symptom severity, more past suicide attempts, and more sleep, appetite, and energy
disturbances (Blanco et al. 2012; Altemus et al. 2014). Moreover, clinical data
suggest that women with depression express different biomarkers and cognitive
symptoms of depression than men, while presenting more commonly the anxious
and atypical subtypes of depression (Marcus et al. 2005; Penninx et al. 2013; Labaka
et al. 2018). Regarding comorbidities, women with depression are more likely to
suffer from anxiety, whereas men have higher rates of comorbid substance use
disorders (Marcus et al. 2005; Schuch et al. 2014). A plethora of biological factors,
such as genetic and epigenetic predisposition, stress, immune system,
neuroplasticity, and hormonal milieu, are considered as contributors in the patho-
physiology of depression, and in particular, in women’s vulnerability to depression
(Wainwright and Galea 2013; Nestler 2014; Gold et al. 2015; Miller and Raison
2016; Wray et al. 2018; Rubinow and Schmidt 2019).
On the other hand, anxiety disorders are described as the most common class of
mental disorders, with a lifetime prevalence of 28.8%, according to National
Comorbidity Survey Replication in the United States (Kessler et al. 2005). Similarly
to MDD, a systematic review estimated an increase of 25.6% in global prevalence
for 2020 of anxiety disorders, due to COVID-19 (COVID-19 Mental Disorders
Collaborators, 2021). Common anxiety disorders include panic disorder, with or
without agoraphobia, social phobia, generalized anxiety disorder, and specific pho-
bias (Thibaut 2017). Several studies have pinpointed adolescence as the onset age of
anxiety, and evidence so far does not support a sex difference at age of onset
(McLean et al. 2011; Lijster et al. 2017). Furthermore, a significant link is found
between anxiety and depression and there is high comorbidity between these disor-
ders. To that end, current guidelines recommend, in combination with psychother-
apy, primarily selective serotonin reuptake inhibitors (SSRIs) and selective serotonin
norepinephrine reuptake inhibitors (SNRIs) as the first-line treatment for anxiety
disorders. Until now, the precise etiology of anxiety disorders still remains elusive,
although neuronal circuits of fear and anxiety are relatively well-understood; how-
ever, the most prominent hypotheses suggest an interaction of genetic vulnerability
and psychosocial factors, i.e., stressful events during childhood or adolescence
(Bandelow et al. 2017).
Similar to depression, women are twice more likely to suffer from anxiety
disorders than men. In detail, the lifetime and 12-month male to female prevalence
ratios of anxiety disorders were 1:1.7 and 1:1.79, respectively (McLean et al. 2011).
The disparity in prevalence between the two genders is seen in stress, anxiety-
related, and fear-based disorders, such as generalized anxiety disorder, panic disor-
der, social anxiety disorder, specific phobias, and posttraumatic stress disorder
(PTSD) (Tolin and Foa 2006; Kessler et al. 2009). This sex bias is mainly attributed
to women’s greater sensitivity to stressful experiences, and studies have shown that
women exhibit more severe, frequent, and persistent symptoms, while experiencing
more intense emotions of fear and panic than men (Holbrook et al. 2002; Donner and
Lowry 2013; Altemus et al. 2014). Fear extinction network regions, such as medial
prefrontal cortex, amygdala, and hippocampus, exhibit sexual differentiation, pos-
sibly explaining the sex differences seen in fear conditioning, extinction, and thus in
anxiety disorders in general (Maeng and Milad 2015). Indeed, numerous studies
focus on the interrelation between stress and fear mechanisms and their modulation
by sex steroids, aiming to elucidate the pathophysiology of anxiety disorders, as well
as to facilitate the development of new and improved treatments (Graham and Milad
2013).
Based on the above, this chapter aims to discuss the pathophysiology and
treatment of depression and anxiety, while focusing on reported sex differences
and considering sex as a biological factor.
2 Sex Differences in Depression and Anxiety
2.1 Sex Differences in Transcriptomic and Genetic Evidence

of Depression and Anxiety
Transcriptome analysis of the brain relies on the identification of differences in the

regulation and expression of genes between normal tissues of numerous brain
regions or between normal tissues and diseased tissues (Wang and Wang 2019).
The study of brain transcriptome has allowed the identification of sex differences in
gene expression and RNA transcripts that contribute to the development of depres-
sion and stress susceptibility (Seney et al. 2022). Indeed, an extensive transcriptomic
study of gene expression changes concluded that men and women with depression
exhibited distinctive transcriptional profiles with minimum overlap across cortical
and subcortical brain areas (Labonté et al. 2017). Gene expression has been found to
even be in opposite directions in men and women with depression (Seney et al.
2018).
Similar to transcriptome studies, genome-wide association studies (GWAS) have
also contributed to the elucidation of genetic loci that explain the sex-specific
susceptibility, while stating the significant genetic heterogeneity in depression and
highlighting the importance of environmental influence (Flint and Kendler 2014;
Hyde et al. 2016). Post-mortem studies indicate that women have more alterations in
gene expression of serotonin and glutamate receptors than men. In contrast, frequent
variations in the expression of brain-derived neurotrophic factor (BDNF) genes and
the BDNF receptor TrkB have been observed in men (Tripp et al. 2012). Interest-
ingly, Seney et al. in (2018) performed gene-ontology analyses and concluded that
synapse-related genes are upregulated in depressed women and downregulated in
men, whereas the opposite relation described the microglia- and oligodendrocyte-
related genes (Seney et al. 2018).
As commonly used, rodent models of depression rely on stress exposure to
elucidate depression-like behavioral indices and investigate possible sex differences
in the phenotype and genetic background. Such paradigms include numerous
stressors, i.e., chronic psychosocial stress, chronic unpredictable stress, social defeat
stress, often applied for a significant amount of time, aiming to cause depressive-like
behaviors in rodents (Italia et al. 2020) and several of these animal models of
depression and anxiety present sex differences (Kokras and Dalla 2014). The chronic
variable stress paradigm elicits depression-like behavior similarly in male and
female mice; however, there is less than 30% overlap in stress-induced gene
expression in the nucleus accumbens (NAc) and vmPFC in both sexes (Labonté
et al. 2017). Interestingly, various stress-inducing paradigms utilized by Hodes and
colleagues induced depressive-like behavior only in female mice. The sex difference
in this behavioral susceptibility to stress was significantly associated with specific
gene expression patterns in females, such as increased expression of the DNA
methyltransferase 3a (Dnmt3a) in the female NAc (Hodes et al. 2015, 2017). Sex
differences in stress-induced gene expression were also reported after applying the
chronic restraint stress paradigm. In particular, upregulation of the corticotropin-
releasing hormone (CRH) and downregulation of plasticity- and opioid-related
genes in the hippocampus were significantly correlated with stress only in males
(Randesi et al. 2018).
As discussed, epigenetic mechanisms have also been proposed as potential
contributing factors to depression, which are associated with sex differences
(Hodes et al. 2017). Studies investigating the implication of epigenetics in sexual
differentiation of the brain suggest DNA methylation, and other epigenetic mecha-
nisms, as key processes for brain feminization and establishment of male and female
behavior. Furthermore, a relatively new concept proposes the transgenerational
transmission of stress susceptibility. For instance, female children of depressed
women have higher chances to develop depression (LeMoult et al. 2015).
Such extensive sex differences at the transcriptional level, gene expression
patterns, and epigenetic mechanisms highlight the significance of developing
sex-specific treatments for MDD.
2.2 Sex Differences in the Immune System Response

in Depression and Anxiety
The response of the immune system has been proposed as a critical contributor to sex
differences in mood and anxiety disorders (Nestler et al. 2002). Several studies have
presented brain inflammatory cytokines as important contributors to depression and
anxiety, suggesting that continuous neuroimmune activation during adulthood may
affect brain health in general, and more specifically contribute to the manifestation of
depression in women (Dantzer and Kelley 2007; Schwarz and Bilbo 2012; Remus
and Dantzer 2016). In particular, patients with depression displayed immune acti-
vation and had increased levels of interleukins (IL) and cytokines, such as IL-12,
IL-1b, IL-8, and Tumor Necrosis Factor Alpha (TNF-a) (Ganguly and Brenhouse
2015). Even in elderly patients with cognitive decline, improvement of depressive
symptoms correlated with a reduction in pro-inflammatory interleukins (Kokras et al.
2018b). In parallel with the dysregulation of the immune system in depression, sex
differences in anti- and pro-inflammatory biomarkers have also been reported. For
instance, increased levels of pro-inflammatory IL-8, interferon-γ, and leptin, as well
as lower levels of the anti-inflammatory adiponectin and the pro-inflammatory IL-5,
were recently found in depressed women (Birur et al. 2017).
Furthermore, inflammatory challenges, such as administration of bacterial endo-
toxins, have been shown to induce depressogenic effects in healthy women, but not
in men. Such challenges cause augmentation of depressed mood and social discon-
nectedness only in women, an effect associated with TNF-α and IL-6 levels (Moieni
et al. 2015). Several preclinical studies utilizing immunological models of depres-
sion, which induce sickness behavior, have revealed sex differences in biological
and behavior responses (Pitychoutis et al. 2009; Jovicic et al. 2015; Sens et al. 2017).
For instance, Sens et al in (2017), reported increased anhedonia- and depression-like
behaviors, evidenced as reduced sucrose consumption and enhanced immobility in
the forced swim test (FST), in mice of both sexes after acute LPS administration.
Interestingly, female mice exhibited prolonged reductions in grooming behavior,
and increased serotonergic activity in prefrontal cortex, striatum, amygdala, hypo-
thalamus, and hippocampus, whereas in males food consumption was significantly
lower over an extended time period (Sens et al. 2017). Furthermore, the association
between the immune system and depression has been investigated via stress models,
which focus almost entirely on sex differences in inflammatory consequences of
stress (Hudson et al. 2014; Bollinger et al. 2016). Most of these studies focus on the
microglia as the leading cell population of neuroinflammatory responses in the brain,
triggered by pathophysiological and environmental changes (Kreutzberg 1996;
Kettenmann et al. 2013). Stress is considered as an essential factor that causes
microglia activation, changing eventually the cells’ functionality state and facilitat-
ing the expression of stress-dependent depressive-like behaviors (Kreisel et al.
2014). Bollinger et al. reported that both acute and chronic restraint stress reduced
activation of microglia in the mPFC of female rats only (Bollinger et al. 2016),
whereas, another study reported that only male animals exhibited increased
microglial activation (Calcia et al. 2016). Recent data (Rainville et al. 2018;
Tsyglakova et al. 2019) show that sex differences also appear from the close
interaction of the immune system with the hypothalamic–pituitary–adrenal (HPA)
axis, whose role in sex differences will be discussed in the following section.
2.3 Sex Differences in Neuroendocrine Aspects of Depression

and Anxiety
Stress-related abnormal activation or regulation of the HPA axis is an important

factor in the neurobiology of depression and anxiety disorders (Kokras et al. 2019).
Sex differences in the HPA axis of rodents have been reported both at baseline and
following stress stimuli (Goel et al. 2014). Under basal and stress-free conditions
female rats, exhibit higher plasma corticosterone levels throughout the circadian
cycle, compared to males (Atkinson and Waddell 1997; Weinstock et al. 1998).
Regarding Adrenocorticotropic hormone (ACTH), no sex differences have been
reported in rodents at baseline; however, investigation of basal HPA output confirms
a differential corticosterone sensitivity to ACTH release in females compared to
males (Atkinson and Waddell 1997). Stress-induced activation of the HPA axis has
been reported to be more robust in female rats, however, this activation does not
appear fully correlated with the female behavioral response to stress (Kokras et al.
2012, 2019, 2021). Overall, corticosterone levels are higher in female rats after the
delivery of a stressor, compared to males (Mitsushima et al. 2003). Such sex
difference has been reported in rodent studies using either physical or psychological
stressors.
The literature regarding sex differences in baseline and stress-induced levels of
cortisol and ACTH in humas is less consistent due to the diversity in experimental
designs, i.e., time and source of hormone sampling, age, and health status of sub-
jects. In particular, data from human studies suggest that the nature of the stressor,
i.e., physical or psychological, constitutes an important factor for the male and
female response (Goel et al. 2014). Interestingly, a human study, which evaluated
cortisol responses to daily events in MDD reported that women with depression had
larger cortisol responses to negative events than men with depression (Peeters et al.
2003). It seems that progesterone and estradiol regulate cortisol output and circula-
tion to contribute to the net effect of higher cortisol in the follicular phase versus the
luteal phase of women’s menstrual cycle and the cortisol-sex hormones interaction
plays an important role in premenstrual symptoms (Roca et al. 2003; Hamidovic
et al. 2020). Although sex differences in cortisol and ACTH have long been
observed in human studies on depression and stress-related disorders (Young et al.
2001; Young and Breslau 2004), translation of rodent findings to human should be
cautious and for a more detailed comparison of human-rodent studies, the reader is
directed to (Kokras et al. 2019).
Sex differences have also been reported in corticotropin-releasing factor receptor

(CRFr) signaling and trafficking. In particular, a study by Bangasser et al. (2010),
showed that female rats present robust cellular signaling to low levels of CRF and
less adaptation to high CRF levels. These findings suggest that dysfunction in the
CRFr signaling may contribute to the increased vulnerability of females to develop
stress-related pathologies (Bangasser et al. 2010). Extending the above mentioned
finding, the same group described that the locus coeruleus (LC)-norepinephrine
arousal system of females is significantly affected by increased CRF levels due to
abnormalities of the receptor trafficking, suggesting this cellular mechanism as a
possible contributor to the increased susceptibility of stress-related disorders in
females (Bangasser et al. 2013).
2.4 Sex Differences in Cognition During Depression

and Anxiety
Both clinical and preclinical studies have associated depression with disruption of
complex mood-related neuronal circuitry, size reduction of certain brain regions, and
synaptic loss (Kang et al. 2012; Duman et al. 2016). Furthermore, typical, i.e.,
SSRIs, and fast-acting, i.e., esketamine, antidepressants have been reported to
alleviate the effects of depression on neuronal plasticity, partially reinstating the
synaptic function and spine density at least in male rats (Duman and Aghajanian
2012). It is well established that in females, the fluctuation of estrogens has an
extensive effect on synaptic plasticity, and such effects have been linked with
increased risk of developing depression in women compared to men (Bloch et al.
2003; Borrow and Cameron 2014). In a study where cyclicity was considered as a
factor, females with low ovarian hormones, i.e., during diestrus 1, froze more during
extinction recall than females in proestrus and male rats (Rey et al. 2014). Such sex
differences were absent in a similar study, where the phases of the estrous cycle were
not monitored (Milad et al. 2009). The effect of estrous cycle on fear extinction
memory was also shown after administration of estrogens during diestrus 1, before
and after extinction training, as treated females exhibited reduced freezing during
extinction recall (Zeidan et al. 2011). Similarly, Galvin and Ninan (2014) supported
that circulation of endogenous estrogens reinforces synaptic potentiation and
strengthens the extinction circuitry in female rats (Galvin and Ninan 2014). More-
over, estrogens’ administration to ovariectomized female rats and mice enhances
contextual fear extinction and fear conditioning (Morgan and Pfaff 2001; Chang
et al. 2009). Similar to rodent studies, prescribed hormonal contraceptives were
found to impair fear extinction in women (Graham and Milad 2013). In parallel to
estrogens’ effects, testosterone, and its non-aromatizable metabolites, have also been
reported to affect anxiety and fear processes (McHenry et al. 2014), via alleviating
anxiety behaviors and enhancing cognition in male rodents (Frye et al. 2008; Hodosy
et al. 2012; McDermott et al. 2012).
3 Sex Differences in Treatment
Treatment of depression and anxiety requires a multifaced approach tailored to

individual patient’s needs, involving supportive psychosocial interventions,
psychoeducation and counseling, psychotherapy, and pharmacological treatment
where applicable (National Collaborating Centre for Mental Health (UK) 2010;
Andrews et al. 2018). When pharmacotherapy for depression and anxiety is deemed
necessary, the most commonly prescribed class of medications is the selective
serotonin reuptake inhibitors (SSRI), followed by serotonin-noradrenaline reuptake
inhibitors (SNRI), whereas tricyclic antidepressants and monoamine oxidase inhib-
itors have been almost phased out. These classes of medications, although com-
monly and sometimes misleadingly referred as “antidepressants” (Zohar et al. 2015)
are equally effective in treating anxiety, obsessive-compulsive disorder and other
brain diseases involving the monoaminergic neurotransmission. In the following
sections, notable sex differences in the pharmacological treatment with these med-
ications are presented.
3.1 Sex Differences in Pharmacokinetics
Several sex differences in antidepressants’ pharmacokinetic properties have been

reported, such as in absorption, distribution, metabolism, and excretion of these
drugs (Khan et al. 2005; Kokras et al. 2011; Sramek et al. 2016). In fact, it is
suggested that some inconsistent findings regarding sex differences in the pharma-
codynamics of antidepressants might actually be due to sex differences in pharma-
cokinetics. In particular, it has been shown that gastric acid secretion is significantly
more restricted in women and that the transit time in the gastrointestinal tract is also
prolonged, thus significantly reducing the maximum concentration of drugs in the
blood (Hutson et al. 1989; Kokras et al. 2011). Furthermore, antidepressants’
bioavailability is considerably increased in females (Nicolas et al. 2009; Marazziti
et al. 2013). Similarly, increased body fat mass in women causes more rapid and
extensive distribution of lipophilic molecules, such as antidepressants, lower redis-
tribution rate and lower clearance, and overall, antidepressants’ absorption and
distribution are significantly affected by hormonal fluctuations during women's
menstrual cycle (Anderson 2005; Nicolas et al. 2009). Interestingly, it has been
shown that antidepressants’ binding to proteins is weakened in women, increasing
the fraction of unbound drug molecules (Kristensen 1983).
Sex differences in pharmacokinetics have also been reported due to proteins’
levels in the blood-brain barrier (BBB) of the P-glycoprotein (P-gp) transporter. P-gp
is part of the efflux mechanisms of antidepressants from the brain (Meibohm et al.
2002). Two studies suggest that decreased levels of P-gp’s substrates, such as
fluoxetine, fluvoxamine, and venlafaxine, in the brain are due to the active role of
the transporter in antidepressant’s efflux (Uhr et al. 2007; Sarginson et al. 2010).
Furthermore, sex hormones have been detected to cause decreased P-gp levels by
affecting the expression of multidrug resistance protein 1 (MDR1) gene expression,
although simultaneously they were found to increase drug absorption, via inhibition
of P-gp’s actions in the gastrointestinal tract (Nicolson et al. 2010). Similar to drug
absorption and distribution, sex differences in the metabolism of antidepressants
have been attributed to genetic polymorphisms, confounding diseases, hormonal
effects, such as hormonal fluctuation during the menstrual cycle, interactions with
concomitant prescribed treatments, and dietary, as well as epigenetic factors
(Waxman and Holloway 2009; Pitychoutis et al. 2010). Sex differences are also
present in drug excretion. The excretion of drug molecules is dependent on their
polarity, with polar molecules been excreted through renal function, whereas
non-polar molecules via the liver (Nicolson et al. 2010). It is known that women
have decreased blood flow in the organs mentioned above, in comparison to men,
affecting the volume of hepatic parenchyma and renal microtubular infiltration. Such
differences have been proposed to furnish reduced metabolism of antidepressants in
women (Schwartz 2007). Overall, it seems that women are exposed to higher levels
of antidepressants than men, when they are prescribed the same dose, but no
guidelines have ever been issued regarding different dosing of antidepressants in
men and women.
3.2 Sex Differences in Pharmacodynamics
Similar to pharmacokinetics, sex differences have also been reported for the phar-
macodynamics of antidepressants (Bigos et al. 2009; Kokras et al. 2011). In general,
the majority of clinical studies point out that men respond better to TCAs and that
women respond better to SSRIs (Kornstein et al. 2000; Khan et al. 2005; Berlanga
and Flores-Ramos 2006; Young et al. 2009). However, there is no consensus on this
matter, as the literature on sex differences in antidepressant effects still remains
inconclusive (Parker et al. 2003; Kornstein et al. 2014). Many researchers identify
the lack of stratification in clinical trials, according to age and women’s hormonal
status, as one of the main reasons behind the variability in findings on sex differences
in antidepressants (Thase et al. 2005). When age and hormonal status were taken into
account postmenopausal women were found to respond better to TCAs, such as
imipramine, whereas younger women responded more favorably to MAOIs and
SSRIs (Kornstein et al. 2000). Moreover, another study by Naito et al. (2007)
reported that older women (44 years of age) and men with MDD respond less to
fluvoxamine compared to younger female patients (Naito et al. 2007). Interestingly,
a study by Thase et al. (2005) reported that administration of hormonal replacement
therapy enhances the effects of SSRIs in older women (50), leading to comparable
remission rates among men and women of all age groups (Thase et al. 2005).
Furthermore, Williams and Trainor (2018) recently proposed that estrogens enhance
antidepressants’ efficacy in menopausal women and that the co-administration of
estrogens and antidepressants may be more efficacious than antidepressants alone
Fig. 1 Sex differences in sertraline’s effect in the immobility duration during the Forced Swim
Test. Male (a) rats have a modest response to sertraline, as reflected by the overlap of 66% (light
blue area) between vehicle-treated (blue curve) and antidepressant-treated (white curve) male rats.
Female (b) rats have a higher response to sertraline than males, indicated by the reduction of
immobility duration (shift of the white curve toward the left of the corresponding darker gray
curve). In females, there is an overlap of only 13% (lighter pink area) between vehicle-treated (pink
curve) and antidepressant-treated (white curve) rats. After treatment, 20% of males still present
more immobility than the average vehicle-treated male rat, whereas none of the antidepressant-
treated females presents more immobility than the average vehicle-treated female rat. Adapted
under license from (Kokras and Dalla 2017)
(Williams and Trainor 2018). Another study also proposed that administration of
17-β estradiol to perimenopausal women with depression exert a prolonged antide-
pressant effect as a stand-alone treatment (Soares et al. 2001). Similar results have
also been reported for menopausal women with MDD (Keating et al. 2011). This
synergistic effect of hormones and antidepressants has also been reported by some
clinical studies demonstrating improved effects of sertraline, fluoxetine, and imip-
ramine, in combination with estrogens’ treatment in perimenopausal women with
depression (Schneider et al. 1997, 2001; Stahl 1998; Richardson and Robinson
2000). Unfortunately, few other studies fail to recapitulate such synergistic effect
(Shapira et al. 1985; Amsterdam et al. 1999), maybe due to administration of
different types of estrogens. Interestingly, Frokjaer et al. (2015) suggested that the
sex-steroid hormone fluctuations can provoke depressive symptoms, which were
associated with increased cerebral serotonin transporter (SERT) binding (Frokjaer
et al. 2015). Such findings imply a differential response to antidepressants that target
SERT between women with regular menstrual cycles and men.
In preclinical studies, behavioral tests such as the Forced Swim Test (FST) and
the Tail Suspension Test (TST) are commonly used to study the effects of antide-
pressants in animals. In these behavioral tests, which are sensitive in sex differences,
antidepressants reduce immobility levels (Dalla et al. 2010; Saland et al. 2017;
Williams and Trainor 2018). Evidently, at baseline, female Wistar rats present
significantly higher levels of immobility and lower counts of head shakes during
FST in comparison to males (Dalla et al. 2008; Kokras et al. 2012, 2015) (see Fig. 1).
Furthermore, female rats and mice respond more favorably to acute administration of
fluoxetine, paroxetine, or sertraline, compared to males, since lower doses are
required to cause an antidepressant effect (David et al. 2001; Gómez et al. 2014;
Kokras et al. 2015; Fernández-Guasti et al. 2017). Similar to SSRIs, few studies have
described sex differences in the antidepressant response to ketamine, with female
animals presenting higher sensitivity to ketamine than males (Carrier and Kabbaj
2013).
Sex differences in antidepressant effects have also been reported in animal studies
using chronic drug administration to alleviate already established depressive-like
phenotypes [for review (Eid et al. 2019)]. In various preclinical models of depression
or stress exposure, chronic SSRI administration exerts in the FST a more restricted
behavioral effect in female animals compared to males (David et al. 2009; Mahmoud
et al. 2016; Wainwright et al. 2016). However, Mahmoud et al. (2016) reported a
small difference in treatment effects of SSRIs between sham-operated (controls) and
ovariectomized animals both on behavior and neuronal phenotypes (Mahmoud et al.
2016). Interestingly, in the social isolation stress model, female rats required lower
doses of ketamine for the alleviation of depressive-like symptomatology compared
to male rats (Sarkar and Kabbaj 2016). Lastly, another study reported longer-lasting
antidepressant-like effects of ketamine in male mice relative to females after expo-
sure to chronic mild stress (Franceschelli et al. 2015).
Finally, regarding the potential synergistic effect of hormones with antidepres-
sants, a preclinical study showed that co-administration of ineffective doses of
fluoxetine and desipramine with sub-threshold doses of an estrogenic compound,
such as 17β-estradiol, ethinylestradiol or diethyl-stilbestrol, in ovariectomized
female rats resulted in antidepressant-like effects in the FST (Estrada-Camarena
et al. 2004). Similarly, exogenous testosterone administration in control and chron-
ically stressed male rats has been shown to improve anxiety and depressive-like
behaviors (Frye and Walf 2009; Wainwright et al. 2016).
3.3 Sex Differences in Side Effects of Antidepressants
Sex differences are also evident in side effects following antidepressant treatment.
Women tend to experience more frequent and more severe side effects compared to
men (Kando et al. 1995). For example, women are more susceptible to drug-induced
arrhythmias due to pharmacotherapy compared to men (Makkar et al. 1993; Drici
and Clément 2001). Moreover, women with dysthymia present a more intense
withdrawal syndrome after paroxetine discontinuation than men (Bogetto et al.
2002), whereas such sex differences also extend to nausea and reduced libido
(Kornstein et al. 2000). On the contrary, men are more prone to develop indigestion,
sexual dysfunction, and urinary tract disorders during antidepressant treatment
(Montejo-González et al. 1997).
3.4 Hormonal Milieu and Antidepressants
Several studies attribute the existence of sex differences in depression and anxiety to
the effects of sex hormones. For instance, the hormonal status of female patients with
depression, due to aging, has been directly linked with differential effects of
antidepressant treatment (see above in Sect. 3.2). Furthermore, substantial fluctua-
tions and reduction in circulating levels of ovarian hormones in women are associ-
ated with increased risk of depression, in particular during the postpartum, peri-, and
postmenopausal periods (Hendrick et al. 1998; Cohen et al. 2006; Soares 2014).
The most common hypothesis for the development of perimenopausal depression
is estradiol’s withdrawal. Specifically, it has been suggested that the fluctuating and
at the same time declining levels of estradiol are likely to trigger depressive episodes
(Schmidt 2005). Another hypothesis supports that a subset of women is more prone
to perimenopausal depression due to abnormally elevated estradiol levels (de Wit
et al. 2021). On the other hand, it has been suggested that women with postmeno-
pausal depression do not exhibit alterations in the Hypothalamic-Pituitary-Ovarian
(HPO) axis (Woods et al. 2008). Furthermore, older women with depression have
lower testosterone and DHEA-S levels than younger women. Interestingly, depres-
sive symptoms in younger women were associated with increased levels of DHEA-S
(Morrison et al. 2001). Low circulating testosterone levels have also been associated
with increased risk of developing anxiety- and stress-related disorders in men
(Shores et al. 2004; DiBlasio et al. 2008; Zarrouf et al. 2009). Moreover, hormonal
withdrawal has also been described as a key factor in the development of depression
in men, particularly in men with low testosterone (Westley et al. 2015).
Many preclinical studies commonly perform a bilateral removal of the ovaries, in
order to drastically reduce estrogens and progesterone, in order to simulate meno-
pause and mimic the postmenopausal period (da Rocha et al. 2012). As such,
gonadectomy in male and female rats has been associated with depressive-like
behaviors in FST (Li et al. 2011; Carrier and Kabbaj 2012). Similar to clinical
data, the effect of total deprivation of ovarian hormones has also been associated
with low response to antidepressants in rodent studies (Estrada-Camarena et al.
2010; Keating et al. 2011). Our group investigated whether letrozole, an aromatase
inhibitor that blocks the conversion of androgens to estrogens, has an effect on
depressive-like symptomatology and interferes with the antidepressant effect of
fluoxetine in normally cycling female rats. Interestingly, subacute letrozole admin-
istration exerted an antidepressant effect in FST, similar to the antidepressant effect
of chronic fluoxetine treatment (Kokras et al. 2014). Specifically, both subacute
letrozole and chronic fluoxetine treatments significantly decreased immobility dura-
tion and increased swimming behavior in cycling female rats. As swimming behav-
ior in the FST has been associated with serotonergic activity in the prefrontal cortex
(Mikail et al. 2012), it is possible that letrozole exerts its behavioral effects via
modulation of the serotonergic system.
Interestingly, sustained (7 days) and chronic (21 days) letrozole administration in
female rats did not affect depressive-like symptomatology in the FST (Kokras et al.
Fig. 2 Sex difference in head-shaking frequency during the rat Forced Swim Test. Male rats (blue
curve) exhibit a higher head shake frequency than female rats (pink curve). The purple area
indicates the overlap of only 37% between sexes in this behavior. Only 6% of male rats are more
female-like than the average female, and only 2% of females are more male-like than the average
male. Adapted under license from (Kokras and Dalla 2017)
2014; Chaiton et al. 2019). Age could also play a role in female’s response to
letrozole, and it is worth mentioning that Chaiton et al. (2019) used middle-aged
rats, in comparison to younger females used in Kokras et al. (2014), (2018a) studies
(Kokras et al. 2014, 2018a; Chaiton et al. 2019).
However, both acute and sustained letrozole administration increased head-
shaking frequency during the FST, a behavior correlated with testosterone levels
(Kokras et al. 2014). Previous work from our group indicated that at baseline males
shake their heads significantly more than females, and that head shaking is increased
during the estrous and proestrous phases of the estrous cycle in females (Kokras et al.
2009, 2012) (see Fig. 2). As head shaking in the open field has been linked to 5-HT2
receptor activity (Wieland et al. 1993), these data could support the association
between this FST behavior with the effect of sex hormones on the serotonergic
system.
Similar to peri- and postmenopausal depression, both low and high levels of
estradiol have been associated with postpartum depression. Specifically, the hor-
mone withdrawal hypothesis of postpartum depression was tested by mimicking the
profound reduction in estradiol levels via pharmacological manipulations in healthy
women. All women that were pharmacologically treated with a gonadotropin-
releasing hormone agonist developed depressive symptoms (Frokjaer et al. 2015).
Moreover, Bloch et al proposed that both estradiol and progesterone withdrawal
contribute to the pathophysiology of postpartum depression (Bloch et al. 2000). On
the other hand, studies investigating women with postpartum depression report high
levels of estradiol compared to controls (O'Hara et al. 1991; Heidrich et al. 1994).
Although several clinical studies support each hypothesis for the development of
peri- and postmenopausal and postpartum depression, the contradicting results can
be attributed to the lack of consistency in the experimental designs among studies.
Interestingly, recently certain recommendations and guidelines on how to accurately
assess the effects of menstrual cycle and how to evaluate depression in women
during the menopausal transition and postmenopausal period were published (Maki
et al. 2018; Schmalenberger et al. 2021).
3.5 Neuroestrogens in Depression and Anxiety
Besides the effects of peripheral sex hormones, “neuroestrogens” are produced

locally in the brain and have been proposed as potent modulators of neuronal
functions relevant to synaptic plasticity, cognition and neuroprotection in both
males and females (Srivastava et al. 2013; Arevalo et al. 2015). A recent study by
our group shout to differentiate the effects of gonadal- and extra-gonadal estrogens
(neuroestrogens) on FST behaviors. This study was performed in male and female,
sham-operated and gonadectomized rats. Although chronic peripheral administra-
tion of letrozole did not cause any effects on FST behaviors, castration in males and
letrozole treatment in ovariectomized females reduced head-shaking frequency.
Altogether these results implicate testosterone and extra-gonadal estrogens in the
expression of the head-shaking behavior during the FST (Kokras et al. 2018a).
Recent studies associate estrogen signaling with depression and anxiety disor-
ders. It has long been known that estrogens, as lipophilic steroid hormones, diffuse
through the plasma membrane and bind to intracellular estrogen receptors
(ER) alpha (ERα) and beta (ERβ) that function as transcription factors. Both these
receptors have been proposed to contribute to the pathophysiology of mood disor-
ders and especially in depression (Guintivano et al. 2014). Variants in the gene that
encodes the ERα have been linked with higher risk of developing depression in
women (Ryan et al. 2011; Levey et al. 2021).
In addition, activation of ERα via estradiol in rats was found to normalize
depressive-like behaviors during FST (Furuta et al. 2013). Furthermore, activation
of ERβ in female rodents has been also found to decrease immobility during the FST,
suggesting an antidepressant effects only in females (Walf et al. 2004; Hughes et al.
2008). However, a different receptor, G protein-coupled estrogen receptor 1 (GPER1,
also known as GPR30), which is a transmembrane receptor, was discovered in 2005
(Vrtačnik et al. 2014). Rapid neuroestrogen effects, necessary for the fine-tuning of
neuronal circuits, are now considered to be mediated via the newly-identified GPER1
(Tang et al. 2014; Alexander et al. 2017). GPER1 activation results in the
non-genomic rapid activation of multiple intracellular cascades, including the ERK,
mTOR pathways and others (Sellers et al. 2015; Wang et al. 2017). These findings
represent a significant paradigm-shift from the classical estrogen signaling model.
Indeed, preliminary evidence supports the GPER1 involvement in anxiety in male
and female mice, as well as in humans (Kastenberger et al. 2012; Kastenberger and
Schwarzer 2014). Regarding depression, one human study reported higher serum
GPER1 levels in male and female patients with depression, in comparison to
controls, as well as a positive correlation between GPER1 levels and depression
scores (Findikli et al. 2017). However, there are only a few animal studies investi-
gating a mood-elevating effect of GPER1 activation. Briefly, it is suggested that
GPER activation exerts antidepressant effects in intact male mice and ovariecto-
mized female rats, an effect that can be blocked by G15, which is a GPER1’s
antagonist (Dennis et al. 2009; Wang et al. 2021). Interestingly, GPER1-related
downstream signaling pathways implicated in rapid neuroestrogen effects (e.g.,
mTOR, ERK, PI3-kinase/Akt) are also implicated in ketamine’s rapid antidepressant
effects (Sarkar and Kabbaj 2016) (see Fig. 3). Overall, all the abovementioned
118
Fig. 3 Effects of typical antidepressants and key components of rapid-acting antidepressants’ mechanism on cellular and synaptic plasticity. Block of
P. Pavlidi et al.
norepinephrine (NE) reuptake transporter by SNRIs and serotonin (5-HT) reuptake transporter by SSRIs activate downstream signaling pathways, which
include the activation of a2 adrenergic receptors, serotonergic receptors, G-coupled proteins, adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP),
protein kinase A (PKA), and mitogen-activated protein kinases (MAPK) cascades, resulting in increased expression of CREB (cyclic AMP response element
binding protein), brain-derived neurotrophic factor (BDNF), and TrkB receptor. AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor)
receptor and NMDA (N-methyl-D-aspartate) receptor potentiation by glutamate upregulate the expression of BDNF and its release to the synaptic cleft,
respectively. Glutamate-dependent release of BDNF stimulates the TrkB-Akt-mTOR signaling pathway and causes a rapid increase in synaptic protein synthesis
and new synapse formation, while increasing the presentation of GluA1 receptor and presynaptic density protein 95 in the membrane. Stressed induced
augmentation of corticotropin-releasing hormone (CRH), glucocorticoids (GCs) and glucocorticoids receptor (GR) inhibits the cellular plasticity and resilience.
Similarly, stress acts as an inhibitor of upregulation of BDNF and TrkB expression (Agid et al. 2007; Duman 2018)
Sex Differences in Depression and Anxiety
119
findings support the significance of neuroestrogens’ rapid actions and their involve-
ment in depression and mood disorders.
3.6 Effect of Antidepressants on Hormonal Levels
Despite the multiple interactions of hormones and antidepressants, most preclinical

and clinical studies omit to investigate antidepressants’ influence on circulating
levels of sex hormones (Pavlidi et al. 2021). Only a small number of clinical studies
measures the levels of testosterone and estrogens prior to and after the administration
of antidepressants. Although some studies describe the impact of antidepressants’
administration on testosterone and estrogens’ levels, the exact effect of each antide-
pressant on hormonal levels is not known. Furthermore, most of the reported effects
of antidepressants on hormonal levels are conflicting, and this can be attributed to
variable treatment schemes and sampling methods.
Nevertheless, most studies converge that testosterone levels are more frequently
affected by antidepressant administration, compared to estrogens’ levels. Moreover,
preclinical studies suggest that SSRIs exert the highest influence on hormonal levels
compared to the rest of antidepressant drug classes [for review (Pavlidi et al. 2021)].
These findings could be associated with the well-known adverse effects of SSRIs on
sexual behavior. However, medications affecting the serotoninergic neurotransmis-
sion may impair sexual function without necessarily reducing the levels of sex
hormones, and sexual dysfunction affects both men and women (Lorenz et al.
2016). Moreover, sexual dysfunction may appear in different areas (i.e., erectile
dysfunction, anorgasmia, and decreased libido) and involve diverse neurobiological
mechanisms (Clayton et al. 2014). Therefore, more research is needed, ideally using
appropriate preclinical models (Kyratsas et al. 2013; Olivier and Olivier 2019).
4 Conclusion: Sex as a Biological Variable
Several clinical and preclinical studies confirm the existence of various sex differ-
ences in neurobiological mechanisms of depression and anxiety, along with sex
differences in antidepressant treatment response (Kokras and Dalla 2014). Unfortu-
nately, only recently, most preclinical studies started to include both sexes in animal
models of anxiety and depression. A significant emphasis has been given by the
National Institutes of Health (NIH) policy to consider sex as a biological variable in
preclinical investigation (Miller et al. 2017). Funding agencies, such as the NIH and
the European Union commission, vigorously request the inclusion of sex and gender
in experimental designs, aiming to support accurate investigations and facilitate
better understanding of disease and treatment (Clayton and Collins 2014). Further-
more, inclusion of sex and gender as a biological variable may significantly increase
the translation of findings from preclinical to clinical set ups, which in turn can lead
Fig. 4 The effects of considering sex as an important biological variable for the development of
novel treatments. (a) Novel investigational therapies tested preclinically only in male rodents (blue),
are more likely to fail in clinical studies, which include only or mainly one sex (e.g., women). (b)
On the contrary, novel investigational treatments that are tested in preclinical studies in both male
and female rodents (blue and pink) have enhanced probabilities to show a successful outcome in
clinical trials. In cases where male animals differ from female animals in the drug response,
subsequent clinical trials should be stratified by sex. On the other hand, in cases where males and
females do not differ, clinical investigations should have equal representation of both sexes.
Adapted under license from (Kokras et al. 2019)
to the development of more efficacious treatment for depression and anxiety disor-
ders (Kokras et al. 2019; Shansky 2019; Pawluski et al. 2020; Butlen-Ducuing et al.
2021) (see Fig. 4).
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Sex Differences in Psychosis: Focus
on Animal Models
Andrea Gogos and Maarten van den Buuse
Contents
1 Sex Differences in Schizophrenia Epidemiology, Symptomatology,
and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
1.1 Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
1.2 Sex Differences in Schizophrenia Symptoms and Epidemiology . . . . . . . . . . . . . . . . . . . 135
1.3 Sex Differences in Schizophrenia Pathophysiology: Post-Mortem Studies . . . . . . . . . 136
2 Sex Differences and Sex Hormone Effects in Behavioral Animal Models . . . . . . . . . . . . . . . . 138
2.1 Locomotor Hyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.2 Prepulse Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
2.3 Sex Differences in Genetic Animal Models of Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . 147
3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Abstract Most psychiatric illnesses, such as schizophrenia, show profound sex

differences in incidence, clinical presentation, course, and outcome. Fortunately,
more recently the literature on sex differences and (to a lesser extent) effects of sex
steroid hormones is expanding, and in this review we have focused on such studies
in psychosis, both from a clinical/epidemiological and preclinical/animal model
perspective. We begin by briefly describing the clinical evidence for sex differences
in schizophrenia epidemiology, symptomatology, and pathophysiology. We then
detail sex differences and sex hormone effects in behavioral animal models of
A. Gogos (*)
Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia
Department of Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Parkville, VIC, Australia
e-mail: andrea.gogos@florey.edu.au
M. van den Buuse
School of Psychology and Public Health, La Trobe University, Melbourne, VIC, Australia
Department of Pharmacology, University of Melbourne, Parkville, VIC, Australia
College of Public Health, Medical and Veterinary Sciences, James Cook University,
Townsville, QLD, Australia
https://doi.org/10.1007/7854_2022_305
134 A. Gogos and M. van den Buuse
psychosis, specifically psychotropic drug-induced locomotor hyperactivity and dis-

ruption of prepulse inhibition. We expand on the preclinical data to include devel-
opmental and genetic models of psychosis, such as the maternal immune activation
model and neuregulin transgenic animals, respectively. Finally, we suggest several
recommendations for future studies, in order to facilitate a better understanding of
sex differences in the development of psychosis.
Keywords Developmental models · Estradiol · Female · Genetic models ·

Locomotor hyperactivity · Male · Prepulse inhibition · Schizophrenia
1 Sex Differences in Schizophrenia Epidemiology,

Symptomatology, and Pathophysiology
1.1 Schizophrenia
Schizophrenia continues to be one of the most complex, debilitating neuropsychiat-

ric disorders in which its etiology and pathogenesis remain uncertain, despite
extensive efforts in its research over the past century. The lifetime prevalence of
schizophrenia appears low compared to other neuropsychiatric disorders (0.7% for
schizophrenia, 13% for major depression, 14% for any anxiety disorder (Alonso
et al. 2004; McGrath et al. 2008)). However, factors such as early onset, high
chronicity, poor prognosis, and slow progress in treatment development have
resulted in a tremendously high burden on the patients affected, families involved,
and healthcare systems on a global scale. Schizophrenia debilitates the patient’s
quality of life, with suicide being the single major cause of premature mortality
among patients (Pompili et al. 2009). Schizophrenia is being increasingly acknowl-
edged as a polygenic disorder in which multiple genes and gene variants, each with
small effect size, collectively contribute to disease risk (Risch 2000). Despite the
heterogeneity in symptomatology, schizophrenia is characterized by several clinical
dimensions including positive psychotic symptoms, negative symptoms, disorgani-
zation, and cognitive impairment. This chapter will focus on the positive psychotic
symptoms which comprise delusional beliefs, auditory hallucinations, and thought
disorder (APA 2013; Mueser and McGurk 2004). Although the complex pathophys-
iology of schizophrenia still remains unclear, it is well understood that dysfunctions
in a multitude of neurotransmitter systems are likely to contribute to the pathology
associated with schizophrenia, including the dopamine, glutamate, gamma-
aminobutyric acid (GABA), cholinergic, and serotonin systems (Benes and Berretta
2001; Howes and Kapur 2009; Moghaddam and Javitt 2012; Raedler et al. 2007).
Sex Differences in Psychosis: Focus on Animal Models 135
1.2 Sex Differences in Schizophrenia Symptoms

and Epidemiology
A rich literature elaborately describes sex differences in schizophrenia relating to

disease risk, onset, symptoms, course, and outcome, with an overall finding of a
favorable progression of illness in females [reviewed in Gogos et al. (2015, 2019);
Markham (2012); Ochoa et al. (2012); Sbisa et al. (2017); Sun et al. (2016)]. In
regard to disease onset, the incidence of schizophrenia is greater in males than
females (1.4:1 ratio) (McGrath et al. 2004). Studies have shown a robust sex
difference in the mean peak age-of-onset which for males occurs between the ages
of 18–24, approximately 4 years earlier than females (Eranti et al. 2013; Hafner
2003; Hambrecht et al. 1992; Szymanski et al. 1995). Further, females uniquely have
a second peak age-at-onset at 45–49 years (Hafner 2003; Hambrecht et al. 1992) and
have a higher risk of developing schizophrenia than males in the 50–70 years age
group (Thorup et al. 2007a; van der Werf et al. 2014). Sex differences in the first
clinical presentation of schizophrenia have been shown in a number of studies,
highlighting that these differences are consistent across countries and cultures
(Hambrecht et al. 1992; Goldstein and Link 1988; Zhang et al. 2012). Sex differ-
ences in symptomology have also been documented. Generally, males have been
reported to exhibit more severe negative symptoms, such as withdrawal, social
isolation, and poorer functioning, whereas females express more severe positive
and general psychopathological symptoms, such as impulsivity, paranoia, affective/
depressive symptoms, and obsessive thinking (Goldstein and Link 1988; Zhang
et al. 2012; Koster et al. 2008; Thorup et al. 2007b). Some studies, but not all (Gogos
et al. 2010a), have found that females with schizophrenia have fewer cognitive
deficits than males, particularly in memory domains (Han et al. 2012). It has been
proposed that in females, a later age-at-onset and presentation of affective symptoms
predict a better prognosis whereas in males, their earlier onset and prominent
negative symptoms predict a worse course of illness (Abel et al. 2010).
Sex differences in disease progression and prognosis have been observed, with
females showing shorter, less frequent hospitalizations (Szymanski et al. 1995;
Cotton et al. 2009; Usall et al. 2003) and less comorbid substance abuse (Thorup
et al. 2007a; Koster et al. 2008; Cotton et al. 2009) than males. Females experience
higher rates of remission and recovery than males (Carpiniello et al. 2012; Grossman
et al. 2008) and have better social functioning, including being married, employed,
and maintaining interpersonal relationships (Hafner 2003; Thorup et al. 2007a;
Cotton et al. 2009; Vila-Rodriguez et al. 2011). Determining the optimal antipsy-
chotic treatment dose is complex and differs based on sex and age, because of
various factors such as pharmacokinetics, pharmacodynamics, symptom profile,
adherence, hormone status, and comorbidities (Seeman 2004). Although, some
studies suggest that females may have a better treatment response than males
(Szymanski et al. 1995; Gattaz et al. 1994) and require lower doses of antipsychotics
for therapeutic effect (Seeman 2004; Smith 2010). Further, pre-menopausal women
responded better than post-menopausal women, regardless of treatment type or
illness chronicity (Ochoa et al. 2012). Males with schizophrenia experience more
brain morphological abnormalities than females, including greater ventricular
enlargement (Narr et al. 2001; Nopoulos et al. 1997), more severe frontal and
temporal lobe atrophy (Narr et al. 2001; Bryant et al. 1999), and greater abnormal-
ities in white matter microstructure (Kanaan et al. 2012; Kelly et al. 2018).
In light of these sex differences, it has become increasingly evident that sex
steroid hormones play an important role, namely that estradiol may protect females
against the development and severity of the illness [reviewed in Gogos et al. (2015,
2019); Sun et al. (2016)]. However, it should be recognized that sex differences may
be independent of sex hormone effects (Gogos et al. 2019; Davies and Wilkinson
2006). There are several biological, cultural, and environmental factors that may
underlie these sex differences, for example, sex-specific differences in comorbid and
psychosocial factors and use of social supports may explain some of the sex
differences observed. These issues highlight the need for further investigation into
sex differences using a systematic approach to control for confounding variables
(Gogos et al. 2019; Bale and Epperson 2017; Joel and McCarthy 2017). Animal
models provide a means to better understand the complex interaction of genetic,
environmental, and hormonal factors involved in the development of psychosis.
1.3 Sex Differences in Schizophrenia Pathophysiology:

Post-Mortem Studies
Few studies have addressed sex differences in brain pathophysiology in patients who
had schizophrenia. This may be due to the methodology used rather than a lack of
sex difference, although future studies are needed to clarify this. For example, while
most post-mortem studies use sex-matched controls and report no effect of sex, these
studies generally have two-thirds male to one-third female subjects, and thus may be
under-powered to detect a sex difference. Addressing the problem of low statistical
power in individual studies, a meta-analysis on sex differences in gene expression in
schizophrenia prefrontal cortex comprised data from six independent studies totaling
89 male controls, 90 male schizophrenia patients, 35 female controls, and 32 female
schizophrenia patients (Qin et al. 2016). This combined analysis found a sex by
diagnosis interaction where 23 genes were up-regulated and 23 genes were down-
regulated in the male group, but there were no changes in gene expression in the
female group. Changes included genes related to energy metabolism (ATP5B,
ATP5A1, ABCG2), GABAergic neurotransmission (GABARAPL1) and glutathi-
one redox mechanisms (GSTA4). However, the problem of low power was not
entirely circumvented as the authors suggested that the apparent lack of gene
expression changes in females was likely due to the smaller female sample size.
When the male sample was randomly reduced to 30 subjects per group there were no
significant differences in expression level of any gene and only showed significant
results when at least 60 subjects were included (Qin et al. 2016).
A recent, large study compared RNA-sequencing data from the dorsolateral

prefrontal cortex from 437 controls and 341 (122 female) patients who had schizo-
phrenia (collected from two distinct cohorts derived from four brain banks)
(Hoffman et al. 2022). While these authors found robust signatures of differentially
expressed genes for diagnosis (2,209 genes), and similarly for sex (686 genes), they
did not find a genome-wide statistically significant diagnosis by sex signature. It was
suggested that the effect size of sex differences in schizophrenia gene expression is
small, highlighting the challenge of identifying robust sex by diagnosis interactions
in even smaller samples. However, when performing network analysis, the authors
did find a diagnosis by sex signature of differentially expressed genes. Specifically,
they identified gene modules involving diverse pathways including neural nucleus
development, neuron projection morphogenesis, and regulation of neural precursor
cell proliferation (Hoffman et al. 2022). Unfortunately, there is no further description
given regarding the sex-specific genes/pathways implicated.
Some individual, smaller studies have reported on sex differences in post-mortem
findings in schizophrenia. In the anterior cingulate cortex from schizophrenia
patients, there was evidence of sex-specific regulation of the expression of small
non-coding RNA (Ragan et al. 2017). Specifically, they found that the expression of
a number of microRNAs and small nucleolar RNAs differed between male and
female schizophrenia cases or between female cases and controls (Ragan et al.
2017). Another study measured the expression of several GABAergic genes in the
anterior cingulate cortex from schizophrenia patients and found that males who had
schizophrenia had lower expression of GABA-Aα5, GABA-Aβ1, and GABA-Aε
compared to controls, whereas females who had schizophrenia had higher expres-
sion of GABA-Aβ1 and GAD67 compared to controls (Bristow et al. 2015). Others
have found that sex differences in gene expression occur regardless of psychiatric
diagnosis (control, schizophrenia, bipolar disorder, or major depressive disorder).
For example, cortical levels of the metabotropic glutamate 2 receptor (GRM2) gene
were higher in males compared to females (Dean et al. 2019) and catechol-o-
methyltransferase (COMT) gene expression in the cerebellum was higher in females
(Dempster et al. 2006). Thus, while data are limited, there is some evidence of
sexually dimorphic gene expression that warrants further research in schizophrenia.
Future post-mortem studies should aim to increase their sample size to have enough
power to detect a sex difference, use multiple brain regions to determine whether
changes are localized or widespread, and measure samples from various psychiatric
groups to determine whether changes are diagnosis-specific.
2 Sex Differences and Sex Hormone Effects in Behavioral

Animal Models
While there are challenges in attempting to model human disorders in rodents,

preclinical studies using animal models have vastly widened our options in explor-
ing novel therapeutics and exploring in greater depth the pathophysiological mech-
anisms that underpin schizophrenia (van den Buuse et al. 2005). There is currently
no single animal model that can encapsulate all facets of the illness, but instead there
are an estimated 20+ animal models, each reflecting certain phenotypes of the
disorder (Jones et al. 2011; Sotiropoulos et al. 2021). Typically, an ideal animal
model of any given human condition would possess robust evidence for three sets of
criteria or validity domains: face validity, predictive validity, and construct validity
(van den Buuse et al. 2005; Sotiropoulos et al. 2021; Willner 1986). Face validity
refers to the similarity in the animal’s behavior, etiology, biochemistry, symptom-
atology, and/or treatment with the human condition of interest. Predictive validity
refers to analogous outcomes of an intervention on the animal model/behavior and
human symptom; and construct validity addresses whether there is comparable
etiology or central pathophysiological mechanisms (van den Buuse et al. 2005;
Willner 1986). For example, psychotropic drug-induced locomotor hyperactivity
and disruption of prepulse inhibition are valid animal behavioral models of psycho-
sis (van den Buuse et al. 2005; van den Buuse 2010). Historically, the majority of
studies on these and other behavioral animal models have only used males or did not
analyze for male–female differences (Beery and Zucker 2011). Here we will focus
on studies which include analysis of sex differences and/or sex hormone effects.
2.1 Locomotor Hyperactivity
Locomotor activity of rats or mice can easily be quantified using technology such as
automated photocell cages or video-tracking. In its simplest form, assessment of
locomotor hyperactivity measures horizontal distance moved in an open field, with
more complex models adding individual behaviors such as zone preference, rearing,
and grooming. To fulfill the criterium of face validity, rodent locomotor hyperactiv-
ity is often compared to psychotic agitation. However, psychosis in humans does not
always coincide with behavioral hyperactivity. Instead, rodent locomotor hyperac-
tivity is better suited to study underlying neurochemical and neuropharmacological
mechanisms which overlap with those implicated in psychosis in humans,
i.e. construct validity. This is particularly the case for dopaminergic hyperactivity
which for many decades has been implicated as one of the main underlying mech-
anisms in psychosis. The common underlying mechanism of action of most anti-
psychotic drugs is dopamine receptor antagonism, highlighting dopaminergic
hyperactivity as a fundamental mechanism in psychosis (Howes and Kapur 2009;
Kapur and Mamo 2003). More recently, extensive imaging studies have confirmed
3000 SD rats Meth Meth 3000 C57Bl/6 mice Meth

Meth
Distance moved (cm)
saline
2000 saline 2000
1000 1000
0 0
-15 0 15 30 45 60 -60 -30 0 30 60 90 120
SD rats MK-801 MK801 C57Bl/6 mice MK-801

2000 2000
MK801
saline
Distance moved (cm)
1500 1500
saline
1000 1000
500 500
0 0
-15 0 15 30 45 60 -60 -30 0 30 60 90 120
Time after injection (min) Time after injection (min)
Fig. 1 Sex differences in locomotor hyperactivity induced by methamphetamine (Meth, top

panels) or MK-801 (bottom panels) in Sprague-Dawley (SD) rats or C57Bl/6 mice. Injection of
0.5 mg/kg of Meth or 0.05 mg/kg of MK-801 (left panels) induce markedly greater locomotor
hyperactivity in female than in male rats. In contrast, female mice only show modest and short-
lasting greater hyperactivity than male mice following injection of 2 mg/kg of methamphetamine
(top right panel) and show lower hyperactivity to 0.25 mg/kg of MK-801 treatment (bottom right
panel) compared to male mice. Graphs were compiled from unpublished data (n ¼ 32/sex for rats,
n ¼ 24/sex for mice). Drugs were injected subcutaneously in rats and intraperitoneally in mice
such a role and shown hyperactivity and hyperreactivity of subcortical dopaminergic

pathways in schizophrenia as well as other forms of psychosis (Abi-Dargham et al.
1998; Laruelle et al. 1999; McCutcheon et al. 2019; Weidenauer et al. 2017).
The best way to model dopaminergic hyperactivity in rats and mice is by studying
the relative effects of dopamine-releasing drugs such as amphetamine and metham-
phetamine, which can elicit profound locomotor hyperactivity in these animals
(Fig. 1). Thus, if a genetic or pharmacological model of psychosis shows enhanced
or reduced effects of these drugs on locomotor activity, this can be taken as indirect
evidence of dopaminergic hyperactivity or hypoactivity, respectively, in these
models, without the need for complex imaging protocols or direct measurements
of dopamine release such as by microdialysis (Rivera-Garcia et al. 2020). This
approach can be expanded by using other pharmacological challenges which also
introduces a level of neurotransmitter specificity that cannot be deduced from simply
studying baseline locomotor activity, which may be mediated by any number of
brain factors and neurocircuitry. While amphetamine-induced locomotor
hyperactivity is strongly dependent on dopamine transmission in the nucleus

accumbens (Kelly and Iversen 1976; Ouagazzal et al. 1994) and can be blocked
by typical antipsychotic dopamine receptor blockers, such as haloperidol, these
drugs do not have a similar specific effect against locomotor hyperactivity induced
by targeting other neurotransmitter systems. For example, the effect of MK-801 on
locomotor activity does not require dopamine (Ouagazzal et al. 1994; Chartoff et al.
2005) and was antagonized by the atypical antipsychotic, clozapine, but only by
doses of haloperidol which also reduce baseline activity, i.e. are sedative (O’Neill
and Shaw 1999). Another strategy to assess the importance of specific genes and
drugs for psychosis is to use alternative behavioral models, including prepulse
inhibition and similar techniques to measure information processing (see Sect.
2.2), as well as other approaches including brain connectivity imaging, induced
pluripotent stem cells, and big data approaches (Sotiropoulos et al. 2021).
2.1.1 Sex Differences and Hormone Treatments
Acute psychotropic drug-induced locomotor hyperactivity is also suited to explore

the effects of sex hormones. An important caveat here is that several studies have
shown that female rats display significantly greater baseline locomotor activity and
psychotropic drug-induced locomotor hyperactivity than males ((Gogos et al. 2017;
Milesi-Halle et al. 2007); see also Fig. 1) and that these sex differences may be
related to reduced clearance of methamphetamine and amphetamine in females
compared to males (Becker et al. 1982; Milesi-Halle et al. 2005). A similar effect
of sex is observed in mice although the difference is much less pronounced than in
rats (Fig. 1). Indeed, we recently showed no sex differences in brain methamphet-
amine levels following injection of a behaviorally active dose of the drug in mice
(Greening et al. 2021). It should be noted that it is common to require different doses
in mice than rats to see a comparable behavioral response (Fig. 1). However, further
studies examining sex differences in locomotor hyperactivity in rats and mice should
use a range of psychotomimetic drug doses.
Human studies on the effects of amphetamine have shown conflicting results with
some showing increased dopamine release and greater subjective effects following a
challenge dose of amphetamine in females vs. males (Riccardi et al. 2006; White
et al. 2002), although others failed to find consistent evidence for sex differences in
the effects of amphetamine on dopamine release (Rivera-Garcia et al. 2020; Smith
et al. 2019). Furthermore, one study found dopamine release to be higher upon an
amphetamine challenge in males than in females (Munro et al. 2006). Some of the
inconsistencies in the human literature may be caused by experimental variables
such as stage of the menstrual cycle of the females in the studies, the subregions of
the brain investigated, and downstream factors, such as GABAergic and
metabotropic glutamatergic activity (Zachry et al. 2021). To the best of our knowl-
edge, there are no reports on sex differences in the metabolism and pharmacokinetics
of psychostimulant drugs, such as methamphetamine and amphetamine, in humans
(Volkow et al. 2010).
Historically, striatal dopaminergic hyperactivity has been a prevailing theory to

explain psychotic symptoms (Howes and Kapur 2009; Kapur and Mamo 2003;
Laruelle and Abi-Dargham 1999). However, alongside it, the theory of
glutamatergic hypoactivity, particularly through N-methyl-D-aspartate (NMDA)
receptors, has received widespread attention. This was initially based on the obser-
vation that NMDA receptor antagonists, such as phencyclidine and ketamine, could
induce psychosis-like behavioral changes (Balu 2016). More recently, post-mortem
studies have shown molecular evidence for reduced NMDA receptor functionality in
schizophrenia (Balu 2016; Weickert et al. 2013). Interestingly, these molecular
deficits occurred independent of the sex of the subjects (Weickert et al. 2013) even
though several other studies have reported sex differences in NMDA receptor
expression and sex-specific effects of polymorphisms in glutamate related genes
on schizophrenia risk (for references, see Wickens et al. (2018)). An autoimmune
disease associated with high levels of anti-NMDA receptor antibodies and rapidly
progressive psychiatric symptoms including psychosis is four times more prevalent
in women than in men (Dalmau et al. 2019).
Similar to amphetamine and methamphetamine, some studies have reported
greater locomotor hyperactivity induced by acute treatment with NMDA receptor
antagonists, such as MK-801, in female rats ((Andine et al. 1999; Honack and
Loscher 1993); see also Fig. 1) although others showed similar responses to phen-
cyclidine (Gogos et al. 2017). These apparently discrepant findings could be related
to the challenge drug used to test sex differences in NMDA receptor-mediated
behavioral responses. Andiné et al. (1999) found markedly higher serum and brain
concentrations of MK-801 in female rats compared to male rats. This could mean
that apparent sex differences in the behavioral effects of MK-801 could simply be
due to reduced metabolism of the drug in female rats (Andine et al. 1999). Additional
studies using micro-injections of MK-801 into the brain (De Leonibus et al. 2001)
may circumvent this problem but so far such studies have not compared male and
female animals. In contrast to MK-801, Shelnutt et al. (1999) found that, while the
half-life of phencyclidine was longer in female rats than in male rats due to slower
metabolism, there were no sex differences in initial serum concentration and renal
clearance of the drug. These findings illustrate that in behavioral animal models, the
choice of drug challenge may markedly influence the results in terms of sex
differences. Moreover, the enhanced effect of MK-801 on locomotor hyperactivity
in female rats was not matched by a similar sex difference in mice ((van den Buuse
et al. 2017a) and Fig. 1)). Instead, here MK-801 induced locomotor hyperactivity
was found to be greater in male than in female mice, a difference which could be
reduced by castration in male mice (van den Buuse et al. 2017a).
We and others have previously studied the effects of a variety of sex steroid
hormones on locomotor hyperactivity acutely induced by amphetamine/metham-
phetamine or NMDA receptor antagonists in rats and mice (Table 1). These have
included estradiol, estrogen receptor modulators (SERMs), as well as testosterone.
For example, as shown in Table 1, male and female rats tend to have opposite
responses to estrogens (increased hyperactivity in males and attenuated hyperactivity
in females), although these studies do not compare both males and females in the
Table 1 Selected studies showing the effect of sex steroid hormones on locomotor hyperactivity
induced by amphetamine (Amph), methamphetamine (Meth), or NMDA receptor ligands (MK-801,
phencyclidine)
Female/
Species Model/finding male Reference
Rat E2, not T or DHT, increased Amph hyperactivity Male (Menniti and
only Baum 1981)
Rat E2 did not alter Amph hyperactivity Female (Gogos et al.
only 2012)
Rat T attenuated and RAL increased Amph hyperactivity Male (Purves-Tyson
only et al. 2015)
Rat E2, but not RAL or TAM, attenuated meth Female (Sbisa et al.
hyperactivity only 2018)
Mouse Castration reduced MK-801 hyperactivity, OVX had Male and (van den Buuse
no effect female et al. 2017a)
Mouse Amph hyperactivity reduced in male ERα KO, Male and (Georgiou et al.
enhanced in female ERα and ERβ KO female 2019)
Mouse Amph and phencyclidine hyperactivity reduced in Male and (Chavez et al.
female ArKO mice; only minor genotype differences in female 2009)
males
Mouse DHEA decreased Amph hyperactivity Female (Kilic et al.
only 2014)
Amph amphetamine, ArKO aromatase knockout, DHEA dehydroepiandrosterone, DHT dihydrotes-
tosterone, E2 estradiol, ERα estrogen receptor alpha, ERβ estrogen receptor beta, KO knockout,
Meth methamphetamine, OVX ovariectomy, RAL raloxifene, T testosterone, TAM tamoxifen
same experiment. Complementary studies have used removal of the main source of
circulating sex steroid hormones by gonadectomy or genetic knockout of aromatase
(converts testosterone to estradiol) or of estrogen receptors (Table 1). A study
measuring cocaine-induced locomotor hyperactivity concluded that ovarian hor-
mones, particularly estradiol, enhanced the locomotor response to cocaine in female
rats (Sell et al. 2000). Specifically, they found that hyperactivity was greater in
(a) females than males, (b) in ovariectomized females treated with estradiol or both
estradiol and progesterone than ovariectomy alone, and (c) during proestrus and
estrus (high estradiol/high progesterone phases) than diestrus in normally cycling
female rats (Sell et al. 2000). Several studies have extended these paradigms to
include animals self-administering these drugs (e.g. Hu and Becker 2003) but these
findings are not included here. Mechanisms underlying sex differences and sex
hormone effects in psychosis-like behavior may include dopamine transporter,
dopamine D2 autoreceptor and postsynaptic receptor expression, and vesicular
monoamine transporter (VMAT) 2 (Gogos et al. 2015; Zachry et al. 2021; Becker
et al. 2001; Chavez et al. 2010). The complexity of interaction of all these factors in
the living brain prevents a simple explanation of sex differences and hormone effects
on behavioral responses. Therefore, recent studies have started to use more compre-
hensive approaches such as RNA-sequencing or proteomics (Greening et al. 2021;
Carboni and Domenici 2016; Chalkiadaki et al. 2019; Humphreys et al. 2014; Huo
et al. 2018; Wesseling et al. 2013). In addition, an overview of the available
preclinical data (Table 1) shows that the role of sex steroid hormones may differ
between rats and mice. Although comprehensive species comparison studies will
need to be done to confirm this, species specificity of the preclinical data may have
implications for the interpretation of animal model findings for drug development
and clinical practice.
2.1.2 Locomotor Hyperactivity Studies in Developmental Models

of Psychosis
In addition to acute drug-induced psychosis-relevant behaviors, several chronic

developmental models have been studied. As with acute models, relatively few
studies address sex differences and even fewer investigate the effect of sex steroid
hormones. Prenatal administration of the DNA synthesis inhibitor,
methylazoxymethanol acetate (MAM), induces a range of behavioral and neuroan-
atomical effects with relevance to schizophrenia in the offspring (Lodge and Grace
2009). In contrast to male rats, following MAM administration at gestational day
(GD) 17 female offspring did not show enhanced amphetamine-induced locomotor
hyperactivity, suggesting a protective effect of the female sex against the develop-
ment of this schizophrenia endophenotype (Perez et al. 2019). In both control and
MAM offspring, the effect of amphetamine was greater during the metestrus stage of
the estrous cycle than during the proestrus stage, but this was not dependent on
prenatal pretreatment (Perez et al. 2019). These data show that even in the metestrus
stage of low circulating estradiol/progesterone levels, female MAM rats did not
show the enhanced amphetamine-induced locomotor hyperactivity seen in male
offspring. This is in contrast to midbrain dopamine neuron population activity in
the ventral tegmental area which is enhanced in female MAM offspring compared to
female control offspring (Perez et al. 2014), similar to what is observed in male rats
(Lodge and Grace 2009). Interestingly, injection of a progesterone antagonist, but
not of an estrogen receptor antagonist, into the ventral hippocampus attenuated
dopamine neuron population activity in the ventral tegmental area in female MAM
offspring (Perez et al. 2014). Although it remains unclear if similar effects are found
in control female offspring or similar hormone effects can be observed in male
MAM offspring. These effects of a progesterone antagonist are in line with previous
studies suggesting a role for progesterone, in addition to estrogen, in psychosis
development in females (Sun et al. 2016).
Effects of prenatal MAM have also been studied in mice (Chalkiadaki et al. 2019;
Huo et al. 2018). Female offspring exposed to MAM on GD16 showed greater
locomotor hyperactivity following an acute MK-801 challenge than control female
offspring, but this was not studied in males. This effect was not observed following
MAM treatment on GD17. Tests of hippocampal function were affected similarly in
male and female MAM offspring but prefrontal cortex function was impaired only in
males (Chalkiadaki et al. 2019). This study also included a proteomic analysis of
prefrontal cortex protein expression which revealed a greater number of proteins
down-regulated in male MAM offspring than in females, particularly genes related
to glutamatergic function. However, in mice exposed to MAM on GD17, spontane-

ous hyperlocomotion was more severely affected in females than in males and
transcriptomics analysis showed that twice as many genes were differentially
expressed in female MAM offspring than male MAM offspring (Huo et al. 2018).
Taken together, these studies (Chalkiadaki et al. 2019; Huo et al. 2018) suggest that
sex differences in the effect of a developmental insult like MAM are strongly
dependent on the timing of the treatment, consistent with well-known sex differ-
ences in neurodevelopment.
Similar conclusions regarding sex differences in the effect of neurodevelopmental
insults on psychosis-like behavior have emerged from models focusing on maternal
infection and immune responses (Hill 2016), i.e. the maternal immune activation
(MIA) model. The most commonly used of those includes injection of the viral
mimetic, poly(I:C), on a specific GD, with widespread behavioral and neurochem-
ical consequences in the offspring (for references, see Hill (2016); Meyer and Feldon
(2012); Meyer et al. (2009); Reisinger et al. (2015)). However, many of the available
studies use only male offspring or, when using both males and females, do not
systematically analyze for sex differences. Where males and females are compared,
variable results have been obtained regarding sex differences in drug-induced
locomotor hyperactivity in this model and some of these discrepancies may be
related to factors such as the choice of the acute drug challenge, (sub)strain of the
rats or mice tested, environmental housing and testing details, and even the batch of
poly(I:C) used. For example, in Long-Evans rat offspring following intravenous
treatment with poly(I:C) at GD15, enhanced methamphetamine-induced locomotor
hyperactivity was found similarly in males and females (Gogos et al. 2020). These
findings were in line with earlier studies in Sprague-Dawley rats (Richtand et al.
2011). In contrast, in Wistar rat offspring following intravenous injection of poly(I:
C) on GD15, MK-801-induced locomotor hyperactivity was markedly enhanced in
males (Lins et al. 2018) but not females (Lins et al. 2019), although different doses of
MK-801 were used between the sexes to account for differences in MK-801 metab-
olism (Andine et al. 1999). However, in Long-Evans rats, while Howland et al. also
used a higher acute dose of MK-801 in males than in females and the degree of
hyperactivity was comparable between the sexes, GD15 poly(I:C) had no effect in
either (Howland et al. 2012). In Sprague-Dawley rats, intraperitoneal injection of
poly(I:C) on days GD14–18 did not affect baseline locomotor activity but induced
enhanced sensitivity to amphetamine-induced locomotor hyperactivity in both male
and female offspring (Vorhees et al. 2015). In contrast, the effect of MK-801 was
attenuated in male and female poly(I:C) offspring (Vorhees et al. 2015). These
contradictory results have instigated a call to more carefully report and, where
possible standardize, a range of experimental variables which may influence the
rigor of the MIA model, including whether sex differences occur (Kentner et al.
2019).
2.2 Prepulse Inhibition
There are several cognitive processes aimed at focusing attention and minimizing
distraction by irrelevant sensory stimuli. Collectively, these processes of sensory
filtering prevent sensory overload and allow the individual to attend to the most
relevant and salient stimuli from the environment (Cromwell et al. 2008). Several
brain regions are involved in sensory gating, including the thalamus and frontal
cortex (Swerdlow et al. 1992, 2016). Experimentally, a range of neurophysiological
protocols are used to study different aspects of sensory gating, including paired-
pulse, P50 gating and mismatch negativity, which can all be measured in humans by
scalp electrophysiology. Prepulse inhibition (PPI) of acoustic startle is a measure of
sensorimotor gating which has been described as the ability to shield from sensory
over-stimulation (Light and Braff 1999). It can be studied similarly in rodents and
humans by measuring whole-body startle or eye-blink, respectively (Light and Braff
1999). While originally described as an endophenotype of schizophrenia, it has
become increasingly clear that several neurological and psychiatric conditions that
include psychotic features or cognitive deficits are associated with sensorimotor
gating deficits (Geyer 2006; Gogos et al. 2009). PPI has been widely used to study
sex differences and the effect of sex steroid hormones on sensory information
processing deficits in psychosis.
2.2.1 Sex Differences and Hormone Treatments
Several studies have shown that PPI is lower in women than in men (for references,
see Kumari et al. (2004)). Moreover, PPI in women varies with the menstrual cycle,
with highest levels (although still lower than men) seen in the follicular phase
(Swerdlow et al. 1997). The well-described reduction of PPI in schizophrenia is
much more prominent in males than in females (Kumari et al. 2004) and this is
unlikely to be simply the result of the lower baseline PPI in women compared to men
(Kumari et al. 2004). Rather, these results could indicate an inherent protective
action of female sex steroid hormones limiting sensorimotor gating deficits in
schizophrenia in women. We investigated this hypothesis in a cohort of healthy
women, who were tested during the low-estradiol/progesterone stage of their men-
strual cycle (Gogos et al. 2006). Acute treatment with the serotonin-1A receptor
partial agonist, buspirone, induced a schizophrenia-like decrease of PPI. While acute
treatment with estradiol had no significant effect on baseline PPI, it prevented the
buspirone-induced reduction of PPI (Gogos et al. 2006). These results in humans
were corroborated by more extensive studies in rats and mice (Table 2). Similar to
humans, changes of PPI are also found across the estrous cycle in rats (Koch 1998),
although the changes are modest and not found in mice (Plappert et al. 2005).
Removal of sex steroid hormones by gonadectomy or aromatase knockout (ArKO)
does not alter baseline PPI (Table 2). However, chronic treatment with estradiol or
the SERM raloxifene reduced disruption of PPI by a number of pharmacological
Table 2 Selected studies in rats and mice on the effect of sex steroid hormones on PPI and drug-
induced disruption of PPI
Female/
Species Model/finding male Reference
Rat • Ovariectomy did not alter PPI Female (Van den Buuse
• Acute E2 increased PPI; effect mimicked by T but only and Eikelis 2001)
not DHT
• Acute E2 increased PPI following APO or MK-801
Rat High dose E2 or low dose E2 plus P prevented dis- Female (Gogos and Van
ruption of PPI by 8-OH-DPAT in OVX rats only den Buuse 2004)
Rat E2 prevented disruption of PPI by APO and Female (Gogos et al.
8-OH-DPAT in OVX rats only 2010b)
Rat E2 reduced and T increased MK-801 induced disrup- Male (Gogos et al.
tion of PPI and 2012)
female
Rat E2 plus P prevented ketamine-induced disruption of Female (van den Buuse
PPI in OVX rats only et al. 2015)
Rat E2 treatment prevented MIA-induced disruption of Female (Sbisa et al.
PPI in intact rats only 2020)
Mouse MK-801 disrupted PPI in intact and castrated male Male (van den Buuse
mice and OVX female mice, but not in intact female and et al. 2017a)
mice female
Mouse Female ArKO mice showed reduced disruption of PPI Male (Chavez et al.
by APO and Amph; no genotype effect in males and 2009)
female
Mouse • ERα agonist increased and ERα antagonist Male (Labouesse et al.
decreased baseline PPI only 2015)
• ERβ modulators had no effect on baseline PPI
• Both ERα and ERβ modulators inhibited Amph-
induced PPI disruption
Mouse • Female ERα KO had reduced PPI Male (Georgiou et al.
• Female ERβ KO had enhanced PPI and 2019)
• Male ERα or ERβ KO showed no difference in PPI female
8-OH-DPAT serotonin-1A receptor agonist, Amph amphetamine, APO apomorphine (dopamine
D1/D2 receptor agonist), ArKO aromatase knockout, DHT dihydrotestosterone, E2 estradiol, ERα
estrogen receptor alpha, ERβ estrogen receptor beta, KO knockout, MIA maternal immune activa-
tion, OVX ovariectomized, P progesterone, T testosterone
challenges, including apomorphine, 8-OH-DPAT, and MK-801 in rats (Gogos et al.

2010b, 2012; Sbisa et al. 2018; Gogos and van den Buuse 2015).
2.2.2 PPI Studies in Developmental Models of Psychosis
Sex-specific effects on PPI have been studied in only a few chronic schizophrenia
animal models. For example, MAM treatment on GD17 resulted in a PPI deficit in
both male and female mice (Huo et al. 2018), similar to what was seen following
GD16 treatment in rats (Lodge and Grace 2009; Perez et al. 2019). However, the
possible restoration of disrupted PPI in these animals (or other developmental
models of schizophrenia (Hill 2016)) by concurrent hormone treatment has not
been studied. Similar to the MAM model, some studies have shown that the degree
of disruption of PPI in the MIA model was not different between male and female
offspring (Gogos et al. 2020; Howland et al. 2012; Ratnayake et al. 2014). However,
this may be related to the timing of the poly(I:C) treatment. Meehan et al. (2017)
showed that PPI was reduced in male Wistar rat offspring following maternal poly(I:
C) treatment at either GD10 or GD19, representing early gestation and very late
gestation, respectively. This disruption of PPI was not observed in female offspring
and, instead, PPI tended to be increased in the female GD10 group while no effect of
MIA was observed in the female GD19 group (Meehan et al. 2017). Also others have
shown a lack of effect of MIA on PPI, either in males or females (Lins et al. 2018,
2019) and overall this has emphasized the importance of factors such as the
gestational timing of the MIA insult, as well as rat or mouse strain, and poly(I:C)
dose, in determining differential effects on PPI in male vs. female offspring (Hill
2016; Meyer and Feldon 2012; Kentner et al. 2019; Choudhury and Lennox 2021).
In offspring from GD15 treated Long-Evans rats (Gogos et al. 2020), we found
significantly reduced PPI in both males and females. Here, chronic estradiol treat-
ment effectively reduced the disruption of baseline PPI found in this model (Sbisa
et al. 2020) and similar effects were seen after treatment with the SERM, raloxifene
(Sbisa 2017). In C57Bl/6 mice, raloxifene treatment in female offspring following
poly(I:C) treatment on GD17 also reduced deficits in gamma power during a
cognitive task (Schroeder et al. 2019). These findings are in line with clinical effects
of these steroids to reduce schizophrenia symptoms (for references, see Gogos et al.
(2015); Sbisa et al. (2017); Kulkarni et al. (2019)). Importantly, the advantage of
SERMs, such as raloxifene, over estradiol is that these compounds have a lower
endocrine side effect profile and may therefore also be useful in men with schizo-
phrenia (de Boer et al. 2018; Weickert et al. 2015).
2.3 Sex Differences in Genetic Animal Models

of Schizophrenia
Post-mortem studies and genome-wide association studies have identified a plethora

of genes potentially involved in schizophrenia development and symptoms. This has
led to the development of many genetic animals with constitutive global or localized
knockout of such factors in the brain. It is beyond the scope of this paper to
comprehensively list these models and the reader is referred to recent reviews for
references (e.g. van den Buuse (2010); Hill (2016); Chen et al. (2006); Powell et al.
(2009)). Of these, Hill (2016) specifically reviewed sex differences in a number of
genetic mouse models. Here we will only add an update on some of the most widely
used genetic mouse models of schizophrenia: reelin, neuregulin, DISC1, and BDNF.
While there are sex differences in the influence of some of these genes on locomotor
hyperactivity and PPI, further studies are required to confirm the role of each sex.
2.3.1 Reelin
Reelin has been implicated by several studies to be involved in the development of

schizophrenia and other neuropsychiatric illnesses (Ishii et al. 2016). In humans,
common variants of the reelin gene were shown to increase risk of schizophrenia
development in females, but not males (Shifman et al. 2008) and reelin levels have
been reported to be decreased in schizophrenia (Eastwood and Harrison 2006).
Recently, we used reelin heterozygous mice (HRM) to further investigate any
sex-specific genotype differences in psychotropic drug-induced locomotor hyperac-
tivity and disruption of PPI (Hume et al. 2020). HRM are a better model of the
moderate reduction of reelin function in schizophrenia than mice with full knockout
of reelin expression (reeler mice), which have marked disruption of cell migration
during development and cortical layering in adulthood. We observed no male–
female differences in either HRM or controls in their response to acute metham-
phetamine treatment in baseline PPI (Hume et al. 2020). Indeed, other than a small
increase in baseline locomotor activity, neither male nor female HRM were different
from sex-matched wildtype controls (Hume et al. 2020). As is common with
constitutive knockouts, it is possible that compensatory mechanisms diminished
any effect of the reduction in reelin in these mice and that another “insult” is needed
to unmask its effects. This was confirmed by the observation that adolescent
treatment with corticosterone, to simulate chronic stress, induced a number of
selective molecular changes, including downregulation of glucocorticoid receptors
and NMDA receptor GluN1, GluN2B, and GluN2C subunits in female HRM, but
not male HRM, compared to sex-matched wildtype controls (Schroeder et al. 2018).
In contrast, corticosterone treatment reduced baseline PPI in male controls, but not
male HRM, and had no effects on PPI in female mice (Schroeder et al. 2015). The
downregulation of NMDA receptor subunits is consistent with a sex-specific inter-
action of reelin with NMDA receptor-mediated regulation of PPI. Indeed, the
MK-801-induced locomotor hyperactivity was significantly enhanced in male
HRM compared to wildtype controls (van den Buuse et al. 2012), however there
were no differences between female HRM and controls. In contrast to locomotor
hyperactivity, MK-801-induced PPI disruption and [3H]MK-801 binding were not
altered in either male or female HRM (van den Buuse et al. 2012). These and other
(Bosch et al. 2016; Notaras et al. 2020) results suggest selective and strictly
sex-specific effects of reduced reelin levels in mouse models of schizophrenia with
a likely neurochemical mechanism being altered NMDA receptor function, consis-
tent with the NMDA receptor hypoactivity hypothesis of schizophrenia.
Although the effect of treatment with sex steroid hormones on schizophrenia-like
behaviors in reelin-deficient mice has not been reported, it is of interest that
treatment with estradiol increased the number of Purkinje cells in male, but not
female HRM, with no effects in wildtype mice (Biamonte et al. 2009). Conversely,
treatment with anti-estrogens reduced Purkinje cell numbers in both female HRM
and wildtype mice, but not in male HRM or controls (Biamonte et al. 2009). Female,
but not male HRM showed increased expression of brain-derived neurotrophic
factor (BDNF) and reduced phosphorylation of the BDNF receptor, TrkB, in the
ventral hippocampus (Hill et al. 2013). The greater BDNF levels in the ventral
hippocampus of female HRM were significantly reduced by ovariectomy to the
levels of intact wildtype controls. Furthermore, estradiol treatment restored the
increased levels of BDNF in female HRM (Hill et al. 2013). Other studies have
shown that estradiol treatment increased reelin expression in the dentate gyrus
(Bender et al. 2010). These data suggest a gene x gene x environment interaction
of reelin with BDNF and sex steroid hormones, particularly estradiol, ultimately
contributing to its purported role in schizophrenia.
2.3.2 Neuregulin
Following the identification of neuregulin 1 (NRG1) as a potential genetic risk factor

in schizophrenia, several genetically-modified mouse models have been developed
and studied in the context of schizophrenia-relevant behaviors (Hill 2016). The large
number of mouse models is in line with the complexity of this growth factor family
which includes four main paralogs (Nrg1–Nrg4) and a multitude of splice variant
isoforms, as well as a large group of tyrosine kinase receptors through which the
neuregulins signal. Early observations on NRG1 hypomorphs produced varying
results and studies often only included males. More recent studies found that
baseline locomotor activity was moderately enhanced in NRG1 mice (O’Tuathaigh
et al. 2006; van den Buuse et al. 2009) with minor sex-specific shifts of individual
components of exploratory behaviors (O’Tuathaigh et al. 2006). These mice also
showed moderately decreased responsiveness to the acute locomotor hyperactivity-
inducing effect of phencyclidine in males compared to controls, but female mutants
tended to be more sensitive to MK-801 (O’Tuathaigh et al. 2010). A different
transmembrane mutant model showed no genotype effects or sex differences in
locomotor hyperactivity induced by either methamphetamine or MK-801 (see Pei
et al. (2014) for data and comparison with earlier NRG1 mutant models). A
hypomorphic type II NRG1 mutant model showed reduced PPI in females, but not
males, compared to wildtype controls (Taylor et al. 2011). It has been argued that
neuregulin haploinsufficiency does not appropriately model deficits in this protein
family in schizophrenia (Law 2014) and later studies have consequently focused on
overexpression models and specific splice variants. Disappointingly, the majority of
those studies only used males or did not distinguish between male and female mice.
Fortunately, there are some exceptions. For example, in two separate publications,
respectively, the behavioral phenotype of male and female type III NRG1
overexpressing mice was described (Olaya et al. 2018a, b). Compared to wildtype
controls, male mutants did not show altered sensitivity to the locomotor hyperactiv-
ity induced by MK-801 treatment (Olaya et al. 2018b) but this response was reduced
in female mutants (Olaya et al. 2018a). Male (Olaya et al. 2018b), but not female
(Olaya et al. 2018a), type III NGR1 overexpressing mice showed disruption of
baseline PPI. In other studies, this group showed that male type III NRG1
overexpressing mice had a greater disruption of PPI following methamphetamine
challenge whereas there was no significant genotype difference in females
(Chesworth et al. 2021). Conversely, methamphetamine-induced locomotor hyper-
activity was not altered in male type III NRG1 overexpressing mice, but this
response was reduced in females (Chesworth et al. 2021). In type I NRG1
overexpressing mice, there was no difference in baseline exploratory locomotor
activity in males, although females tended to show reduced baseline activity (Deakin
et al. 2009). The effect of either dopaminergic or NMDA receptor drugs was not
studied in these mice. These studies illustrate the importance of comparing males
and females of a genetic model of schizophrenia risk genes.
2.3.3 DISC1
As its name suggests, disrupted in schizophrenia (DISC1) gene has been implicated
in the development of the illness (Chubb et al. 2008; Ishizuka et al. 2006) and this
involvement was particularly strong in females with schizophrenia (e.g. Ram Murthy
et al. 2012). Several studies have addressed the role of DISC1 in animal models (for
references, see Hill (2016); Lipina and Roder (2014); Tomoda et al. (2016)) and a
systematic review recently concluded that there is strong evidence for altered
dopamine transmission in this model (Dahoun et al. 2017). However, there was no
change in amphetamine-induced dopamine release although the majority of studies
reported enhanced amphetamine-induced locomotor hyperactivity, especially in
females (Dahoun et al. 2017). Similar to other schizophrenia risk genes,
endophenotypes of the illness are often revealed or exacerbated by a second devel-
opmental “hit.” For example, while most studies on animal models of deficient
DISC1 function use mice, recently a DISC1 overexpressing rat model was described
(Uzuneser et al. 2019). Female DISC1 overexpressing rats showed enhanced
amphetamine-induced locomotor hyperactivity compared to wildtype controls, but
only if they had been exposed to an adolescent immune challenge in the form of
lipopolysaccharide treatment, suggesting a two-hit like vulnerability caused by the
genetic factor. This effect was not seen in male rats, where DISC1 overexpression
either on its own or in combination with a second “hit” had no effect (Uzuneser et al.
2019). PPI was enhanced in female, but not male DISC1 overexpressing rats
compared to sex-matched controls (Uzuneser et al. 2019). Marked sex differences
were also observed in a rat DISC1 knockout model. Here, female knockouts showed
enhanced spontaneous exploratory locomotor activity in an open field, but little
change in PPI. Conversely, male knockouts showed little change in locomotor
activity but a marked reduction of PPI (Ram Murthy et al. 2012), once again
emphasizing the need to compare both sexes in studies on preclinical behavioral
endophenotypes of schizophrenia.
2.3.4 BDNF
Although BDNF is not found in genome-wide association studies as a risk gene for
schizophrenia (Fabbri and Serretti 2017), its widespread role as a trophic factor
involved in brain development and neuroplasticity (Nieto et al. 2013; Notaras and
van den Buuse 2019), as well as its interaction with dopaminergic and glutamatergic
activity, suggests an important role as an intermediary of multiple pathological
processes in this illness (Angelucci et al. 2005; Notaras et al. 2015). Post-mortem
studies have generally shown reduced BDNF expression. For example, Weickert
et al. (2003) showed significantly reduced BDNF gene expression and protein levels
in the dorsolateral prefrontal cortex of subjects with schizophrenia and further
comparison of 8 females and 9 males in the sample revealed no sex differences in
this change (Weickert et al. 2003). Similarly, Hashimoto et al. (2005) showed a 28%
decrease of expression of BDNF in the dorsolateral prefrontal cortex, with a repli-
cation study confirming this finding and showing a 44% decrease in the schizophre-
nia sample. Further analysis of the combined sample showed that this change was
similarly seen in 7 males vs. 19 females with schizophrenia (Hashimoto et al. 2005).
A meta-analysis showed that BDNF levels in blood were significantly lower in both
males and females with schizophrenia with no difference between the sexes (Green
et al. 2011).
The lack of sex differences in post-mortem and clinical studies on BDNF levels in
schizophrenia is surprising as several other studies have shown an interaction of sex
steroid hormones with BDNF expression (Wu et al. 2013). However, sex differences
in the role of BDNF may be more important for cognitive deficits in the illness rather
than positive symptoms. This is supported by preclinical studies on the effects of
psychotropic drugs on locomotor activity and PPI comparing mice with reduced
BDNF levels or with the common BDNF val66met polymorphism to controls. For
example, in BDNF heterozygous mice, whose BDNF levels in the brain are only
approximately 50% of those in wildtype controls, amphetamine-induced locomotor
hyperactivity was found to be prolonged in one study (Saylor and McGinty 2008)
and enhanced in another (Manning et al. 2016). However, this effect was similarly
found in males and females (Du et al. 2019) and analogous observations were found
in a BDNF val66met mouse model (Greening et al. 2021). With respect to PPI, we
observed no differences between male and female BDNF heterozygous mice and
controls in terms of baseline PPI or its disruption by acute MK-801 treatment (Klug
et al. 2012). Similarly, baseline PPI or its disruption by apomorphine (Gururajan
et al. 2015) or MK-801 (van den Buuse et al. 2017b) was not different between male
and female BDNF heterozygous rats and controls. In other experiments, baseline PPI
was found to be lower in both male and female BDNF heterozygous mice compared
to wildtype controls (Manning and van den Buuse 2013). Baseline PPI was similarly
lower in mice with the BDNF val66met polymorphism although this was only
significant for the val/met genotype and not the more extreme met/met genotype
which may have induced compensatory mechanisms (Notaras et al. 2017). There
were no sex differences in these genotype effects (Manning and van den Buuse
2013; Notaras et al. 2017). These results in behavioral models of psychosis contrast
with several studies showing sex differences in the role of BDNF in cognitive
models. These studies are outside the scope of this review and are summarized
elsewhere (Hill 2016; Wu et al. 2013; McGregor et al. 2017; Scharfman and
Maclusky 2005). Importantly, the effect of sex steroid hormones and SERMs on
cognition in schizophrenia has been suggested to be at least partially mediated via
BDNF-TrkB interactions in the brain (Schroeder et al. 2019; Wu et al. 2015),
emphasizing the need for further studies to clarify this interaction.
3 Conclusions
For many years, preclinical studies on brain mechanisms involved in psychosis

largely used only male subjects or did not analyze for sex differences (Beery and
Zucker 2011). This was similarly found in other fields of neuroscience and has led to
calls for funding to be prioritized to studies which at least use equal numbers of male
and female subjects (Clayton and Collins 2014). Particularly in the field of biological
psychiatry this is of paramount importance as most psychiatric illnesses show
profound sex differences in incidence, clinical presentation, and response to medi-
cation. One frequent reason for the “male focus” in animal model studies has been
concerns about confounding contributions from the estrous cycle. However, while
some variability of behavioral factors clearly exist across this cycle, it is becoming
clear that male–female differences are often independent of the stage of the estrous
cycle or much greater than its effects (Gogos et al. 2019; Davies and Wilkinson
2006).
Fortunately, more recently the literature on sex differences and (to a lesser extent)
effects of sex steroid hormones and related compounds is expanding, and in this
review we have focused on such studies in psychosis, both from a clinical/epidemi-
ological and preclinical/animal model perspective. Evidently there is no simple,
unifying mechanism to explain sex differences or hormone effects in psychosis,
but the available studies provide strong incentive for further investigations. This is
particularly relevant following the discovery that estradiol and sex steroid hormone
analogues, such as SERMs, show promise in a number of symptom domains in
schizophrenia (Gogos et al. 2015; Kulkarni et al. 2019, 2012; de Boer et al. 2018).
Future studies could reveal the molecular and clinical mechanism of these drugs,
with the ultimate aim being more effective and potentially sex-specific treatments
with low side effect profiles. Further study of sex differences may also reveal entirely
new treatment targets which are unique for either females or males, likely in
combination with genetic and non-endocrine environmental factors. To aid the
field, we therefore make the following recommendations: (1) always include
age-matched groups of males and females with sufficient group sizes for adequate
statistical power; (2) if a behavioral output is the main study variable, use acute
pharmacological challenges to identify neurotransmitter mechanisms underlying the
effects of both the primary intervention in the study (genotype, developmental insult,
etc.) but also of any sex differences; (3) recognizing there is no single behavioral test
for psychosis-like behavior, compare animals in more than one relevant behavioral
model; (4) if prominent sex differences are found, follow-up studies could address
underlying hormone involvement, for example by gonadectomy and/or hormone
treatment; (5) in publications, include full analysis of sex differences and interac-
tions between sex and other relevant study parameters; post-hoc tests in male or
female sub-groups are based on these interactions. We believe that these recommen-
dations will facilitate a better understanding of sex differences and the role of sex
hormones in the development of psychosis and will extend to other fields of
neuropsychiatry.
Acknowledgements This research was part-funded by the National Health and Medical Research
Council of Australia (AG CDF 1108098). The Florey Institute of Neuroscience and Mental Health
acknowledges the funding from the Victorian Government’s Operational Infrastructure Support.
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Sex Differences in Neurodevelopmental
Disorders: A Key Role for the Immune
System
Michaela R. Breach and Kathryn M. Lenz
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2 ASD and ADHD Symptomology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2.1 ASD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2.2 ADHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
3 Sexual Differentiation: Why Do Sex Differences Matter? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4 Immune Cells Shape Neurodevelopment and Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
4.1 Microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
4.2 Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4.3 Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
5 Peripheral Inflammation, Neurodevelopmental Disorders, and Brain Development . . . . . . 180
5.1 Peripheral Immune Dysregulation in ASD and ADHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
5.2 Maternal/Prenatal Immune Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
5.3 Early Life Immune Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
6 Concluding Remarks and Ways Forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Abstract Sex differences are prominent defining features of neurodevelopmental

disorders. Understanding the sex biases in these disorders can shed light on mech-
anisms leading to relative risk and resilience for the disorders, as well as more
broadly advance our understanding of how sex differences may relate to brain
development. The prevalence of neurodevelopmental disorders is increasing, and
the two most common neurodevelopmental disorders, Autism Spectrum Disorder
(ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) exhibit male-biases in
prevalence rates and sex differences in symptomology. While the causes of
M. R. Breach
Neuroscience Graduate Program, The Ohio State University, Columbus, OH, USA
K. M. Lenz (*)
Department of Psychology, The Ohio State University, Columbus, OH, USA
Department of Neuroscience, The Ohio State University, Columbus, OH, USA
Institute for Behavioral Medicine Research, The Ohio State University, Columbus, OH, USA
e-mail: lenz.56@osu.edu
https://doi.org/10.1007/7854_2022_308
Published Online: 19 April 2022
166 M. R. Breach and K. M. Lenz
neurodevelopmental disorders and their sex differences remain to be fully under-

stood, increasing evidence suggests that the immune system plays a critical role in
shaping development. In this chapter we discuss sex differences in prevalence and
symptomology of ASD and ADHD, review sexual differentiation and immune
regulation of neurodevelopment, and discuss findings from human and rodent
studies of immune dysregulation and perinatal immune perturbation as they relate
to potential mechanisms underlying neurodevelopmental disorders. This chapter will
give an overview of how understanding sex differences in neuroimmune function in
the context of neurodevelopmental disorders could lend insight into their etiologies
and better treatment strategies.
Keywords Cytokines · Development · Hormones · Inflammation · Microglia
1 Introduction
Most neurological and psychiatric disorders exhibit sex biases in either prevalence
rates, symptomology, or trajectory. Understanding the sex biases in these disorders
can shed light on mechanisms of risk and resilience, as well as advance the
understanding we have of the brain and human behavior. Neurodevelopmental
disorders are a group of conditions usually diagnosed early in development and
characterized by impairments in personal, social, and occupational functioning
(APA 2013). Notably, the prevalence of neurodevelopmental disorders is increasing
(Zablotsky et al. 2019). Potential reasons for the rising prevalence are manifold and
include differences in research methodology or diagnostic criteria (Zaroff and Uhm
2012; Hertz-Picciotto and Delwiche 2009; Polanczyk et al. 2014), earlier age at
diagnosis (Hertz-Picciotto and Delwiche 2009), or an increase in risk factors for the
disorders (Sciberras et al. 2017; Rice et al. 2012). Additionally, reduced stigma/
elevated awareness might be a factor: a recent study found that rates of mental health
literacy for neurodevelopmental disorders were higher than reported previously
(Vovou et al. 2021). Nonetheless, a true increase in prevalence over time cannot
be ruled out (Hertz-Picciotto and Delwiche 2009; Rice et al. 2012), and a near 18%
prevalence rate merits investigation into a better understanding of these conditions
(Zablotsky et al. 2019). The two most common neurodevelopmental disorders,
Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder
(ADHD), exhibit male-biases in prevalence rates (3:1 for ASD and 2:1 for ADHD)
(Zablotsky et al. 2017; Danielson et al. 2018). The 2016 National Survey of
Children’s Health found that of children in the USA aged 2–17 years of age, 8.4%
were diagnosed with ADHD (Danielson et al. 2018), and 3.8% were diagnosed with
ASD (Zablotsky et al. 2017). The causes of neurodevelopmental disorders are poorly
understood, and the reasons underlying their sex biases remain elusive. The immune
system plays a critical role in shaping neurodevelopment, and there is increasing
evidence that shifts in immune activation, and thus differential programming of
Sex Differences in Neurodevelopmental Disorders: A Key Role for the. . . 167
neurodevelopment, are associated with neurodevelopmental disorders. In the fol-

lowing chapter, we will describe symptoms of ASD and ADHD and their sex
differences, review sexual differentiation and the role of immune cells in
neurodevelopment, detail immune dysregulation found in ASD and ADHD, and
discuss findings from rodent models of perinatal immune perturbation as they relate
to potential mechanisms underlying neurodevelopmental disorders.1 Understanding
sex differences in neuroimmune function in the context of neurodevelopmental
disorders could ultimately lend insight into their etiologies and lead to better
treatment strategies.
2 ASD and ADHD Symptomology
2.1 ASD
Autism Spectrum Disorder is a newer diagnostic category established in the DSM-5

to account for what were previously known as four separate diagnoses: Autistic
Disorder, Asperger’s Disorder, Childhood Disintegrative Disorder, and Pervasive
Developmental Disorder Not Otherwise Specified. The impetus for the diagnostic
reconceptualization was to provide a more accurate and scientifically useful diag-
nostic category (NINDS 2013), since these diagnoses were not reliably differentiated
from one another in the clinic (Miller and Ozonoff 2000; Mayes et al. 2001). Using
the DSM-5 criteria, a diagnosis of ASD requires persistent and pervasive deficits in
social communication and interaction, as well as restricted repetitive patterns of
behaviors, interests, or activities. These symptoms must be present in early child-
hood and impair everyday functioning. To account for heterogeneity of support
needs and symptom severity, clinicians can categorize ASD severity ranging from
“requiring support” to “requiring very substantial support.” (APA 2013).
In addition to the 3:1 male-biased sex difference in ASD diagnosis rate, many
studies have documented sex differences in ASD symptomology (see Table 1; for
review, see Werling and Geschwind (2013), though some other studies do not report
such sex differences (Van Wijngaarden-Cremers et al. 2014; Andersson et al. 2013;
Mayes et al. 2020; Wiggins et al. 2021; Nasca et al. 2020). In the studies that have
reported sex differences in symptoms, autistic males exhibit more externalizing
behaviors, such as aggression, hyperactivity, and restricted or repetitive behavior
compared to females (Giarelli et al. 2010; Mandy et al. 2012; May et al. 2014; May
et al. 2016; Szatmari et al. 2012; Kaat et al. 2021; Corbett et al. 2021; Carter et al.
2007; Sipes et al. 2011). The evidence appears strongest for increased restricted and
1
Identity-first language will be used when referring to autistic individuals and person-first language
will be used when referring to individuals with ADHD to represent and respect the wishes of those
communities, respectively (OAR 2020). Additionally, while many of the studies cited in this
chapter refer to “boys,” “girls,” “men,” and “women,” the authors will consistently refer to
“males” and “females” to reflect that these studies examined cis-gender individuals.
Table 1 Summary of sex differences that have been documented in ASD and ADHD
Endpoint Sex difference
Sex differences in ASD
Social skills Males have more severe symptoms as children, but females
have more severe symptoms as adolescents and adults
(McLennan et al. 1993; Mahendiran et al. 2019)
Communication skills Males have more severe symptoms as children, but females
(McLennan et al. 1993; Mahendiran et al. 2019)
Restricted/repetitive behavior Males more severe (Van Wijngaarden-Cremers et al. 2014;
Wiggins et al. 2021; Mandy et al. 2012; Kaat et al. 2021)
Externalizing behavior Males more severe (Giarelli et al. 2010; Mandy et al. 2012;
(hyperactivity, aggression) May et al. 2014)
Internalizing behavior Females more severe (Mandy et al. 2012; May et al. 2014;
(anxiety, depression) Solomon and Herman 2009)
Camouflaging behavior Adult autistic females camouflage more (Lai et al. 2017)
Peripheral immune markers IL-1β, IL-8, MIP-1β, and VEGF negatively correlate with
symptom severity in females but not males. PDGF negatively
correlates with symptom severity in both sexes (Masi et al.
2017).
Males primarily have elevations in cytokines relative to con-
trols while females primarily have elevations in growth factors
relative to controls (Schwarz et al. 2011)
Sex differences in ADHD
Sub-types Females more likely to have inattentive subtype, while males
are more likely to have the combined subtype (Willcutt 2012;
Biederman et al. 2002)
Age at symptom onset Females diagnosed later (Martin et al. 2018; Murray et al.
2019)
Social skills Males have more severe symptoms as children, but females
(Mahendiran et al. 2019)
Communication skills Males have more severe symptoms as children, but females
(Mahendiran et al. 2019)
Externalizing behavior (hyper- Males more severe (Gershon 2002; Carlson et al. 1997)
activity, aggression)
Internalizing behavior Females more severe (Gershon 2002; Ottosen et al. 2019;
(anxiety, depression, Rasmussen and Levander 2009; Rucklidge and Tannock 2001;
suicidality) Chronis-Tuscano et al. 2010)
repetitive behavior in males, as multiple studies that do not find sex differences in
other symptoms still observe this pattern (Van Wijngaarden-Cremers et al. 2014;
Wiggins et al. 2021). Conversely, autistic females are more likely to exhibit inter-
nalizing symptoms, such as anxiety and depression (Mandy et al. 2012; May et al.
2014; Solomon and Herman 2009). While one study in autistic children found better
verbal, motor, and social skills in autistic males relative to females (Carter et al.
2007), other studies in children have found that females have better communication,
social skills, and social cognition compared to males (Harrop et al. 2019; McLennan
et al. 1993; Mahendiran et al. 2019), but manifest more severe symptoms as they age
(Kaat et al. 2021; McLennan et al. 1993). This is possibly because females’ social
skills appear to decline with age while males’ social skills improve (Mahendiran
et al. 2019). Moreover, the trajectory of ASD is influenced by sex. Collapsing across
the ages eliminated sex differences in social and communication abilities
(Mahendiran et al. 2019), which may underlie null findings in some studies that do
not find sex differences in social or communication domains.
Females may also be underdiagnosed with ASD due to higher rates of camouflag-
ing behavior, whereby they hide or “mask” symptoms that are considered socially
unacceptable or performatively offer socially acceptable behaviors (Lai et al. 2017).
Adult autistic women camouflage more than men (Lai et al. 2017), adolescent
autistic females have been shown to be better at using subtle language skills
compared to males, and a separate study in children aged 5–10 found autistic females
used more vivid gestures compared to males (Corbett et al. 2021; Rynkiewicz et al.
2016). Teachers also report fewer concerns regarding the social skills of autistic girls
relative to autistic boys (Hiller et al. 2016). These phenomena are likely influenced
by gendered expectations and perceptions regarding the behaviors of girls and boys.
More work must be done to assess how common camouflaging is, the way that we
measure ASD-like symptoms, and gender-related behavioral expectations as they
may contribute to reduced prevalence rates in females.
In summary, the findings regarding sex differences in ASD symptomatology are
heterogeneous, with the most conclusive evidence supporting higher levels of
restricted and repetitive behaviors in males relative to females, and studies are
beginning to show that symptoms may worsen with age in females.
2.2 ADHD
For Attention-Deficit/Hyperactivity Disorder, the DSM-5 requires a persistent pat-

tern of inattention, hyperactivity, and/or impulsivity that interferes with functioning
or development for diagnosis. These symptoms must be present prior to age 12, and
they must manifest in multiple settings to produce significant impairment. There are
three sub-classifications of ADHD: predominately inattentive, predominately hyper-
active/impulsive, or a combined type (APA 2013). In the general population, males
are more likely to be diagnosed with any of the ADHD subtypes (Willcutt 2012). Of
those who receive a diagnosis of ADHD, males are more likely to meet criteria for
the combined subtype (Willcutt 2012), while females are more likely to be diagnosed
with the inattentive subtype (Willcutt 2012; Biederman et al. 2002; Rucklidge 2010).
ADHD severity is generally worse in males (Gershon 2002; Arnett et al. 2015), and
this may be explained by the fact that males in the general population display more
ADHD symptoms and by the fact that males have greater overall variance in ADHD
symptoms compared to females (Mayes et al. 2020; Arnett et al. 2015).
A recent cross-sectional study in children ages 7–13 years old found that younger
females with ADHD generally outperformed their male counterparts in terms of
social and communication skills, though the relationship for communication was
reversed in older children, where older males outperformed females in communica-
tion skills (Mahendiran et al. 2019). As with ASD, males are more likely to display
externalizing behaviors like aggression (Gershon 2002), and they have higher rates
of comorbid conduct disorder and oppositional defiant disorder compared to females
(Biederman et al. 2002; Ottosen et al. 2019; Carlson et al. 1997); however, individ-
uals with ADHD have higher likelihood of being diagnosed with oppositional
defiant disorder and this association is higher in females with ADHD compared to
males (Ottosen et al. 2019). Males with ADHD also show higher rates of learning
disability (Biederman et al. 2002) and perform worse on tasks involving processing
speed, inhibition, and working memory compared to females (Arnett et al. 2015).
These trends continue into adulthood, as adult males with ADHD display higher
rates of criminality (Rasmussen and Levander 2009) and have greater impairment on
complex cognitive tasks (Balint et al. 2009). Conversely, females with ADHD tend
to display more internalizing problems such as anxiety, depression, and suicidality
(Gershon 2002; Ottosen et al. 2019; Rasmussen and Levander 2009; Rucklidge and
Tannock 2001; Chronis-Tuscano et al. 2010), though a recent study did not replicate
this finding (Mayes et al. 2020).
The sex bias in diagnosis may also be driven by symptomology, such that the
disruptiveness of males in classroom, social, or home settings may mean that they
are more likely to be referred for diagnosis by teachers or caregivers (Gaub and
Carlson 1997). Studies in clinical samples of children with ADHD have found large
male-biases (10:1) while studies in non-referred community-based samples have
shown less of a male-bias in prevalence (3:1) (Willcutt 2012; Arnett et al. 2015).
Additionally, males receive their diagnoses and manifest symptoms earlier relative
to females (Martin et al. 2018; Murray et al. 2019). Individuals with the inattentive
subtype, the subtype females are more prone to, also tend to manifest their symptoms
at later ages (Willcutt 2012), and females are also more likely to manifest hyperac-
tivity symptoms at later ages (Murray et al. 2019). This discrepancy may contribute
to the differing and reduced prevalence ratios (1:1 and 2:1) seen in studies of adults
with ADHD (Williamson and Johnston 2015) relative to those found in children.
Moreover, as with ASD, not much is known regarding the developmental trajectory
of symptoms of ADHD, but evidence suggests that both disorders are influenced
by sex.
3 Sexual Differentiation: Why Do Sex Differences Matter?
To better understand how and why sex differences in ASD and ADHD come about,
we must understand the basics of sexual differentiation. In early mammalian devel-
opment, every embryo contains a bipotential structure called the gonadal ridge that
can differentiate into ovaries or testes. The process by which the gonadal ridge
becomes either ovaries or testes, known as sex determination, depends on the

combination of chromosomes inherited from the parents. Mothers can pass on one
of two X chromosomes, whereas fathers may pass down an X or a Y chromosome to
the embryo. The presence of a Y chromosome, specifically the sex-determining
region of the Y chromosome (SRY), will lead to synthesis of a secreted protein,
called testis-determining factor (TDF). TDF causes the bipotential gonads to differ-
entiate into testes which then initiate the process of male-specific sexual differenti-
ation via secretion of androgens. The major mammalian androgens are testosterone
and its metabolite, dihydrotestosterone (DHT). Testosterone and DHT induce devel-
opment of male-specific accessory structures and external genitalia, respectively, in a
process known as masculinization. Meanwhile, the testes also secrete anti-Mullerian
hormone, which causes regression of female-specific accessory structures in a
process known as defeminization. In the absence of a Y chromosome and thus an
absence of SRY/TDF and androgenic hormones, the bipotential gonads will develop
into ovaries via a process known as feminization, and the male accessory structures
will regress (demasculinization) to initiate female-specific sexual differentiation
(Breedlove 1994). However, while feminization does not depend on hormones, it
is still an active process. Carefully orchestrated genetic programs direct female-
specific brain organization, and perturbations in this programming can shift the
trajectory of brain feminization. Additionally, feminization also requires active
repression of male-specific genetic programming, such that higher DNA methylation
levels in the developing female brain maintain feminization by silencing genes
implicated in brain masculinization (Nugent et al. 2015).
Sexual differentiation of the brain is likewise driven by steroid hormones. In
humans, sexual differentiation takes place prenatally during the second trimester of
gestation (Smail et al. 1981). In rodents, this critical period begins when the testes
start to secrete androgens on embryonic day (E)18 and extends into neonatal life
(< postnatal day (P)10), even after androgen secretion stops around the day of birth
(McCarthy 2008). Given their nonpolar structure, sex steroid hormones can readily
cross the blood brain barrier (BBB) to produce enduring cellular and molecular
changes during this period of brain development. Once in the brain, testosterone can
be converted into DHT via the enzyme 5α-reductase, or into 17-β-estradiol via the
enzyme p450 aromatase (Lephart 1996). Androgens like testosterone and DHT are
important for masculinization in primates, whereas estradiol is the main masculin-
izing hormone in rodents (McCarthy 2008; Wallen 2005), thus the aromatase
enzyme is highly expressed in the rodent brain during the critical period for sexual
differentiation to convert androgens into estrogens (George and Ojeda 1982; Lauber
1994). Later in life, steroid hormones, including androgens, estrogens, and pro-
gestins, act on the previously organized brain circuitry to permit expression of, or
“activate” sex-specific aspects of brain and behavior (Pheonix et al. 1959). In this
way, early life exposure to sex-typical hormones prepares the brain for later life
hormonal surges that drive sex differences in a variety of behaviors, from cognition
to social and sexual behavior.
Differences in typical sex steroid exposure during this early life organizational
period have been linked to neurodevelopmental disorder risk. For example, females
with polycystic ovary syndrome, a condition associated with elevated testosterone

and other sex steroids, are more likely have children with ADHD or ASD (Cesta
et al. 2020; Cherskov et al. 2018). Likewise, individuals with congenital adrenal
hyperplasia (CAH), another difference of sexual development (DSD) characterized
by high levels of androgen exposure in utero, exhibit higher rates of autistic traits
(Knickmeyer et al. 2006). Studies that have measured levels of sex steroid hormones
in the amniotic fluid suggest that testosterone correlates positively with parent-
reported autistic traits (Auyeung et al. 2009; Auyeung et al. 2010). Another study
showed that combined elevations in androgens, progesterone, and cortisol in amni-
otic fluid, dubbed a “latent steroidogenic factor,” were statistically associated with
individuals going on to develop autism in childhood (Baron-Cohen et al. 2015).
Further studies implicate higher levels of prenatal estrogens as a risk factor for an
ASD diagnosis and internalizing behaviors (Baron-Cohen et al. 2020; Day et al.
2020). Sex steroids may impact brain development and later life behavior in a
sex-dependent manner, as high levels of testosterone in the amniotic fluid were
associated with reduced functional connectivity in the social brain default mode
network during adolescence in males but not females (Lombardo et al. 2020).
Nonetheless, not all studies support the idea that elevated levels of prenatal steroids
program an increased likelihood of ASD. For example, conditions associated with
reduced levels of sex steroids, like Klinefelter syndrome, are also associated with
increased risk of both ASD and ADHD-like traits (Ross et al. 2012), and some
studies have failed to find a relationship between perinatal sex steroids and autism
(Kung et al. 2016; Jamnadass et al. 2015; Whitehouse et al. 2012). Overall, the take-
home point from these steroid hormone studies is that deviations in either direction
from average levels of prenatal steroids may be associated with higher risk of ASD.
More work must be done to establish whether or not, and, if so, how prenatal sex
steroids may contribute to the etiology of ASD at a mechanistic, rather than purely
correlational, level.
There is also evidence for persistent differences in steroid hormone-related
function in the ASD brain outside of the prenatal period. Aromatase, the enzyme
responsible for converting testosterone to estradiol, is reduced in the frontal cortex of
brains of autistic adults (Sarachana et al. 2011). Likewise, retinoic acid-related
orphan receptor-alpha (RORA), a transcription factor that regulates expression of
the gene for aromatase along with 438 other autism candidate genes, is
downregulated in the postmortem frontal cortex of autistic adults (Sarachana et al.
2011). Testosterone itself downregulates RORA expression (Sarachana et al. 2011),
suggestive of a positive feedback loop leading to further levels of elevated testos-
terone in the ASD brain. However, estrogens upregulate RORA and may provide a
protective buffering mechanism in females who have higher levels of these hor-
mones (Sarachana et al. 2011). Indeed, control females express marginally higher
levels of RORA in the frontal cortex, and this trend disappears in ASD brains
(Hu et al. 2015). The percent reduction in aromatase protein is higher in ASD
males, suggesting that females may offset aromatase deficiency and lead to the
male-bias in ASD prevalence (Hu et al. 2015). Whether these trends are unique to
adulthood or also exist prenatally must be parsed out in animal studies.
While steroid-mediated mechanisms of neurodevelopmental disorders is an area

of active research interest, much remains to be established. Particularly of interest is
how basic mechanisms of brain sexual differentiation, including hormone secretion
and sensitivity, may be shifted in the context of ASD risk genes, early life pertur-
bations, or combined genetic and environmental risk exposure. However, steroid
hormones do program the function of many cells throughout the brain during early
life, including brain-resident immune cells called microglia. In the next section, we
discuss how the immune system regulates neurodevelopment, sex differences in
neuroimmune cell function, and evidence for neuroimmune cell dysregulation in
ASD and ADHD.
4 Immune Cells Shape Neurodevelopment and Behavior
4.1 Microglia
Microglia are macrophages located in the central nervous system (CNS), though
they are not derived from neural stem cells. Microglia are derived from myeloid
progenitor cells from the yolk sac and infiltrate the brain during early embryogen-
esis, on E9.5 in rodents and gestational week 4.5 in humans (Ginhoux et al. 2010;
Verney et al. 2010). Once microglia progenitors have entered the developing brain
they differentiate, proliferate, and migrate widely (Bennett et al. 2016). After
microglia have seeded the brain, they form a long-lived and self-sustaining cellular
population, where they provide local host defense and perform a variety of homeo-
static and developmentally relevant functions (Frost and Schafer 2016).
Microglia secrete factors such as cytokines and chemokines, which are small
proteins that enable recruitment of and communication between immune cells,
respectively, though both cytokines and chemokines also have neuromodulatory
functions (Ratnayake et al. 2013; Vezzani and Viviani 2015). In the developing
brain of both sexes, microglia populate proliferative and neurogenic zones
(Shigemoto-Mogami et al. 2014; Nelson et al. 2017) and regulate cell genesis and
survival through secretion of cytokines and growth factors (Shigemoto-Mogami
et al. 2014; Deverman and Patterson 2009; Ueno et al. 2013). Such microglial
support of cell genesis likely happens throughout the forebrain, as neonatal depletion
of microglia reduces cell genesis in the rat cortex, hippocampus, and amygdala of
males and females (Nelson et al. 2021). Microglia also participate in the cell death
process, sensing cell death signals and releasing reactive oxygen species to advance
apoptosis (Marín-Teva et al. 2004; Wakselman et al. 2008; Ravichandran 2011).
Microglia also phagocytose (e.g., engulf and digest) viable precursor cells and
dead and dying cells in the developing brain of both sexes (Sierra et al. 2010;
Cunningham et al. 2013). Microglia phagocytosis is also crucial for developmental
synaptic patterning. During normal brain development, neurons form an excess of
excitatory synapses, and maturation of brain circuitry is marked by pruning of excess
synapses (Molliver et al. 1973; Huttenlocher and Dabholkar 1998). Microglia
facilitate the synaptic pruning process, though most of the work supporting this role
for microglia has been done in males, so confirmational studies to show similar, or
perhaps differential, effects occurring in females must still be performed. Microglia
engulf pre- and postsynaptic elements in the juvenile hippocampus, and mice lacking
the receptor for fractalkine (CX3CL1), a “find me” signal released by neurons to
attract microglia, had a transient increase in dendritic spines and immature synapses
in the third postnatal week, indicative of reduced synaptic pruning (Paolicelli et al.
2011). Microglia also sense “eat me” signals from the complement cascade, an
immune pathway traditionally involved in marking pathogens and apoptotic cells
for phagocytosis and elimination, to direct synaptic pruning activity. This happens in
the neonatal thalamus, where microglia prune less active retinogeniculate axons via
detection of the protein complement component (C)3 (Schafer et al. 2012). Con-
versely, microglia can also partially engulf or nibble at (“trogocytose”) presynaptic
elements to alter their shape, and microglia-dendrite contact can induce spine head
and filopodia formation (Miyamoto et al. 2016; Weinhard et al. 2018a). Microglia
signaling may also affect synaptic activity, as microglial-derived brain-derived
neurotrophic factor (BDNF) influences glutamate receptor subtype composition,
and microglial depletion decreases learning-dependent formation of dendritic spines
in the cortex (Parkhurst et al. 2013). Perturbation of embryonic microglia via
knockout of either fractalkine receptor, complement receptors, or DAP12 (a protein
important for microglial activation) altered the outgrowth of forebrain dopaminergic
axons and disturbed laminar organization of neocortical interneurons (Squarzoni
et al. 2014).
Studies in rodents indicate that microglia undergo several stages of development,
reaching maturity during the juvenile period, around P28 (Bennett et al. 2016;
Matcovitch-Natan et al. 2016; Thion et al. 2019). As microglia mature, they express
higher levels of immune-response genes, at least in the hippocampus (Hanamsagar
et al. 2017), and adult cortical and hippocampal microglia exhibit sex differences in
their gene expression (Guneykaya et al. 2018). Furthermore, various sex differences
in microglia in the developing brain have been documented (see Fig. 1) (Han et al.
2021a). Sex differences in microglia morphology or number in the developing brain
can be dramatic. However, such sex differences are not static over development, as
patterns vary dynamically across developmental stage and brain region.
Microglia sex differences are organized by early life steroid hormone exposure
(e.g., estradiol in rodents) and may have long-term effects on development and
behavior. For example, microglia phagocytose more viable neural progenitor cells in
the neonatal hippocampus of female rats compared to male rats, and this sex
difference is eliminated by administration of a masculinizing dose of estradiol to
females (Nelson et al. 2017). Likewise, neonatal ablation of microglia reduces sex
differences in hippocampal cell genesis (Nelson et al. 2021). Male rats also have
more ameboid microglia in the neonatal preoptic area (POA), and microglia deple-
tion during the neonatal period prevents the development of male-specific POA syn-
aptic patterning and adult sexual behavior (Lenz et al. 2013). Female synaptic
patterning in the POA and sexual behavior can be masculinized via neonatal
administration of estradiol or prostaglandin E2, an inflammatory mediator that can
Fig. 1 Summary of known sex differences in microglia number and morphology during rodent
neurodevelopment. On E15, males have more total and ameboid microglia in the basal ganglia
compared to females (Breach et al. 2021). During the first 3 days of life, males have more total and
ameboid microglia in the medial preoptic area (mPOA) (Lenz et al. 2013), while females have more
ameboid and phagocytic microglia in the hippocampus (HPC) (Nelson et al. 2017; Nelson et al.
2021) and amygdala (AMY) (VanRyzin et al. 2019). Conversely, males have more total, ameboid,
and “reactive” microglia in the HPC and AMY on P4 (Schwarz et al. 2012). On P8, females display
more phagocytic microglia in the HPC (Weinhard et al. 2018b). Subsequently, on P21, males have
more microglia in the HPC, while females have more microglia in the AMY (Guneykaya et al.
2018). By P28, males have more phagocytic microglia in the HPC (Weinhard et al. 2018b), while
females have more total and “reactive” microglia in the HPC and AMY (P30) (Schwarz et al. 2012).
Collectively, these data suggest there are dynamic fluctuations in sex differences of microglia
number and phenotype throughout development, particularly in the hippocampus and amygdala,
though this may be reflective of greater experimental focus on these limbic regions across time
relative to other brain regions
be released by immunocompetent cells, neurons, and astrocytes (Lenz et al. 2013;

Amateau and McCarthy 2004). Microglia inhibition prevents this masculinization
(Lenz et al. 2013). Microglia also regulate social play behavior in a sex-dependent
manner. Juvenile social play is an adaptive behavior conserved across mammalian
species important for social, cognitive, and neural development (Pellis et al. 2010;
Vanderschuren and Trezza 2014). Moreover, complement-dependent phagocytosis
of dopamine (D)1 receptors in the adolescent nucleus accumbens programs social
play in male rats, as inhibition of microglial phagocytosis increased play behavior in
a D1r-dependent manner (Kopec et al. 2018). Conversely, while inhibition of
complement-dependent phagocytosis also increased play in female rats, it was not
dependent on D1r (Kopec et al. 2018). A separate study found that microglial
phagocytosis of viable newborn astrocytes in the neonatal amygdala programs sex
differences in juvenile play, and administration of testosterone to females during the
neonatal period masculinizes play, along with phagocytosis and the number of
newborn cells (VanRyzin et al. 2019). Meanwhile, neonatal inhibition of phagocy-
tosis in males feminizes juvenile play behavior (VanRyzin et al. 2019). Because
social behavior is a key domain impacted in neurodevelopmental disorders, these
studies suggest that abnormal microglia activity may be involved in shaping abnor-
mal social behavior found in ASD and ADHD.
Studies that inhibit, deplete, or knockout microglia during development recapit-
ulate behavior associated with neurodevelopmental disorders. For example, embry-
onic depletion of microglia via maternal dietary treatment with a colony stimulating
factor receptor 1 (CSF1R) inhibitor produced hyperactivity in adolescent female
mice (Rosin et al. 2018). Transient microglia depletion in the neonatal period leads
to locomotor hyperactivity, increased risk assessment behavior, impaired cognition,

reduced juvenile social play, and increased adult social avoidance in rats (VanRyzin
et al. 2016; Nelson and Lenz 2017). Genetic knockout of fractalkine receptor
(CX3CR1), which reduces neuron-microglia communication and causes a transient
reduction of microglia during development, impairs juvenile social behavior and
increases repetitive grooming behavior in mice (Zhan et al. 2014). Beyond the
possible relevance to neurodevelopmental disorders, these rodent studies make
clear that microglia are important mediators of brain development and resultant
behavioral programming.
4.1.1 Microglia Are Dysregulated in ASD
While human studies on microglia in ADHD are lacking, there is evidence for
abnormal microglia activation in the brains of autistic individuals, at least in
males. Positron emission tomography (PET) imaging of 18 kDa translocator protein
(TSPO), a protein found in activated microglia and astrocytes, revealed decreased
expression in several cortical brain areas of ASD males (Zürcher et al. 2021).
Conversely, a different PET study analyzing a radiocarbon that specifically binds
to activated microglia ([11C])-labeled (R)-(1-[2-chrorophynyl]-N-methyl-N-
[1-methylpropyl]-3 isoquinoline carboxamide) found elevated microglia activation
in the cerebellum, midbrain, pons, and cingulate and orbitofrontal cortices of ASD
males (Suzuki et al. 2013). This result complements other work in postmortem
autistic tissue finding elevated microglia number, density, and/or activation in the
cerebellum (Vargas et al. 2005), dorsolateral prefrontal cortex (Morgan et al. 2010),
and fronto-insular and visual cortices of ASD males (Tetreault et al. 2012) compared
to controls.
Studies investigating gene expression in the cortex of ASD versus control brains
generally find downregulations in expression of genes associated with neuronal and
synaptic function (Voineagu et al. 2011; Gupta et al. 2014; Parikshak et al. 2016) and
upregulations in expression of genes associated with inflammatory and immune
responses, including genes associated with microglia and activated microglia
(Voineagu et al. 2011; Gupta et al. 2014; Parikshak et al. 2016; Edmonson et al.
2014). The expression of genes associated with activated microglia was also nega-
tively correlated with the expression of genes associated with neuronal activity,
suggesting that dysregulation of microglia operates in tandem with altered neuronal
activity (Gupta et al. 2014). Likewise, a transcriptomic profiling and comparison of
major psychiatric disorders discovered that microglial genes were uniquely
upregulated in ASD cortex while multiple neuron-associated modules were
downregulated (Gandal et al. 2018). Alterations in gene expression may be region-
specific, as another study found decreased microglial markers in the cerebellum
(Edmonson et al. 2014). A more recent study using single nucleus RNA sequencing
found that the most differentially expressed genes in ASD versus control brains were
found in cortical layer 2/3 excitatory neurons, VIP interneurons, and microglia, and
changes in these neurons and microglia were predictive of clinical severity

(Velmeshev et al. 2019).
One study found that when a microglial maturity index was applied to the human
brain transcriptome, the male microglial transcriptome was more developmentally
mature compared to that of females (Hanamsagar et al. 2017), and autistic brains
displayed markers of exaggerated microglial maturity, suggesting greater
neuroinflammation (Hanamsagar et al. 2017). Exaggerated protein synthesis in
microglia leads to autism-like behavior in male but not female mice (Xu et al.
2020), and a genetic mouse model of autism revealed differentially expressed
genes in juvenile male brains were positively enriched for glia-specific gene sets,
whereas those in the female brain were generally negatively enriched for glia-
specific gene sets (Jung et al. 2018). Despite this, the above studies in humans are
all biased toward male participants, and none analyzed sex differences. However, a
study on samples from fetal and adult brains from non-autistic individuals of both
sexes found that ASD-elevated glial and immune molecules were more likely to be
expressed in the cortex of fetal and adult males compared to females, whereas
ASD-reduced neuronal/synaptic genes were more likely to be expressed in the
adult female cortex (Werling et al. 2016). This finding suggests that males and
females may be differentially vulnerable to different mechanisms of ASD. Said
another way, males may be more susceptible to neuroinflammatory-associated risk
factors, whereas females may be more susceptible to neuronal dysfunction-
associated risk factors. Seemingly conflicting with this, ASD candidate genes were
more highly co-expressed in the female fetal brain compared to the male fetal brain,
and autistic females displayed higher numbers of de novo mutations compared to
autistic males, even after controlling for ASD severity (Zhang et al. 2020). This
finding supports the “female protective effect” theory of autism – the idea that
mutation-induced gene deficiencies may be compensated for more readily in
females, and that females may require higher etiological or genetic load to manifest
autistic symptoms (Szatmari et al. 2012; Jacquemont et al. 2014). Overall, while
there appear to be sex differences in expression of genes dysregulated in ASD, more
work needs to ascertain whether specific ASD candidate genes are expressed or
co-expressed in a sex-dependent manner.
4.2 Astrocytes
Another immunocompetent cell located in the CNS is the astrocyte. Unlike

microglia, astrocytes are neural progenitor cell-derived glial cells. Their primary
functions in the brain include serving as a main component of the BBB, regulat-
ing glutamate homeostasis, and providing metabolic and trophic support (Khakh and
Deneen 2019). They can also receive and relay immune signals, particularly through
communication with microglia (Matejuk and Ransohoff 2020). Like microglia,
astrocytes display sex differences in number, differentiation, and function. For
example, in the POA, male rats have more complex astrocytes with increased
process number and length relative to females, and this can be masculinized in
females with estradiol treatment (Amateau and McCarthy 2002). Primary astrocytes
cultured from cortical samples of male mice release more inflammatory cytokines
compared to females following immune stimulation with bacterial endotoxin lipo-
polysaccharide (LPS), and this was masculinized in females with neonatal testoster-
one treatment (Santos-Galindo et al. 2011).
Postmortem and genetic studies corroborate the idea that astrocytes may be
involved in neurodevelopmental disorders, although little has been done to investi-
gate sex differences as most of these studies’ subjects consist primarily of males
and/or findings were not analyzed by sex. Some of the previously mentioned studies
on postmortem autistic tissue found increased astrocytic activation in the cerebellum,
middle frontal gyrus, and anterior cingulate gyrus (Vargas et al. 2005), and elevated
astrocytic genes in prefrontal cortex and cerebellum (Edmonson et al. 2014). Like-
wise, both astrocyte and microglia markers are elevated in the autistic cortex
(Voineagu et al. 2011). A separate study found increased number of protoplasmic
astrocytes, a subtype of astrocyte that is highly branched and distributed throughout
the gray matter (Miller and Raff 1984), in autistic cortex (Velmeshev et al. 2019).
This same study observed dysregulation of developmental transcription factors in
protoplasmic astrocytes (Velmeshev et al. 2019). Astrocyte associated gene modules
are also upregulated in ASD, schizophrenia, and bipolar disorder cortical samples of
both sexes (Gandal et al. 2018). As mentioned in Sect. 4.1, ASD-elevated glial genes
are expressed at higher levels in the male brain relative to the female brain, and this
includes astrocytic markers (Werling et al. 2016), suggesting again that males may
be more susceptible to immune-related pathways of ASD. Moreover, astrocytic
cytokine signaling via IL-33 release directs microglial synaptic pruning during
development (Vainchtein et al. 2018), and astrocytes may also release ATP/ADP
to regulate histamine-induced microglial phagocytosis in both sexes (Xia et al.
2021). Given that many potential avenues exist for communication between astro-
cytes and microglia, and given that both cell types are dysregulated in ASD,
astrocytes, too, may contribute to neurodevelopmental disorders. Future research is
needed in this area, particularly within the domains of sex differences, neuroimmune
crosstalk, and brain development in neurodevelopmental disorders.
4.3 Mast Cells
Mast cells are often-overlooked immune cells that play a role in sexual differentia-
tion, neurodevelopment, and behavior. Mast cells are hematopoietic immune cells
found in tissues throughout the body and are primarily known for their role in
allergic and atopic disease (Galli and Tsai 2012). They can also be found in the
brain and leptomeninges during embryonic development (Khalil et al. 2007;
Lambract-Hall et al. 1990), and they are highest in number in the brain during the
perinatal period, where they are located preferentially at sites of blood vessel growth
or on vessels sheathed by astrocytic processes (Khalil et al. 2007). Mast cells house
granules which contain neuroactive and inflammatory mediators, including cyto-

kines, proteases, histamine, and serotonin (Silver and Curley 2013; Wernersson and
Pejler 2014). When activated, mast cells undergo a process called degranulation,
which involves bolus or piecemeal release of their granules into the extracellular
space (Marszalek et al. 1997). Mast cells can also synthesize and release inflamma-
tory mediators such as prostaglandins and cytokines independent of degranulation
(Galli et al. 2005). Like microglia, mast cells are capable of sensing and responding
to complement proteins, neuropeptides, steroid hormones, cytokines, and microbes
(Dong et al. 2014), making them versatile cells. Interestingly, mast cell-mediated
diseases are more common in females (Ballardini et al. 2013; Osman et al. 2007;
Acker et al. 2017). This may stem from the fact that peripheral mast cells synthesize
and store more immune mediators in females compared to males, resulting in more
severe responses to immunological and psychological stress in females (Mackey
et al. 2016). This sex difference is established by perinatal androgen exposure
(Mackey et al. 2020).
Sex differences have also been found in mast cells in the brain. For example, mast
cells are sexually dimorphic in the rodent hippocampus and POA, where males have
more mast cells compared to females (Breach et al. 2021; Lenz et al. 2018; Joshi
et al. 2019). Such sex differences contribute to neurodevelopment and behavior, as
mast cells are the first step in microglia-mediated sex-specific synaptic patterning of
the POA. In the developing POA of males, estradiol binds to estrogen receptors on
mast cells, inducing degranulation and release of histamine. This histamine binds to
histamine receptors on microglia to facilitate microglia synthesis and release of
prostaglandin, which binds to GPCRs on dendrites to facilitate dendritic spine
formation – all of which is required for male-specific adult sexual behavior (Lenz
et al. 2018).
Moreover, many studies in adult rodents have implicated mast cells in behaviors
relevant to neurodevelopmental disorders, at least in males. Blockade of central, but
not peripheral mast cell activity increases anxiety-like behavior (Nautiyal et al.
2008). Mast cell-deficient mice also display elevated anxiety-like behavior (Nautiyal
et al. 2008) and have deficits in hippocampal-dependent spatial learning/memory
and reduced neurogenesis (Nautiyal et al. 2012). Increasing the levels of mast cell
mediator serotonin via chronic treatment with selective serotonin reuptake inhibitor
fluoxetine reverses cognitive and neurogenic deficits in male mast cell-deficient mice
(Nautiyal et al. 2012). Application of histamine into the lateral septum of male rats
decreases anxiety and novelty-suppressed feeding (Chee and Menard 2013; Chee
et al. 2014), and spontaneous locomotor activity correlates with degranulation of
mast cells in the meninges (Larson et al. 2011). Mast cell activity is also important
for social behavior. Intracerebroventricular infusion of a mast cell activator increased
sociability in male mice, while removal of mast cells in a diphtheria toxin inducible
knockout model reduced social preference levels without influencing anxiety- or
depressive-like behavior (Tanioka et al. 2021). Given the sex differences that have
been documented in mast cell number and function, future research determining
whether these findings extend to females is merited.
Although few studies have specifically investigated mast cells in the context of
neurodevelopmental disorders, evidence is mounting for altered peripheral mast cell
activity in neurodevelopmental disorders, primarily due to the mast cell’s role in
mediating allergy and atopy. The incidence of ASD is 10x higher in children with
mastocytosis, a disease caused by mast cell hyperactivity, compared to children in
the general population (Theoharides 2009). Allergic diseases are associated with
psychological and behavioral issues in preschoolers (Chang et al. 2013), and both
ASD and ADHD have been linked to allergic disease (Chua et al. 2020). For
example, allergic rhinitis, atopic dermatitis, asthma, eczema, food allergy, and
urticaria have all been found to associate with either ASD (Chen et al. 2013;
Kotey et al. 2014; Lyall et al. 2015; Zerbo et al. 2015a) and/or ADHD (Roth et al.
1991; Schmitt et al. 2010; Fasmer et al. 2011; Mogensen et al. 2011; Chen et al.
2014; Chen et al. 2017; Wang et al. 2018; Jiang et al. 2018). The development of
atopic dermatitis and asthma in early life increases risk for subsequent diagnosis of
both ASD and ADHD (Chen et al. 2014; Liao et al. 2016). Furthermore, allergic
symptoms may contribute to abnormal behavior, as overall allergy in autistic
children was associated with higher measures of stereotyped behavior (Lyall et al.
2015). A study on children with ADHD found increased levels of allergic markers
(IgE and eosinophils) and decreased levels of 5-HT and hemoglobin in the blood,
and ADHD risk increased with the number of these biochemical factors present
(Wang et al. 2018). Combined with evidence from rodent studies on mast cell
regulation of behavior, this work suggests that peripheral and central mast cell
activity may be involved in ASD and ADHD. Moreover, this work also demon-
strates that we should consider peripheral inflammation when it comes to studying
neurodevelopmental disorders and their rodent models. In the next section, we will
discuss peripheral immune dysregulation documented in ASD and ADHD.
5 Peripheral Inflammation, Neurodevelopmental

Disorders, and Brain Development
The brain was historically thought to be privileged from the immune system, as the
BBB generally bars peripheral immune cells from freely entering the brain under
normal conditions (Bechmann et al. 2007). However, the immune system commu-
nicates with the brain through three general mechanisms: diffusion at the sites of the
brain’s circumventricular organs (CVOs), interactions at the endothelial interface of
the BBB, and direct stimulation of the vagus nerve and sympathetic afferents
(D’Mello and Swain 2016). Cytokines are integral for immune to brain communi-
cation, and peripheral immune perturbation can readily affect this signaling
(D’Mello and Swain 2016). Moreover, given the ability of the immune system to
communicate with the brain, and given the role that the immune system plays in
neurodevelopment, it makes sense that peripheral immune dysregulation and per-
turbation are associated with neurodevelopmental disorders like ASD and ADHD. In
this section, we will detail peripheral immune dysregulation documented in ASD

and ADHD. Then, we will discuss how rodent studies of early life inflammation
model endophenotypes of neurodevelopmental disorders and provide insight into
potential mechanisms of neurodevelopmental disorder-associated alterations in
neurobehavioral development.
5.1 Peripheral Immune Dysregulation in ASD and ADHD
Evidence for immune dysregulation in ASD stems from multiple fields of study,
though note that this work has been conducted primarily in male subjects with
minimal attention paid to sex (see Sect. 5.1.1 for a discussion of sex differences in
peripheral immune dysregulation). Certain haplotypes in the major histocompatibil-
ity complex (MHC), a major gene locus on chromosome 6 that codes for various
immune-related proteins, have been associated with autism (Torres et al. 2012) and
many top gene sets associated with ASD involve immune pathways, including genes
associated with recruitment of immune cells and the inflammatory response to viral
infection (Saxena et al. 2012). Likewise, autistic children exhibit cytokine
dysregulation that is developmentally dynamic. Blood samples from neonates that
subsequently received an ASD diagnosis display decreased levels both pro- and
anti-inflammatory cytokines (Abdallah et al. 2012). Conversely, older autistic chil-
dren have increased levels of proinflammatory cytokines (Ashwood et al. 2011;
Enstrom et al. 2008; Xie et al. 2016; Masi et al. 2015; Saghazadeh et al. 2019) and
reduced levels of the anti-inflammatory cytokine TGFβ (Masi et al. 2015;
Saghazadeh et al. 2019; Ashwood et al. 2008; Okada et al. 2007). Altered cytokine
levels may also contribute to symptomatology, as elevated levels of proinflammatory
cytokines (Ashwood et al. 2011; Xie et al. 2016) and reduced levels of anti-
inflammatory TGFβ (Ashwood et al. 2008) are associated with increased symptom
severity.
T cells, a term for a diverse group of lymphocytes involved in the adaptive
immune response, are also dysregulated in ASD. Helper T cells, a subset of T
cells, are some of the most prolific cytokine generators and can take on multiple
phenotypes, three of which are Th1, Th2, and Th17 (Luckheeram et al. 2012). Th1
and Th17 cells are generally considered proinflammatory, whereas Th2 cells are
generally considered anti-inflammatory. Peripheral immune cells isolated from
autistic children show enhanced production of both Th1- and Th2-like cytokines
(Careaga et al. 2017), as well as Th1-, Th2-, and Th17-associated transcription
factors (Ahmad et al. 2017). Autistic children also display reduced expression of
transcription factors associated with a different class of T cell, the immunosuppres-
sive regulatory T cell (Ahmad et al. 2017). Moreover, specific analysis of T cell
numbers has found that autistic children have reduced numbers of regulatory T cells
(Moaaz et al. 2019) and Th1 cells (Gupta et al. 1998), and increased numbers of Th2,
Th17, and activated Th17 cells (Moaaz et al. 2019; Gupta et al. 1998; Basheer et al.
2018). A high ratio of Th17 cells to regulatory T cells is associated with more severe
autistic symptoms (Moaaz et al. 2019). Increases in helper T cell activity in ASD
may be due to decreased regulation by regulatory T cells, and the balance of T cell
phenotypes in ASD may also be moderated by comorbidity with other syndromes.
Rose et al. found that autistic children with comorbid gastrointestinal (GI) symptoms
displayed elevated Th17 populations whereas autistic children without comorbid GI
symptoms had increased frequency of Th2 populations, and both ASD groups had
reduced regulatory T cell populations (Rose et al. 2020).
Regarding ADHD, a study investigating gene expression in peripheral blood
mononuclear cells found that genes differentially expressed in adults with ADHD
(prior to statistical correction) were enriched for pathways relating to the immune
and inflammatory response, with statistical correction still supporting differential
expression of immune genes such as C1qA, TNFSF8, and IL7R (Mortimer et al.
2020). A recent meta-analysis on DNA methylation studies found six differentially
methylated regions within the major histocompatibility complex, including
complement-related genes C4A and C4B (van Dongen et al. 2019). Notably,
overexpression of human C4A in mice increased microglia-mediated synaptic prun-
ing during development in thalamus and cortex and reduced social preference
behavior, increased anxiety, and impaired spatial working memory (Yilmaz et al.
2021). Moreover, other studies have associated polymorphisms in various genes
coding for proinflammatory cytokines with increased risk for ADHD and/or symp-
tom severity (Drtilkova et al. 2008; Smith et al. 2014), and two polymorphisms in the
IL-16 gene are associated with the inattentive ADHD phenotype (Lasky-Su et al.
2008). One study found a trend for elevated protein levels of pro- and anti-
inflammatory cytokines in the serum of patients with untreated ADHD, but not
medicated ADHD patients (Oades et al. 2010a). Another study found statistically
elevated levels of serum IL-6 and IL-10 in children with ADHD (Donfrancesco et al.
2016), and serum IL-16 is positively associated with hyperactivity/impulsivity
whereas serum IL-13 is positively associated with inattention (Oades et al. 2010b).
Some studies in peripheral blood suggest that the response to oxidative stress, a
process associated with cellular damage, death, and immune activation, may be
insufficient in individuals with ADHD (Joseph et al. 2015). A separate study
examining differential expression of genetic and epigenetic markers in peripheral
blood cells found that expression of three microRNAs was predictive of ADHD
diagnostic status (Sanchez-Mora et al. 2019). MicroRNAs interact with DNA to
repress gene expression, and subsequent downstream analysis of genes normally
targeted by these microRNAs that were differentially expressed in ADHD found
enrichment of genes associated with “proliferation of neuroglia,” among other
annotations (Sanchez-Mora et al. 2019). This finding suggests that epigenetic regu-
lation of neuroglial proliferation may be altered in ADHD, though the ability to
extrapolate findings from peripheral immune cells to the brain is limited.
Evidence also exists for a potential autoimmune component of ASD and ADHD.
Antibodies that react to one’s own tissue, called autoantibodies, have been
documented in both conditions. In ASD, autoantibodies have been detected against
proteins such as Hexokinase-1, BDNF, glial fibrillary acidic protein, myelin pro-
teins, and heat shock protein 90, among others (Mazon-Cabrera et al. 2019).
Autoantibodies for unidentified proteins have also been detected in the hypothala-
mus, thalamus, and cerebellum tissue from the postmortem adult autistic brain
(Cabanlit et al. 2007; Goines et al. 2011). A recent systematic review of studies
examining autoantibodies in serum or CSF concluded that autistic children
expressed higher levels of antibodies reactive to myelin proteins, endothelial cell
proteins, nuclear antigens, and folate receptors, though more work needs to be done
due to significant heterogeneity between studies (Zou et al. 2020).
A small study in ASD and ADHD children documented autoantibodies for
GAD65, an enzyme expressed in GABAergic neurons (Rout et al. 2012), though
another study in older patients could not replicate this finding (Hegvik et al. 2014). In
line with evidence for cerebellar dysfunction in ADHD, two studies on children with
ADHD found antibodies directed against Yo (PCA-1), a protein found in the
cytoplasm of cerebellar Purkinje cells, in most ADHD cases (77.5%) (Passarelli
et al. 2013; Donfrancesco et al. 2020), although again this finding was not replicated
in a subsequent study (Cetin et al. 2019). Another group found that a subset of
individuals with ADHD had autoantibodies against the dopamine transporter (DAT)
(Giana et al. 2015; Adriani et al. 2018), and this correlated positively with ADHD
symptoms in patients with a 10-repeat variant of the DAT1 gene (Giana et al. 2015).
These autoantibodies may be related to neuroanatomical or functional abnormalities
associated with neurodevelopmental disorders, as administration of myelin-specific
antibodies isolated from autistic donors to rodent hippocampal slices reduced
myelination and long-term potentiation in the hippocampus (Gonzalez-Gronow
et al. 2015).
Maternal autoimmune disease is associated with increased risk for ASD in
offspring (Chen et al. 2016), and studies have also documented autoantibodies in
the blood of some mothers of autistic children (Mazon-Cabrera et al. 2019). As with
individual autoantibodies, maternal autoantibodies may have consequences for
offspring brain structure. For example, head and brain enlargement is found in
about 16% and 9% of autistic patients (primarily examined males) (Sacco et al.
2015), and autistic male children from mothers with maternal autoantibodies
displayed exacerbated ASD-associated brain enlargement (females not examined)
(Nordahl et al. 2013). This could be related to behavior, as brain enlargement was
more severe in lower functioning patients (Sacco et al. 2015). Likewise, a mouse
model of maternal autoantibody exposure produced repetitive grooming, reduced
play, and altered vocalization behavior in offspring of both sexes (Jones et al. 2020).
However, there may be sex-specific effects regarding how these maternal autoanti-
bodies causally relate to brain and behavioral deficits. A follow-up study in the same
mouse model found that maternal autoantibody exposure increased brain volume in
female but not male offspring (Bruce et al. 2021). Brain region volumes generally
correlated positively with behavior in maternal antibody-exposed females while
correlating negatively or not at all in exposed males (Bruce et al. 2021). This begs
the question: What other outcomes relating to neurodevelopmental disorder risk and
etiology may vary according to sex that have not yet been systematically assessed?
5.1.1 What about Sex Differences?
As mentioned, a major problem persists in most studies mentioned in this section: no

comparisons have been made by sex. The few studies that have examined sex as a
biological variable in the context of neurodevelopmental disorder-associated
immune dysregulation have found sex differences. For example, decreased levels
of peripheral IL-1β, IL-8, MIP-1β, and VEGF were each associated with higher
autism severity in females but not males (Masi et al. 2017). Conversely, platelet-
derived growth factor (PDGF)-BB was negatively associated with ASD severity in
both sexes (Masi et al. 2017). A separate study examining serum biomarkers in
adults with and without Asperger’s syndrome (AS), an ASD characterized by less
severe symptoms and the absence of language difficulty, found distinct molecular
profiles in males and females. Males with AS primarily displayed increases in
various cytokines whereas females with AS primarily displayed increases in levels
of growth factors like BDNF (Schwarz et al. 2011). These data call into question the
applicability and validity of previous research in neurodevelopmental disorder-
related immune dysregulation and underscore the need for the inclusion of both
males and females in clinical research.
5.2 Maternal/Prenatal Immune Activation
In humans, maternal immune activation (MIA) during pregnancy, whether it be

through infection, allergy, autoimmunity, or pollution, increases risk for
neurodevelopmental disorders in the offspring, including ASD, ADHD, and schizo-
phrenia (see Box 1 for a brief discussion of schizophrenia) (Chen et al. 2014; Zerbo
et al. 2015b; Han et al. 2021b; Instanes et al. 2017; Roberts et al. 2013; Brown et al.
2004). Rodent studies of maternal immune activation (MIA) during pregnancy have
improved our understanding of the underlying physiological mechanisms through
which immune perturbation during pregnancy may confer increased risk for
neurobehavioral and immune abnormalities in the offspring, including in some
cases sex differences in this risk or resilience.
Box 1 Maternal Immune Activation and Sex Differences: Relevance

to Schizophrenia
Schizophrenia is a long-term psychiatric disorder that typically shows onset in
the late teenage years. Despite the latent nature of onset in late adolescence to
early adulthood, there are early life risk factors for schizophrenia that overlap
with the neurodevelopmental disorders covered in this chapter. In particular,
maternal immune activation during pregnancy, such as viral infection, is
associated with, on average, a three-fold increased risk for schizophrenia in
(continued)
Box 1 (continued)
offspring (Brown et al. 2004; Pearce 2001), as are obstetric complications,
preeclampsia, and nutritional deficiencies during pregnancy (for review, see
Müller 2018). There is also clinical evidence of inflammatory differences in
male and female schizophrenic individuals, including elevated peripheral
cytokines and altered TSPO PET labeling (sometimes increased, sometimes
decreased) in schizophrenic individuals that correlate with symptom severity,
and increased markers of inflammation in the postmortem brains of schizo-
phrenic individuals (Müller 2018; Lin et al. 1998; Saetre et al. 2007). Inter-
estingly, in a study that has directly compared gene expression profiles across
psychiatric disorders, the same astrocyte gene modules are seen to be elevated
in both schizophrenia and ASD but noteworthy differences were seen in
microglia-related gene module expression between the two disorders (Gandal
et al. 2018).
Given this relationship between immune activation and schizophrenia,
maternal immune activation rodent models are also relevant to the study of
schizophrenia, and many research groups have assessed neural systems
hypothesized to be implicated in schizophrenia (e.g., dopaminergic system,
excitatory/inhibitory balance) as well as behavioral domains of particular
relevance to schizophrenia (e.g., sensory gating of startle responses, atten-
tional and cognitive flexibility deficits) (e.g. Borrell et al. 2002; Meyer et al.
2009; Canetta et al. 2016) to understand the mechanisms through which MIA
could contribute to the etiology of schizophrenia. Thus, many of the studies we
report on in this chapter as relevant to ASD may also be relevant to our
understanding of schizophrenia, and in fact, were conceptualized that way
by the researchers who performed them.
With regard to sex differences, there is a slight male sex bias in the overall
rate of schizophrenia (1.4:1) that is far less dramatic than that seen with ASD
and ADHD, but there are also sex differences in age of onset, with males
typically showing significantly earlier age of symptom onset than females (late
teens-early twenties in males versus late twenties-early thirties in females)
(McGrath et al. 2008; Abel et al. 2010). Symptom-wise, females tend to show
higher rates of affective symptoms, particularly later in the course of the
disorder as they undergo menopause (which may show a modulatory role for
female-typical hormones in this sex difference), whereas males show more
severe symptoms overall and more positive symptoms, such as hallucinations
and delusions (reviewed in Abel et al. 2010). Regarding the genetics of
schizophrenia, interesting recent work has now implicated the complement
system, in particular the C4 gene locus in the major histocompatibility com-
plex, as highly associated with sex-specific risk for schizophrenia in males
(Kamitaki et al. 2020). While mutations in the C4 allele are associated with
elevated schizophrenia risk in both sexes, they are more pronounced in males,
(continued)
Box 1 (continued)
and the group found that C4 protein levels are higher in schizophrenic
individuals, and higher in males in CSF during earlier ages (twenties versus
fifties), which may explain male-biased risk for earlier onset schizophrenia
(Kamitaki et al. 2020). Interestingly, this same group demonstrated collabo-
ratively that C4 is necessary for developmental synaptic pruning by microglia
to occur in the brain (Sekar et al. 2016). Thus, overactive C4 function in
schizophrenia, particularly in males, may in part be responsible for the onset of
the disorder. Maternal immune activation may in turn further perturb this
developmental function of the neuroimmune system and be relevant to
sex-specific schizophrenia risk or onset.
Two models of prenatal infection have been used in the majority of preclinical
MIA studies in rodents: LPS, which models bacterial infection, and the synthetic
double stranded RNA polyinosinic:polycytidylic acid (Poly I:C), which models viral
infection (Meyer 2014). LPS binds to toll-like receptor (TLR) 4 while Poly I:C binds
to TLR3 on immune cells to mimic the acute-phase response to infection, and both
cascades converge on the nuclear factor kappa B (NFkB) pathway to produce strong
Th1-like cytokine responses (Meyer 2014). While LPS is a potent inducer of TNFα,
Poly I:C is a potent inducer of interferons (Meyer 2014). However, these infection
models are not the only rodent models of MIA. Given the increasing links between
pollution, allergy, and neurodevelopmental disorders (Chua et al. 2020; Chen et al.
2014; Roberts et al. 2013), other more recently developed MIA paradigms have
focused on pollution-induced inflammation or allergy. These models generally
involve inflammation beginning in the airway that results in the expansion of the
Th2-like cytokine response, as opposed to the Th1-like cytokine response
(Schwartzer et al. 2015; Sharkhuu et al. 2010). Generally, MIA studies across
models reproduce behavioral endophenotypes associated with neurodevelopmental
disorders.
Most MIA studies have been performed exclusively in male rodents. These
studies have found that MIA impairs communication behavior, either reducing
(Kirsten et al. 2012; Malkova et al. 2012) or increasing (Choi et al. 2016) neonatal
ultrasonic vocalizations. MIA paradigms have found reduced social behavior
(Malkova et al. 2012; Choi et al. 2016; Smith et al. 2007; Kirsten et al. 2010a),
impaired learning and memory (Canetta et al. 2016; Kirsten et al. 2012), anxiety-like
behavior (Canetta et al. 2016; Smith et al. 2007; Penteado et al. 2014), and altered
sensorimotor gating (Smith et al. 2007; Mattei et al. 2014). Additionally, some MIA
studies have found increased repetitive behaviors, as assessed by self-grooming or
marble burying (Kirsten et al. 2012; Malkova et al. 2012; Choi et al. 2016; Kirsten
and Bernardi 2017). At the neurochemical level, these male-focused studies have
found evidence for reduced dopaminergic activity, which aligns well with hypoth-
eses of reduced motivation and reward in human neurodevelopmental disorders.
Specifically, levels of the dopamine-synthesis enzyme tyrosine hydroxylase, as well
as dopamine and dopamine metabolite levels in are reduced in the striatum of adult
offspring (Kirsten et al. 2012; Kirsten et al. 2010b). Dopamine hypofunction has also
been documented in the hypothalamus (Kirsten and Bernardi 2017). Reduced levels
of GABA synthesis enzyme, GAD67, in medial prefrontal cortex interneurons in
adult male offspring have also been seen after MIA (Canetta et al. 2016), suggestive
of altered excitatory-inhibitory balance in regions critical for cognition.
In terms of immune mechanisms, two peripheral cytokines, IL-6 and IL-17, are
necessary for the behavioral effects of Poly I:C-induced MIA, at least in males.
Poly I:C injection on E12.5 reduces sensorimotor gating, exploration, and social
behavior in adult offspring, and genetic knockout or blockade of IL-6 action pre-
vents these behavioral alterations as well as MIA-induced changes in cortical gene
expression (Smith et al. 2007). A different group found that the same paradigm
increased levels of serum and placental IL-17a and IL-17 receptor subunit A
(Ra) expression in the fetal brain in an IL-6-dependent manner, suggesting that
IL-17a acts downstream of IL-6 (Choi et al. 2016). These offspring displayed
abnormal cortical lamination, increased ultrasonic vocalizations, repetitive behavior,
and impaired sociability, and maternal treatment with an IL-17a blocking antibody
protected offspring from these effects (Choi et al. 2016). Th17 cells may be the
drivers of poly I:C-induced behavioral deficits, as expression of RORγt, a transcrip-
tional regulator of Th17 cell development, in maternal T cells was required for the
abnormal behavioral phenotype in the offspring (Choi et al. 2016). Importantly, the
evidence for elevated IL-17Ra levels in the fetal brain suggests increased IL-17a or
Th17 activity in the fetus as well. IL-17 activity downregulates both Th1 and Th2
responses (Ajendra et al. 2020). Thus this data goes well with research from humans
showing that blood samples from neonates that would eventually receive an ASD
diagnosis had decreased levels of both Th1-like and Th2-like cytokines (Abdallah
et al. 2012). Meanwhile, a separate study found that Poly I:C offspring display
decreased regulatory T cells and hyper responsive helper T cells in the periphery of
adult offspring (Hsiao et al. 2012), which also goes along with work in older autistic
patients that was discussed above.
Microglia changes have been examined in the context of MIA, but findings are
heterogenous and thus challenging to make broad conclusions about. Most MIA
studies that have investigated microglia examine effects on juvenile and adult male
offspring, with some studies finding increased microglial activation or density and
others finding the opposite or no change (Smolders et al. 2018). Some studies on
adult offspring have found no changes in microglia markers, number, or density in
multiple brain regions (Kirsten et al. 2012; Mattei et al. 2014; Missault et al. 2014).
However, other studies have found elevated microglia number and activation in
the neonatal and adolescent amygdala (O'Loughlin et al. 2017), as well as the
juvenile hippocampus and striatum (Juckel et al. 2011), all regions important for
social behavior, cognition, and locomotor activity. Additionally, lack of change in
microglia number or staining density does not necessarily reflect a lack of change in
function. Studies in a Poly I:C model have found impaired hippocampal
neurogenesis, reduced expression of inflammatory response genes, and impaired
microglial phagocytosis in adult male offspring (females not examined) (Mattei et al.
2014; Mattei et al. 2017). Notably, microglial inhibition with chronic minocycline
treatment in adulthood after MIA rescued neurogenesis, gene expression, phagocy-
tosis, and behavior in these males, suggesting a causal role for microglia in the
neurogenic deficits and behavioral phenotype (Mattei et al. 2014; Mattei et al. 2017).
Evidence suggests that microglia may also be affected proximally to the immune
insult, as Poly I:C MIA alters the transcriptome of early-stage microglia, indicating
increased maturity and inflammatory capacity, at least in males (females not exam-
ined) (Matcovitch-Natan et al. 2016). This study goes along with other work
suggesting accelerated microglial development in ASD (Hanamsagar et al. 2017).
Thus, microglia may be affected proximally to the immune insult during a critical
period of development, programming long-term changes in brain and behavior.
Furthermore, MIA-induced microglia alterations may vary according to paradigm,
age at immune challenge, and age at assessment.
All the above-mentioned studies were conducted in males so it is unknown the
extent to which these particular findings apply in females, which will hopefully be
addressed in future work. Studies that examine both sexes enable scientists to tease
out potential sex differences in vulnerability to prenatal inflammation. Some studies
across paradigms have documented inflammation-induced behavioral changes in
both sexes, including decreased sociability (Breach et al. 2021; Schwartzer et al.
2015; Schwartzer et al. 2017; Hui et al. 2018), altered sexual behavior (Lenz et al.
2019), increased rearing and/or repetitive behavior (Schwartzer et al. 2015;
Schwartzer et al. 2017; Hui et al. 2018; Makinson et al. 2017; Gzielo et al. 2021),
disrupted sensorimotor gating (Borrell et al. 2002), increased baseline startle
response (Van den Eynde et al. 2014), and impaired cognitive flexibility (Breach
et al. 2021). Studies have also found increased anxiety-like behavior (Meyer et al.
2006) and increased (Breach et al. 2021; Schwartzer et al. 2017; Makinson et al.
2017; Gzielo et al. 2021; Makinson et al. 2019) or decreased (Van den Eynde et al.
2014) locomotor activity in both sexes. However, other MIA studies suggest that
males may be preferentially affected by MIA, which would make sense given the sex
bias in prevalence rates, though findings vary across paradigms (See Table 2 for a
summary). For example, LPS-induced MIA reduces juvenile social play behavior in
male but not female Wistar rats (Kirsten et al. 2012; Kirsten et al. 2010a; Taylor et al.
2012; Vitor-Vieira et al. 2021). Additionally, these MIA males displayed altered
vasopressin expression in the amygdala (Taylor et al. 2012), blunted play-induced
neuronal activation in PFC, NAc, and striatum, and potentiated play-induced neu-
ronal activation in the amygdala (Vitor-Vieira et al. 2021). Likewise, one study
found that Poly I:C preferentially impaired social play in male mice (Gzielo et al.
2021), while another found social deficits in both sexes (Hui et al. 2018). However,
male mice were more susceptible to Poly I:C-induced anxiety-like behavior and
sensorimotor gating impairments (Hui et al. 2018). A combined model of diesel
exhaust particle exposure and maternal stress (DEP + MS) increases anxiety-like
behavior only in male offspring (Bolton et al. 2013). This same paradigm preferen-
tially impairs neonatal ultrasonic vocalizations and fear conditioning in male mice
(Bolton et al. 2013; Bilbo et al. 2018), yet increases juvenile locomotor behavior and
neophobia in both sexes (Bolton et al. 2013). In summary, MIA appears to affect
Table 2 Summary of behavioral findings in studies of prenatal inflammation. Studies cited include
rat and mouse models
Offspring behavior Findings from studies with both sexes
Vocalizations – DEP+MS MIA increases vocalization number and decreases call
frequency in male but not female neonates (Bilbo et al. 2018)
– LPS MIA decreases vocalization number in male but not female
neonates (Vitor-Vieira et al. 2021)
– Poly I:C MIA reduces number of vocalizations emitted during
juvenile play in both sexes (Gzielo et al. 2021)
Social behavior – LPS and Poly I:C MIA reduce juvenile play in males and not
females (Kirsten et al. 2012; Kirsten et al. 2010a; Gzielo et al. 2021;
Taylor et al. 2012; Vitor-Vieira et al. 2021)
– Poly I:C, Acute allergic MIA, and Chronic allergic MIA reduce
sociability in both sexes (Breach et al. 2021; Schwartzer et al. 2015;
Schwartzer et al. 2017; Hui et al. 2018)
Sex behavior – Acute Allergic MIA masculinizes female sex behavior and reduces
male olfactory preference for females (Lenz et al. 2019)
Repetitive behavior – Chronic Allergic and Poly I:C MIA increase marble burying in both
sexes (Schwartzer et al. 2015; Schwartzer et al. 2017; Hui et al. 2018;
Gzielo et al. 2021)
Locomotor activity – In-utero LPS, DEP + MS, and Allergic MIA produce hyperactivity
in both sexes (Breach et al. 2021; Schwartzer et al. 2017; Makinson
et al. 2017; Makinson et al. 2019; Bolton et al. 2013)
– Poly I:C MIA produces hypoactivity (Van den Eynde et al. 2014)
or hyperactivity (Gzielo et al. 2021) in both sexes
Cognition – DEP+MS MIA impairs fear conditioning only in males (Bolton

et al. 2013).
– Acute Allergic MIA reduces cognitive flexibility in both sexes
(Breach et al. 2021)
DEP diesel exhaust particle exposure, LPS lipopolysaccharide, MIA maternal immune activation,
MS maternal stress
behavior in both males and females, though some research points to sex-specific
vulnerabilities, particularly in males.
While causal manipulations are lacking, studies examining MIA effects in both
sexes have found alterations to microglia in combination with such impaired behav-
ior discussed above. Adult offspring exposed to Poly I:C on E15 display increases in
microglia number and/or activation in the pons, thalamus, corpus callosum, and
hippocampus (Van den Eynde et al. 2014). Notably, differential mast cell and
microglia number/activation in the neonatal preoptic area is responsible for
programming adult sex behavior (Lenz et al. 2013; Lenz et al. 2018), and acute
prenatal allergic inflammation produces female-specific increases in mast cell num-
ber and ameboid microglial tone in the neonatal POA, and this is followed by
reversed sexual differentiation of POA dendritic spine density and masculinized
sexual behavior in adulthood (Lenz et al. 2019). A follow-up in the same paradigm
found impaired juvenile social play, late adolescent hyperactivity, and adult cogni-
tive inflexibility in male and female offspring, and these behaviors were preceded by
decreases in neonatal microglia colonization in brain regions relevant to these
behaviors in both sexes (Breach et al. 2021). While only conducted in females, a
separate study in a model of maternal allergic asthma found that differentially
methylated regions in adolescent offspring were enriched for transcription factor
binding sites related to regulation of microglial development and microglial inflam-
mation (males not examined) (Vogel Ciernia et al. 2018). Further analysis revealed
that differentially expressed and methylated genes in this model significantly
overlapped with a list of ASD risk genes (Vogel Ciernia et al. 2018), indicating
that alterations to microglia function may be causally implicated in ASD.
Other work suggests that males and females may be differentially susceptible to
certain immune-related consequences of prenatal inflammation. Generally, males
may be predisposed to an elevated immune profile following MIA. For example,
while prenatal Poly I:C on E9.5 increases C3 protein expression in the dentate gyrus
of adult offspring of both sexes, it only produces microglia activation in males,
despite female-specific increases in microglial CD68 and CD11b in the hippocam-
pus (Hui et al. 2018). Prenatal DEP exposure increases inflammatory tone in the fetal
male brain, decreasing brain IL-10 and elevating the number of ameboid microglia in
the cortex and hippocampus (Bolton et al. 2013). The opposite is true in the fetal
female brain, where prenatal DEP elevates brain IL-10 and decreases the number
ameboid microglia in the dentate gyrus (Bolton et al. 2013; Bilbo et al. 2018; Bolton
et al. 2017). As juveniles, DEP males have elevated levels of cytokines, increased
ameboid microglia, and also display increased neuron-microglia overlap (Bolton
et al. 2013; Bolton et al. 2017). Combined DEP + MS increases IL-1β/IL-10 ratio in
the brain of adult male offspring, but decreases it in females (Bolton et al. 2013;
Bilbo et al. 2018). Notably, as mentioned above, these studies found more male-
specific behavioral alterations in anxiety-like behavior as adults, but both males and
females exhibited hyperactivity and reduced social exploration as juveniles (Bilbo
et al. 2018).
Such an elevated immune profile may predispose males to have a hyperactive
immune response following an acute challenge. A separate study employing an LPS
model of in utero inflammation found reduced myelination of the corpus callosum
and increased microglia number in adult offspring of both sexes (Makinson et al.
2017; Makinson et al. 2019). However, some proximal and ultimate immune
consequences were disparate between the sexes. IL-1β and TNFα were elevated in
fetal male brains 48 h following prenatal LPS challenge, a trend that was absent in
females, and male offspring also displayed increased levels of TGFβ, C1q, and C3 in
the brain as neonates, whereas female neonates had decreased levels of C3
(Makinson et al. 2017). Interestingly, these male offspring had potentiated central
cytokine responses to acute administration of LPS in adulthood that were absent in
females (Makinson et al. 2017). Collectively, these findings suggest that males may
be more susceptible to both inflammatory risk factors for and consequences of
prenatal inflammation, which would make sense given genetic data on inflammatory
markers discussed above (Werling et al. 2016). Additionally, given that LPS
advances microglial maturity in males but not females (Matcovitch-Natan et al.
2016; Hanamsagar et al. 2017), exposure to immune challenges throughout life
may explain why adult male brains have higher indices of microglial maturity, and
why the brains of autistic individuals also display signs of elevated microglial
maturity (Hanamsagar et al. 2017). It is therefore imperative that MIA studies should
examine sex as a potential moderator of outcomes, given that (1) in humans, both
males and females develop neurodevelopmental disorders, (2) MIA may induce
sex-specific behavioral deficits, and (3) MIA may induce similar behavioral deficits
in the sexes, but through different mechanisms.
5.3 Early Life Immune Activation
Immune activation during the postnatal period also increases risk for
neurodevelopmental disorders. Preterm born children who experience systemic
inflammation in the first postnatal month have increased risk for ADHD or
ASD-related symptoms (Allred et al. 2017; Korzeniewski et al. 2018). In line with
data showing that the development of atopic dermatitis and asthma in early life
increases risk for subsequent diagnosis of both ASD and ADHD (Chen et al. 2014;
Liao et al. 2016), a rodent model of chronic allergic asthma beginning in the neonatal
period found repetitive behavior and impaired sociality in adult (but not juvenile)
animals (Saitoh et al. 2021). These animals also had impaired microglial engulfment
of postsynaptic markers and reduced expression of engulfment-related genes during
the peak pruning period in the hippocampus, and this was followed by an excess of
excitatory synapses in the hippocampus (Saitoh et al. 2021). A separate model of
early life infection with E. coli increases cytokine and microglial expression in the
hippocampus of neonatal males (females not examined) (Bilbo et al. 2005a), causes
hypomyelination of subcortical white matter and motor cortex in juvenile males
(Lieblein-Boff et al. 2013), and increases levels of microglial markers and
hyperreactive astrocytes in the adult hippocampus (Bilbo et al. 2005a; Bilbo et al.
2005b). Behavioral sequelae include locomotor hyperactivity and impaired coordi-
nation as juveniles and adults (Lieblein-Boff et al. 2013), as well as impaired
learning and memory following acute LPS challenge (Bilbo et al. 2005a; Bilbo
et al. 2005b). Blockade of IL-1β synthesis or microglial activation prior to LPS
challenge prevented the combined effects of neonatal infection + LPS on learning
and memory (Bilbo et al. 2005a; Williamson et al. 2011), collectively suggesting
that microglia-mediated mechanisms may underlie impairments induced by neonatal

inflammation, at least in males.
Notably, diets that are high in fat induce systemic and chronic low-grade inflam-
mation in both brain and body (Guillemot-Legris et al. 2016), and gestational obesity
is a risk factor for ADHD (Rodriguez et al. 2008). Thus, perinatal exposure to high
fat diet (HFD) through manipulation of maternal chow during pregnancy and
lactation is yet another relevant model of early inflammation. As such, perinatal
HFD produces a hypodopaminergic condition in the mesocorticolimbic pathway in
adult males (females not examined) (Vucetic et al. 2010). This paradigm also
decreases motivation to earn reward and increases impulsivity in adult male and
female offspring (Grissom et al. 2015). Male and female HFD offspring also display
elevated concentrations of cysteine in the prefrontal cortex, consistent with oxidative
stress (McKee et al. 2018). Notably, many of these deficits can be rescued in both
sexes through dietary supplementation with methyl donors, which are nutrients that
supply methyl groups important for cellular function and protein synthesis (McKee
et al. 2017; Carlin et al. 2013). However, some effects of perinatal HFD appear to be
male-biased. Examination of gene expression in the prefrontal cortex of male
offspring found that impulsivity related to overexpression of DNMT1 (females not
examined) (Grissom et al. 2015), and this may represent a compensatory mechanism
for decreased DNA methylation in these males (females not examined) (Vucetic
et al. 2010). A follow-up study found that this was a male-specific effect, as perinatal
HFD reduced DNA methylation in the male but not female prefrontal cortex, and this
could be rescued with dietary supplementation of methyl donors (McKee et al.
2018).
Other studies also suggest there may be sex differences following neonatal
inflammation. Specifically, neonatal challenge with LPS increased anxiety- and
depressive-like behaviors, risk-taking, and working memory deficits in peri-
adolescent male mice, with the anxiety- and depressive-like behaviors persisting
into adulthood. Conversely, at either timepoint, female mice only displayed deficits
in sensorimotor gating (Custodio et al. 2018). As adults, males also had increased
nitrite levels and decreased parvalbumin in the hippocampus, while both sexes
experienced increased levels of proinflammatory cytokines in the brain (Custodio
et al. 2018). A similar paradigm found impaired spatial memory in adult males but
not females, though both sexes displayed social impairments as adolescents (Macrae
et al. 2015). Conversely, a recent study found that neonatal inflammation with LPS
produces female-specific impairments in adult sociability and social discrimination,
and this was combined with increased somatostatin cell number in the anterior
cingulate cortex (Smith et al. 2020). As a whole, these studies suggest that males
may be more susceptible to emotional and cognitive related impairments following
neonatal inflammation, whereas females may or may not be more susceptible to
social impairment. More work needs to be done to ascertain the consistency of sex
differences in these domains, as well as whether they may vary by age of inflam-
mation, age at assessment, or other factors.
6 Concluding Remarks and Ways Forward
Autism and ADHD are the two most common neurodevelopmental disorders diag-
nosed in children. Both conditions are diagnosed more commonly in males, and sex
differences in symptomology, onset, comorbidities, and immunological abnormali-
ties have been documented. Despite this, most clinical and preclinical studies on
immune-related mechanisms and endpoints of neurodevelopmental disorders have
not examined sex as a biological variable (see Box 2 for our summary of the major
knowledge gaps and high priority future goals). Overall, research that has investi-
gated sex differences reveals that males and females may be differentially suscep-
tible to certain neurodevelopmental disorder-associated risk factors, with males
potentially being more vulnerable to immune-related risk factors for these condi-
tions. Likewise, mechanisms underlying ASD and ADHD in males and females may
be different and merit further investigation. Only by studying both males and
females and considering how sex interacts with gender norms and societal expecta-
tions to lead to diagnostic biases will we gain a complete picture of how
neurodevelopmental disorders arise and grasp the full complexity of the etiology,
symptomology, and treatment strategies for these complex conditions.
Box 2 Major Knowledge Gaps

Throughout this chapter, we have discussed what research has taught us
regarding ASD, ADHD, and animal models. However, whether many well-
established research findings apply to females is still unknown. Given that
neurodevelopmental disorders continue to affect both males and females, we
believe the following questions should be priorities for scientific research
moving forward.
1. To what extent does sex affect the developmental trajectory of ASD and
ADHD, and how does this change our understanding of symptomology and
prevalence? (Sect. 2).
2. Are males and females differentially susceptible to specific genetic risk
factors or polygenic interactions for ASD and ADHD? (Sects. 4.1 and 4.2).
3. Do the patterns of central and peripheral immune dysregulation in ASD and
ADHD apply to females with these conditions or are they sex-specific?
(Sects. 4 and 5).
4. Does the maternal response in utero to males and females during gestation
modulate the developmental etiology of ASD or other NDDs?
5. Developing animal models that better reflect sex-specific genetic risk and
protective factors for NDDs to dissect mechanisms at play in NDD etiol-
ogy, and studying them across the lifespan and in combination with
environmental risk exposures (Sect. 5.2).
Funding
This material is based upon work supported by the National Science Foundation Graduate Research
Fellowship Program. Any opinions, conclusions, or recommendations expressed in this material are
those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Conflict of Interest The authors declare no competing financial interests.
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Sex Differences in Social Cognition
Pietro Paletta, Noah Bass, Dario Aspesi, and Elena Choleris
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
2 Sex Differences in Social Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.1 Estrogens and Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2.2 Oxytocin and Vasopressin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
3 Sex Differences in Social Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
3.1 Birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
3.2 Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
3.3 Non-human Primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
4 Sex Differences in Aggressive Behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
5 Conclusions and Relevance to Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Abstract In this review we explore the sex differences underlying various types of
social cognition. Particular focus will be placed on the behaviors of social recogni-
tion, social learning, and aggression. Known similarities and differences between
sexes in the expressions of these behaviors and the known brain regions where these
behaviors are mediated are discussed. The role that the sex hormones (estrogens and
androgens) have as well as possible interactions with other neurochemicals, such as
oxytocin, vasopressin, and serotonin is reviewed as well. Finally, implications about
these findings on the mediation of social cognition are mediated and the sex
differences related to humans are considered.
Keywords Aggression · Androgens · Estrogens · Social Learning · Social

Recognition
P. Paletta, N. Bass, D. Aspesi, and E. Choleris (*)

Department of Psychology and Neuroscience Program, University of Guelph, Guelph, ON,
Canada
e-mail: echoleri@uoguelph.ca
https://doi.org/10.1007/7854_2022_325
Published Online: 24 May 2022
208 P. Paletta et al.
1 Introduction
Social cognition refers to behaviors which involve communication and/or interaction

between at least two individuals (Chen and Hong 2018). There are many types of
behavior that can fall under social cognition because their expression relies on
interactions with others, such as reproduction, where social information is used to
choose the best possible mate (Choleris et al. 2018). Social recognition, social
learning, and aggression are other examples of behaviors that fall under social
cognition and will be discussed in this review. These behaviors can be seen in a
wide range of species, from highly social ones like humans or rodents to more
solitary species like tigers. From an evolutionary perspective, these behaviors
developed and continue to be expressed because they provide an important evolu-
tionarily advantage (Burkart 2017; Chen and Hong 2018). For example, the social
behavior of parenting helps ensure the survival of the next generation, by protecting
the offspring from threats and providing them resources they are not yet capable of
acquiring, so that they can also reach an age where they can reproduce themselves
(Chen and Hong 2018). In highly social species like humans, the formation of
communities where individuals live in groups also contributes to their survival as
they can learn from each other, have greater access to resources, and defense from
threats (Silk 2007; Chen and Hong 2018).
The neural mechanisms underlying social behaviors are complex, because there
are many types, as mentioned above, but also because they are mediated by multiple
interconnected brain regions and neurotransmitters, that can function differently in
males and females, unsurprisingly perhaps, given that brain development is sexually
differentiated in a number of regions and social behaviors are regulated by hormones
(Choleris et al. 2018). For example, regions that play roles in social behaviors, such
as the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis
(BNST), are also larger and contain more neurons in male than in female mice and
rats (Forger et al. 2004; Campi et al. 2013; del Abril et al. 1987; Gorski et al. 1978;
Ogawa et al. 2020). This sex difference is driven by the sex hormones. Testosterone
(T), a type of androgen, masculinizes the brain during development, as shown, for
example, by the finding that neonatal administrations of T caused a decrease in the
volume of the anteroventral periventricular nucleus in female rats, a region that is
known to be larger in females than in males (Ito et al. 1986; Ogawa et al. 2020).
Similar effects are seen by the administration of 17β-estradiol (E2), suggesting that
the masculinizing effect is also due to T being converted into E2 by the enzyme
aromatase (Hisasue et al. 2010; Patchev et al. 2004; Ogawa et al. 2020). This
masculinization effect occurs by T being released from the testes as well as the
conversion of T to E2 during the perinatal period and further masculinization occurs
by increased T release during the peripubertal period (Schulz et al. 2009; Ogawa
et al. 2020). In females, during the perinatal period the brain is protected from the
masculinizing effect of high E2 levels by the α-fetoprotein which has a high binding
affinity for estrogens and prevents it from crossing the blood-brain barrier (Bakker
and Baum 2008). Subsequently, in the peripubertal period, there is a decrease in
Sex Differences in Social Cognition 209
α-fetoprotein and an increase in E2 levels released from the ovaries which acts to
feminize the brain, causing the increased volume in regions that are larger in females
than in males (Raynaud 1973; Bakker and Baum 2008; Ogawa et al. 2020). The
effect of these hormones on the sexual differentiation of various regions seems to be
dependent on the estrogen and androgen receptors (ER and AR, respectively). For
example, in studies where the genes for ER alpha (ERα), aromatase, or AR are
knocked out (KO), there is abnormal development of regions that are usually larger
in males, such as the BNST and the MPOA (Tsukahara et al. 2011; Kanaya et al.
2014; Ogawa et al. 2020). Similar to the effects of the steroid receptors, epigenetic
mechanisms play a role in the sexual differentiation of the brain. For example, the
increased size of the BNST in males has been linked to decreased apoptosis in males
compared to females (McCarthy et al. 2018). This effect has been found to be
epigenetically mediated through histone deacetylation repressing the genes that
trigger apoptosis in this region for males but not females, as neonatal administrations
of a histone deacetylation inhibitor to males prevented the masculinization of the
BNST (Matsuda et al. 2011; Murray et al. 2009; McCarthy et al. 2018). The findings
from these and other studies demonstrate a critical role for the sex hormones in the
sexual differentiation of the brain.
Sex hormones also play an important role in the regulation of social behaviors,
and this combined with their effects on sex differences in various brain regions,
likely explains why sex differences in social cognition are common (Choleris et al.
2018). The effects of these hormones can occur through two mechanisms of action:
the genomic and rapid mechanisms. The genomic mechanisms occur following the
binding of estrogens to ERα or ER beta (ERβ) or androgens to the AR, which then
act as transcription factors and bind to estrogen response elements or androgen
response elements of the DNA (Nilsson et al. 2001; Sheppard et al. 2018; Grino
et al. 1990; Lukas-Herald et al. 2017). The rapid effects occur by estrogens binding
to ERα, ERβ, or the G-protein couple ER (GPER) or androgens binding to the AR
and, rather than acting as transcription factors, trigger cell signaling cascades to
affect cell functioning (Sheppard et al. 2018; Lukas-Herald et al. 2017). The
genomic mechanisms tend to be delayed in their onset whereas the rapid mecha-
nisms, as their name suggest, have a much faster onset (Sheppard et al. 2018; Lukas-
Herald et al. 2017). Through these mechanisms, estrogens and androgens can affect
various social behaviors. In addition to the actions of the sex hormones, the sex
chromosomes can also affect behavior, as demonstrated in experiments with a mouse
model that separates the development of the gonads from the sex chromosomes by
randomly expressing the SRY gene onto autosomes, so that of the SRY gene will
cause testes to develop regardless of the sex chromosomes present (De Vries et al.
2002). Studies using this mouse model show that mice with XX chromosomes
investigated an intruder less than XY mice, regardless of their gonadal sex
(McPhie-Lalmansingh et al. 2008). Therefore, both sex hormones and sex chromo-
somes affect social behavior.
This review will highlight the sex differences in the expression of three social
behaviors: social recognition, social learning, and aggression. The role of estrogens
and androgens in males and females, as well as their possible interaction with other
neurotransmitters and neuropeptides will be discussed for each behavior. For social
recognition, the role of oxytocin and vasopressin in relation to estrogens and
androgens will be explored. For social learning, sex differences in various species
and the role of estrogens are reviewed. For aggression, the roles of serotonin and
vasopressin and possible interactions with estrogens and androgens will be
discussed. Lastly, we will conclude with what is known about these sex differences
in these same social behaviors in humans and in selected disorders of social
cognition will be briefly explored.
2 Sex Differences in Social Recognition
2.1 Estrogens and Androgens
Social recognition is defined as the ability to distinguish between conspecifics based

on information that has been acquired from previous encounters or from innate
knowledge (Choleris et al. 2012). Social recognition can be based on various aspects
of the individual being investigated, such as species, sex, hierarchal status, health,
familiarity, and individual-specific features (Choleris et al. 2012). Social recognition
plays a critical role in multiple social behaviors, as the processing of information
about others modulates the social behaviors exhibited toward them. The most
common way of testing social recognition in rodents is the use of a social discrim-
ination paradigm, where the experimental animal is given a binary choice between a
familiar and novel conspecific (Engelmann et al. 1995) (see Fig. 1 for a schematic of
this paradigm).
Fig. 1 Social recognition discrimination paradigm. The paradigm consists of two phases: a sample
phase, performed either once or several times, and a test phase. During the sample phase, two
conspecifics (A and B) are simultaneously presented to an experimental animal, which is free to
explore them. In the test phase, the experimental animal has the choice between one of the two
previously investigated conspecifics (A or B) and a novel individual (C). Recognition of the
previously investigated animal (A or B) should lead the experimental animal to spend more time
investigating the novel one (C). This paradigm is based on the natural preference of mice and rats
for novel over familiar stimuli
In both females and males, estrogens have been found to play an important role in
social recognition. For example, several studies have shown that ovariectomizing
female rats or mice impairs social recognition, and the administration of estrogens or
estrogens and progesterone can recover social recognition in the female rats and
mice (Spiteri and Agmo 2009; Karlsson et al. 2015; Choleris et al. 2018). Similarly,
ERα gene KO in female mice impaired social recognition (Choleris et al. 2003,
2006). Gene KO of ERβ also impaired female social recognition, however the
presence of this impairment is dependent on the method of assessing social recog-
nition (Choleris et al. 2003, 2006). This impairment was seen when a habituation/
dishabituation social recognition paradigm was used. However, when the social
discrimination paradigm was used the impairment became less apparent (Choleris
et al. 2003, 2006). In males, ERα gene KO impaired social recognition, although this
impairment is also dependent on the method of testing, as social recognition was
impaired in a 9-trial habituation/dishabituation paradigm, but not in a 5-trial para-
digm, whereas ERβ gene KO does not impair male social recognition (Imwalle et al.
2002; Sanchez-Andrade and Kendrick 2011). Estrogens seem to be able to mediate
social recognition through both their genomic and rapid mechanisms. For example,
studies showed that implanting a pellet that releases E2 into ovariectomized mice or
the administration of E2 52 h before testing facilitated social recognition (Tang et al.
2005; Spiteri and Agmo 2009). Through estrogens’ rapid mechanisms, it has been
shown that the systemic administration of E2 or specific agonists for ERα (PPT) and
GPER (G-1) to ovariectomized mice 15 min prior to testing facilitated short-term
social recognition memory, whereas the administration of an ERβ agonist (DPN)
impaired it (Phan et al. 2011, 2012; Gabor et al. 2015). Similar effects can be seen
when these treatments are delivered to specific brain regions implicated in social
recognition. For example, in ovariectomized mice when E2, PPT, or G-1 are infused
into the dorsal hippocampus or medial amygdala (MeA) 15 min prior to testing,
short-term social recognition memory is facilitated (Phan et al. 2015; Lymer et al.
2017, 2018). DPN, when infused into the MeA 15 min before testing, can also
facilitate social recognition, however when infused into the dorsal hippocampus it
had no facilitating effect (Lymer et al. 2018; Phan et al. 2015). These results
demonstrate the role estrogens and their receptors, specifically ERα and GPER,
play in regulating social recognition and show that the role ERβ plays is likely
dependent on where in the brain it is activated. In males, the rapid effects of
estrogens and their agonists on social recognition are only beginning to be investi-
gated, with both estrogens and androgens rapidly facilitating it when infused in the
BNST, one of the brain regions shown to be larger in males than females (Ogawa
et al. 2020; Aspesi and Choleris 2021). While more investigations on the hormonal
underpinnings of male social recognition are necessary, the findings from the KO
studies suggest that estrogens do play a role in mediating male short-term social
recognition memory, likely through ERα and not ERβ.
Androgens also affect social recognition, although their role is less clear. Cas-
trated male mice show impaired social recognition 7–10 days following the gonad-
ectomy, whereas 2–4 weeks following the surgery social recognition is similar to
intact males (Aspesi and Choleris 2021; Gabor et al. 2012; Karlsson et al. 2015).
However, in rats, social recognition was not affected by castration when tested every
other day post-surgery even though they were temporarily impaired when tested
1-week post-castration (Bluthe and Dantzer 1990). Similarly, another study showed
that castrating rats impaired social recognition of odor cues of stimulus rats
20–40 days post-surgery (Sawyer et al. 1984). AR gene KO in male mice also
show mixed effects on social recognition because AR KO impairs social recognition
of male, but not female stimulus mice (Karlsson et al. 2016). Based on these and
other findings, it has been suggested that androgens’ effects on social recognition are
also mediated by conversion to estrogens, as disruptions of the enzyme aromatase
impair social recognition in male mice (Pierman et al. 2008). To the best of our
knowledge, it is currently unclear if androgens in females also affect social recog-
nition through estrogenic or direct mechanisms. Overall, while a strong role for
estrogens in female social recognition has been demonstrated, more research needs
to be conducted in males and more investigations are needed to fully elucidate the
role of androgens and their receptors in both sexes.
2.2 Oxytocin and Vasopressin
Estrogens and androgens have also been hypothesized to affect social recognition
through an interplay with oxytocin and vasopressin. Oxytocin (OT) and arginine-
vasopressin (AVP) are structurally similar nonapeptides that underlie many aspects
of social behavior, including social recognition (Song and Albers 2018). Both are
produced in the paraventricular and supraoptic nuclei of the hypothalamus (PVN and
SON, respectively), and AVP can also be produced in several other regions outside
the hypothalamus, such as the posterior BNST (pBNST) and MeA (Stoop 2012).
Research suggests that estrogens interact with OT to affect social recognition,
whereas androgens interact with AVP (Albers 2012; Gabor et al. 2012; Choleris
et al. 2018) For example, estrogens facilitate the production of OT and the OT
receptor (OTR), the mRNA levels of both OT and OTR tend to fluctuate with
circulating estrogens during the estrous cycle, and in the paraventricular nucleus of
the hypothalamus (PVN), where the majority of OT is produced in the brain, 85% of
OT neurons co-express ERβ in female rats (Gabor et al. 2012; Suzuki and Handa
2005). GPER is also highly expressed in the PVN and is also co-expressed on OT
neurons, with male and female rats showing ~50% co-expression and male and
female mice showing ~70% (Hazell et al. 2009). AVP, on the other hand, is
expressed more in male than female brains of both rats and mice and is regulated
by circulating T. AVP levels are reduced by castration and this reduction can be
restored by administrations of T (De Vries et al. 1984; Choleris et al. 2009).
OT affects social recognition in both sexes. Experiments where the gene for OT
was knocked out resulted in impaired social recognition in both males and females as
well as OTR gene KO mice (Ferguson et al. 2000; Choleris et al. 2003, 2006;
Takayanagi et al. 2005). Similarly, intracerebroventricular infusions of an OTR
antagonist impair social recognition in male mice and both male and female rats
(Lukas et al. 2013; Dumais and Veenema 2016). Infusions of OT into the MeA of
male OT KO mice fully recovered social recognition (Ferguson et al. 2001).
Consistently, knocking down the OTR with antisense oligonucleotides in the MeA
of female mice impaired social recognition, suggesting the MeA is a critical region
for oxytocin mediated social recognition in both sexes (Choleris et al. 2007).
Research has also indicated other regions important for oxytocin’s mediation of
social recognition, such as the pBNST, the lateral septum (LS), and the MPOA. For
example, infusing an OTR antagonist into the LS or MPOA impairs social recogni-
tion, and infusing OT into the MPOA facilitates social recognition in male mice and
rats (Smith et al. 2019). Infusing an OTR antagonist into the pBNST of both male
and female rats impaired social recognition, however infusions of OT to this region
facilitated social recognition in males but not in females (Dumais and Veenema
2016). These findings suggest a sex difference in how OT mediates social recogni-
tion in the pBNST. OT’s effects on social recognition in the LS and MPOA in
females have not been investigated yet, so more research is needed in order to
determine if there are also sex differences in these regions. We have also recently
shown an interaction between estrogens and oxytocin in the rapid regulation of
social recognition in female mice, through an interplay between the PVN and the
MeA (Paletta et al. 2018). E2 infused into the PVN was able to rapidly facilitate
short-term social recognition memory and this facilitation was blocked by the
infusion of an OTR antagonist into the MeA, showing support for the interplay
between estrogens’ rapid effects and OT on social recognition (Paletta et al. 2018)
(see Fig. 2a).
AVP, on the other hand, seems to have a more sexually differentiated effect on
social recognition, mainly affecting males (Song and Albers 2018; Choleris et al.
2018). For example, AVP expression is significantly higher in the brains of males
than in females, systemic administration of an AVP antagonist to female rats was not
able to impair social recognition, and intracerebroventricular infusions of AVP or an
antagonist for the AVP 1a receptor (V1aR) did not affect female social recognition
(Choleris et al. 2018; Dumais and Veenema 2016). In males, systemic administration
of AVP was able to facilitate social recognition and this facilitation could be blocked
by an AVP antagonist (Dantzer et al. 1987; Gabor et al. 2012). Similarly, knocking
out the gene for V1aR, and to a lesser extent V1bR, impairs social recognition in
male mice (Wersinger et al. 2002; Bielsky et al. 2004). The BNST and LS also seem
to be important regions for AVP mediated social recognition in males. Deletion of
the BNST AVP cells impaired the recognition of a novel social stimulus in male, but
not female, mice (Whylings et al. 2020). In the LS, infusing AVP can facilitate and
blocking LS V1aRs with an antagonist impairs social recognition in male mice
(Smith et al. 2019). Despite finding no impairing effect on social recognition with
systemic or intracerebroventricular administrations of an AVP antagonist, within the
LS AVP infusions facilitated and a V1aR antagonist impaired social recognition in
female rats (Veenema et al. 2012). The authors of this research suggested that the
difference between systemic/intracerebroventricular and intra LS effects of AVP
antagonism in females on social recognition may be due to AVP having opposing
effect in different brain regions (Veenema et al. 2012). An example of this can be
Fig. 2 Schematic of the hypothesized circuits involved in the hormonal regulation of social
recognition in the brain of female and male mice. (a) in the paraventricular nucleus of the
hypothalamus (PVN) of female mouse brain, social information is processed thanks to the interplay
between estrogen receptors and oxytocinergic neurons projecting to the medial amygdala (MeA).
The MeA, processing the signal from the PVN and the sensory cues from the olfactory bulbs (OB),
can rapidly facilitate social recognition. (b) in the male mouse brain, the interaction between
androgen and estrogen receptors with vasopressinergic neurons in the bed nucleus of the stria
terminalis (BNST) may facilitate social recognition by affecting the release of vasopressin in the
lateral septum (LS). In the male brain, the MeA, as well as the BNST, receives olfactory cues from
the olfactory bulbs, further affecting social information processing in the BNST. The two hypoth-
esized brain circuits highlight that, in both sexes, a complex interaction between sex hormones and
the neuropeptides oxytocin and vasopressin is involved in social recognition. Abbreviations: BNST
bed nucleus of the stria terminalis, LS lateral septum, MeA medial amygdala, OB olfactory bulbs,
PVN paraventricular nucleus
seen with intermale aggression, as AVP in the LS facilitates but in the BNST impairs
intermale aggression (Veenema et al. 2012). However, this has not yet been inves-
tigated for social recognition. We have recently found evidence pointing to a
possible connection between T and AVP in mediating short-term social recognition
memory, as infusions of T or its metabolites, E2 or dihydrotestosterone (DHT), into
the BNST of male mice rapidly facilitated social recognition and increased vaso-
pressin fiber immunodensity in the LS (Aspesi et al. 2021) (see Fig. 2b). These
findings suggest that AVP plays an important role in regulating social recognition in
males, whereas AVP’s effect in females may be more dependent on where in the
brain it is acting.
3 Sex Differences in Social Learning
Early in the development of our species, social connection, including social learning,
was critical for survival. For example, banding together was essential for providing
protection and support to individuals. Social connection is needed for survival and
promotion of gene transmission, and lack of socialization may have severe negative
effects for humans (Cacioppo et al. 2011). Indeed, the feeling of loneliness may act
as a signal to motivate an individual to re-establish social connectiveness in order to
decrease the negative effects that social isolation may have to one’s body and overall
health (Cacioppo et al. 2011). One of the most common and adaptive forms of
learning is social learning which can be defined as “learning that occurs via the
observation of, or interaction with, a conspecific or its products” (Heyes 1994). This
type of learning aids in the mitigation of the risks associated with trial-and-error
learning allowing an individual to capitalize on the experience of others. In many
species, including songbirds, monkeys, and humans, sex differences are observed in
social learning, social learning deficits, and cognitive disorders. However, few
studies have considered the role of sex hormones in social learning and most
research was conducted with either only males (most studies) or only females.
When both sexes are included, most studies fail to compare findings between
sexes thus contributing to the significant gap in the literature on sex differences in
numerous areas of science, including social learning (Choleris and Kavaliers 1999;
Choleris et al. 2018). However, sex differences in clinical and laboratory settings
suggest that the neurobiology of the male and female social brain differ, and these
sex differences may include social learning. Social learning can also occur with
limited or no social interactions. For example, in stimulus or local enhancement an
individual learns when their attention is attracted toward a stimulus/location after
observing either another individual interacts with that stimulus or the consequences
of that interaction on the stimulus/location (Heyes 1994). Researchers have not yet
tested whether there are sex differences in this type of learning but, if sex differences
in social learning are mediated by the “quality” of social interactions, then it could be
predicted that fewer or no sex differences would be seen in types of social learning
that do not involve social interaction. Below we list a few examples of animals in
which sex differences in social learning and a role for the sex hormones have been
shown.
3.1 Birds
In many bird species, complex, species-specific vocal signals, or songs, are used for
territorial defense and mating. Often, these vocal signals develop in the absence of
environmental input, however, vocal signals are learned in some species including
songbirds and humans. In birds, these songs are typically competitively produced by
the male and they attract the female (Beecher and Brenowitz 2005). This often
sex-specific nature of song “transmitters” and “receivers” suggests a hormonal
regulation of song learning.
Indeed, brain volume differs between males and females in two species of
songbirds where males sing more than females: zebra finches (Taeniopygia guttata)
and canaries (Serinus canaria) (MacDougall-Shackleton and Ball 1999). For exam-
ple, significant sex differences were seen in telencephalic nuclei volume, a brain
region critical for song behavior. Specifically, in male zebra finches and canaries,
both the HVC (previously known as the “high vocal center” or HVc) and the robust
nucleus of the arcopallium (previously known as the robust nucleus of the
archistriatum; RA) were found to be five and three times larger than in females,
respectively (MacDougall-Shackleton and Ball 1999). Similarly, the HVC is about
three times larger in male white-crowned sparrows (Zonotrichia leucophrys) than
females, who sing far less than males. Notably, lesion studies revealed that the HVC
and RA are necessary for song production (MacDougall-Shackleton and Ball 1999).
Another brain region known as “area X” is a poorly understood subsection of the
corpus callosum and is approximately four times larger in male zebra finches in
comparison with females (MacDougall-Shackleton and Ball 1999). The sex differ-
ence in song centers appears to not be present in bird species where males and
females sing equally complex songs such as bay wrens (Thryothorus ludovicianus)
and buff-breasted wrens (Thryothorus leucotis) (MacDougall-Shackleton and Ball
1999). These findings suggest that sex differences in the brain appear when there are
sex differences in song learning behavior in many bird species further suggesting a
possible role of sex hormones in these sexual differences (see Fig. 3 for a schematic
of the brain regions involved). Indeed, during social learning, in the brains of
juvenile male and female zebra finches, estrogen synthesis increased when listening
to a tutor song suggesting a role of estrogens in song learning (Bailey and Saldanha
2015). Further, social interaction with females rapidly increased estradiol levels in
the forebrain of male zebra finches (Remage-Healey et al. 2008). These findings
suggest that estrogens are important for social interactions in zebra finches. Unlike
song learning behavior which may be differentially used between sexes, other types
of social behaviors such as socially facilitated drug seeking and the social transmis-
sion of food preference (STFP) are common in both sexes and will be discussed in
the rodent section below.
Fig. 3 Schematic view of the vocal pathways in the songbird brain. The “high vocal nucleus”
(initially hyperstriatum ventrale pars caudale, current acronym used; HVC), involved in auditory
perception, and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) project to
both the nucleus robustus arcopallialis (RA) and the area X of the striatum (Area X). Area X is
connected to the dorsolateral thalamic nucleus (DLM) which can modulate the activity of the
LMAN. The interaction among these brain nuclei affects the activation of motoneurons innervating
the muscles of vocal organs: trachea and syrinx. HVC, RA, and Area X (dashed line) tend to exhibit
sex differences in nucleus volume, with larger nuclei in male than female songbirds. The difference
in volume is not found in songbird species in which both sexes sing complex songs (e.g.,
Thryothorus ludovicianus) and in seasonal birdsongs where seasonal changes in the volume of
song nuclei have been correlated with changes in testosterone concentrations. Abbreviations: Area
X area X of the striatum, DLM medial subdivision of the dorsolateral nucleus of the anterior
thalamus, HVC a letter-based name, LMAN lateral magnocellular nucleus of the anterior
nidopallium, nXIIts tracheosyringeal portion of the nucleus hypoglossus (nucleus XII), RA nucleus
robustus arcopallialis
3.2 Rodents
There are numerous ways rodents can learn socially, and social learning has been
widely investigated in rodents. In some studies females outperform males in social
learning. For example, during a socially facilitated drug seeking task, females self-
administered nicotine more than males (Wang et al. 2014). A common way to test
social learning in rodents is by using the social transmission of food preference
(STFP) paradigm during which the preference for a novel flavored food is transferred
from a “demonstrator” animal to an “observer” during a social interaction (Choleris
and Kavaliers 1999) (see Fig. 4 for a schematic of this paradigm). Using this
paradigm, researchers have used various manipulations prior to the demonstrator-
observer interaction to determine the neurobiological bases of social learning.
A role for estrogens in the STFP has been demonstrated in rodents. Female mice
in the proestrus phase of the estrous cycle, when circulating estrogens are high,
showed a prolonged duration of the expression of the socially acquired food
preference in comparison with mice in other phases of the estrus cycle (Choleris
et al. 2011). Similarly, 24 h after a social interaction with a recently fed demonstra-
tor, observer mice in proestrus, but not estrus or diestrus, showed a preference for the
Fig. 4 Social transmission of food preference (STFP) paradigm. During the STFP, (1) a demon-
strator animal consumes a novel flavored food diet, (2) a demonstrator-observer social interaction
occurs where information about the demonstrator’s diet may be passed to the observer via odor
cues, and (3) an observer choice test occurs where the observer has access to two novel flavored
food diets, one of which the demonstrator consumed before their social interaction. If social
learning occurs, the observer will prefer the demonstrator diet
socially acquired food preference (Ervin et al. 2015a). Moreover, systemic admin-
istrations of E2 and a GPER agonist rapidly restored impaired social learning (Ervin
et al. 2015b). Instead, in studies on delayed (likely genomic) effects with drug
administration 48 and 72 h prior to STFP testing, a selective ERα agonist impaired
social learning whereas a selective ERβ agonist prolonged the duration of the
socially acquired food preference in ovariectomized mice (Clipperton et al. 2008).
These findings suggest that estrogens play an important role in social learning in
female mice in a mechanism- (genomic vs rapid) and receptor- dependent manner.
Whether androgens similarly mediate social learning in females remains to be
investigated. Despite countless studies implicating estrogens in social learning in
females, to the best of our knowledge the role of estrogens in social learning in males
has not been investigated.
3.3 Non-human Primates
Though sex differences in social learning are more prominently discussed in the
human literature, some studies also show sex differences in social learning in
non-human primates. Like most species, in non-human primates, mother-child
relationships tend to be the most important relationship in a child’s life from birth
until nutritional independence. As nutritional independence is reached, individuals
must learn to forage their own food. At this stage in the young non-human primate’s
life, the primary source of social information is their mothers. In comparison with
young males, female chimpanzees spent more time observing their mother termite
fish, which was reflected in greater proficiency in fishing techniques a year later
(Whiten and van de Waal 2018). This finding is consistent with the greater use of
tool-assisted food gathering in females than males who typically rely on hunting for
food (Whiten and van de Waal 2018). Furthermore, in wild vervet monkeys,
regardless of the observer’s sex, observers only learned to open an artificial fruit
when watching an adult female demonstration and not male (Van de Wall et al.
2010). Likewise, during a test of social learning, vervet monkeys copied a female
model significantly more than the male model when there was an equal payoff, but
when there was a payoff bias favoring males (5 times more of the food reward),
monkeys copied the male model (Bono et al. 2018). These findings demonstrate a
role of sex of both the observer and demonstrator in social learning in non-human
primates that is dependent on the behavioral situation and is relevant to learned
feeding behavior. Notably, in female baboons (Papio sp.), variations to the men-
strual cycle were observed as a function of social isolation, exposure to males, and
social attack (Rowell 1970). For example, the follicular phase, during times of rising/
high estrogens, was prolonged during social stress, and perineal swelling increased
during social isolation which was reversed following re-entry to the social setting
(Rowell 1970). Collectively, these findings suggest that social situations may influ-
ence hormonal status in female baboons. Whether the sex steroid hormones mediate
sex differences in social learning in primates remains an avenue for future research.
4 Sex Differences in Aggressive Behaviors
Aggression is a broad term used to define a complex suite of evolutionarily adaptive

behaviors displayed by a variety of organisms. A clear and comprehensive definition
of aggression is hard to find. Traditionally, aggression has been defined as overt
behavior with the intention of inflicting physical damage upon another individual or
goal entity (Soma et al. 2008). However, aggressive behavior has evolved to varying
degrees in different species, social and non-social, to increase survival and repro-
ductive fitness (Hermann 2017). In social species, aggression needs to be expressed
with different degrees of intensity so that damaging physical interactions between
members of the same group that would compromise individual fitness are avoided.
Often, submissive postures displayed by one animal avoid physical harm by a
dominant animal. Other behaviors such as threat displays and ritualized combats
are also used to establish dominance within a group. Thus, non-overt agonistic
behaviors can eliminate or limit the need for fights to obtain dominance status
(Soma et al. 2008).
Given the existence of facets in aggressive behavior, a classification scheme is
needed in order to divide aggression into specific subtypes. Classically, aggression
has been subdivided into defensive and offensive (Blanchard and Blanchard 2003).
However, there exist different subtypes of both offensive and defensive aggression,
including: predatory, intermale, interfemale, maternal, territorial, pain-induced, and
redirect aggression. Moreover, in social species aggression is a major contributor to
the establishment of dominance hierarchies that maintain stability within the group.
The dominant status confers several benefits such as power, access to resources, and
enhanced mating opportunities. Thus, dominance and competitive aggression are
ancestral and critical social strategies used to develop and maintain relationships in
social species (see Fig. 5 for a schematic of the natural occurrences of aggressive
behaviors in social contexts).
Fig. 5 Schematic representation of the natural occurrence of aggressive behaviors in social

contexts. Interaction with conspecific or non-conspecifics modulates the behavioral response of
an individual who can manifest varying degrees of aggression. Environmental/social stimuli (x axis)
can induce an aggressive response of different intensity (y axis). Generally, the emission of
aggressive behaviors is evolutionarily adaptive. Animals express aggression toward a functional
endpoint. For example, individuals of social species must be able to exhibit forms of aggression to
defend their territory, to gain access to resources (food, mates, etc.), or to establish a hierarchy
within the group. Instead, uninhibited and exaggeratedly intense aggression can be considered
violence. Violence is different from aggression in that it is not evolutionarily adaptive and is an out
of context expression of aggression. The manifestation of violent behavior is an abnormal form of
aggression (usually referred as escalated aggression in laboratory conditions) that cannot coexist
with group life, if not in very territorial species or sexes. However, as illustrated in the graph, it is
important to note that the complete absence of any degree of aggressive behavior is not adaptive.
Therefore, aggressive behavior is not only part of the ethological repertoire of social species but is
also of fundamental importance to ensure the survival of the individual
Sex differences in the neural mechanisms regulating the expression of social and
aggressive behaviors are likely since males and females often evolved in response to
different selective pressures (Terranova et al. 2016). In polygamous species, in
males, fighting may be useful to establish a territory and the regulation of space
use; in females, the dominant behaviors could establish a dominance hierarchy that,
in turn, allows sharing the same territory with other females. In humans, aggression
and violence represent a significant societal burden (Rosell and Siever 2015). It is
important to underline that, although aggression and dominance have physiological
and ethological underpinnings, problematic violent outbursts are symptoms of a
pathological condition (Miczek et al. 2007). In humans, aggression and violence
have been observed in adults with borderline and antisocial personality disorders,
schizophrenia, posttraumatic stress disorder (PTSD), bipolar disorder, alcohol/sub-
stance abuse, attention-deficit/hyperactivity disorder (ADHD), and other neuropsy-
chiatric conditions such as dementia (Rosell and Siever 2015). Clinical forms of
aggression may manifest in a chronic and pervasive manner, independent of other
psychiatric disorder, with the syndrome of intermittent explosive disorder (IED),
which is characterized by aggressive manifestations causing distress and functional
impairment (Rosell and Siever 2015). Interestingly, sex differences occur in inci-
dence and clinical course of most of these psychiatric disorders, males being greatly
overrepresented in these populations. However, it is important to highlight that sex
differences occur also in the phenotypic expression of aggression with males more
physically and females more indirectly aggressive (Wranghama 2017). Indirect
aggression showed by females includes behaviors such as spreading rumors about
other individuals (usually about their sexual behavior), social exclusion, and criti-
cizing potential competitors (Vaillancourt 2013).
Rodent studies on aggression in both sexes are limited, mainly because female
laboratory rodents rarely show spontaneous overt aggression. To assess males’
aggressive behaviors, the resident-intruder paradigm is usually employed, where
an intruder is introduced in the home cage of a male, usually isolated, animal (the
resident) (Koolhaas et al. 2013). This paradigm takes advantage of the fact that male
mice often establish and maintain an exclusive territory or dominate a shared
territory in natural conditions (Latham and Mason 2004). Limited research, instead,
has been conducted on aggression or agonistic behavior in female mice. Studies on
female aggression, indeed, have focused on maternal aggression which is displayed
by lactating mothers. Maternal aggression has been suggested to represent an
evolutionary trade-off since it increases the mother’s chance of being harmed,
while, at the same, time dramatically enhancing the survival and fitness of the
offspring (Lonstein and Gammie 2002). Maternal aggression is considered a strategy
used by females to counter the infanticide perpetrated by non-parental conspecifics
(Agrell et al. 1998). However, studies on Swiss albino mice revealed that maternal
aggression is qualitatively different from the intrasexual aggression observed in
males because it is less ritualized and more violent (Al-Maliki et al. 1980).
Several sensory cues may modulate aggression in mother rats and mice, including
stimuli produced by the pups (tactile and olfactory cues, ultrasonic vocalizations,
etc.) and by the opponent (sensory cues, age, size, etc.) (Lonstein and Gammie
2002). Environmental factors further regulate dams’ behavior with lactating mice
being aggressive against male intruders when tested in their home cage with or
without their own pups (Paul et al. 1980). The environmental dependence of the
emission of maternal aggression raised the hypothesis that this kind of aggression is
a form of territorial defense. In support of this theory, it has been observed that
maternal aggression is rarely found outside the home-cage environment, and the
behavioral topography of territorial defense by dominant male and lactating female
rats can be similar (Blanchard et al. 1984; Lonstein and Gammie 2002). In this view,
maternal aggression has developed as a by-product of increased territorial defense in
the dams, and the consequent offspring defense made it a highly adaptive behavior.
However, other studies suggested that maternal aggression is different from territo-
rial aggression because it is mediated by fear and/or anxiety. Administration of the
anxiolytic chlordiazepoxide (CDP) to both aggression-naïve and aggression-
experienced lactating female mice switched their attack strategy against male
intruders from a defensive to an offensive pattern (Palanza et al. 1996).
Non-reproduction related forms of aggressions in females also exist, even if less
frequently studied than in males. For instance, virgin or non-reproductively active
outbred Swiss albino females show aggressive behaviors after isolation, during
group formation, or when the intruder is a lactating female, whereas virgin inbred
strains only rarely attack a same-sex intruder (Morè 2008; Ogawa et al. 2004). Even
though intrasexual aggression has been largely eliminated by domestication, labo-
ratory female rodents may display agonistic behaviors other than direct attacks, such
as chasing or aggressively grooming the intruder, which are not often assessed in
tests for aggression (Grant and Mackintosh 1963; Palanza et al. 2005). These
behaviors do not lead to the intruder’s exclusion from the territory, but rather are
used to establish dominance over the intruder. For this reason, the study of agonistic
interactions in females, but also in males, should involve a comprehensive, etholog-
ical, analysis, based on the full behavioral repertoire of the animal, rather than
measuring only direct attacks (Grant and Mackintosh 1963; Miczek et al. 2001).
This “ethological” approach to the study of aggressive interfemale interactions
provides a more reliable representation, and possible model, of the aggressive
behaviors displayed by human females, who often adopt forms of aggression that
are qualitatively different from those of men, such as “verbal” or “indirect” aggres-
sion (Hay 2007).
Given that the ethological approach to interfemale aggression is not frequently
used, another rodent model to investigate the biological mechanisms underlying
competitive and aggressive strategies in both males and females is provided by
Syrian hamsters. In this species, females naturally display a range of competitive
strategies including the formation of a hierarchical organization and the inhibition of
the reproductive capacity of other females (Rosvall 2011). Syrian hamsters also
engage in social behaviors such as social recognition, social avoidance, and social
communication that are essential to form and maintain hierarchies (Huhman et al.
2003). This animal model allowed the investigation of the neural mechanisms
involved in aggression in both sexes revealing fundamental differences. For exam-
ple, in the anterior hypothalamus (AH), the activation of AVP and OT receptors
increases aggressive behaviors in males, while decreasing aggression in female
Syrian hamsters (Grieb et al. 2021). On the contrary, aggression decreases in
males and increases in females when the serotonin 1A (5-HT1A) receptor is acti-
vated (Grieb et al. 2021).
Several systems and neurotransmitters have been linked to aggression. A well-
known phenomenon is the inhibiting effect of serotonin (5-HT) on impulsive
behaviors, including aggression, in males of various species from invertebrates to
primates (Morrison and Melloni 2014). Low 5-HT levels are usually associated with
higher levels of aggression and impulsivity, whereas increasing 5-HT activity
reduces aggressive behaviors in male mice (Miczek et al. 2001). Specific 5-HT
receptors have been identified that play a role in aggression. The activation of the
5-HT1B receptors seems to be related more to the modulation of impulsiveness than
aggression itself, whereas the 5-HT1A receptors might be directly implicated in the
control of aggressive behaviors (Olivier 2005; Nelson and Trainor 2007). Male mice
exhibiting high levels of aggression, characterized by short latency to attack, showed
increased post-synaptic availability of the 5-HT1A receptors in limbic and cortical
regions (Korte et al. 1996). Interestingly, sex differences in 5-HT regulation of
aggression have been shown. Hypothalamic injections of 5-HT1A agonists

increased aggression in female and reduced it in male Syrian hamsters (Terranova
et al. 2016). Moreover, the activation of 5-HT neurons in the Dorsal Raphe Nucleus
(DRN) has been related to the acquisition of dominance only in female hamsters
(Terranova et al. 2016). In mice, a tryptophan hydroxylase 2 (Tph2) knockout model
has been used to investigate aggression in both sexes. Tph2 is the rate-limiting
enzyme in the synthesis of 5-HT in the brain (Gutknecht et al. 2008). Tph2 KO male
rats showed increased aggressive behavior against intruders when compared to their
wild type counterparts, probably due to decreased 5-HT1A receptor sensitivity
(Peeters et al. 2019). Similar to the males, female Tph2 KO mice displayed high
levels of aggression toward unfamiliar wild type females, suggesting a role of
serotonergic neurotransmission in the regulation of female aggression outside a
reproductive context (Kästner et al. 2019). Yet, it is not possible to conclude a
clear link between 5-HT and aggression with this animal model because Tph2 KO
mice lack 5-HT throughout development and compensatory mechanisms may play a
role in the modulation of aggressive behaviors in adulthood.
In addition to 5-HT, AVP has been implicated in aggression in hamsters, voles,
rats, and mice (Ferris 2000; Stribley and Carter 1999; Fodor et al. 2014; Tan et al.
2019). AVP activation increases aggression in male hamsters, whereas the admin-
istration of V1aR antagonists reduces aggression (Ferris 2000; Potegal and Ferris
1989; Terranova et al. 2016). However, the ability of anterior hypothalamus AVP to
stimulate intermale aggression in hamsters may depend on prior social experiences,
increasing it only in hamsters previously trained to fight or that had been socially
isolated for at least four weeks (Albers 2012). Indeed, V1aR binding in the AH and
aggression are more frequent in socially isolated than group living males (Albers
2012). Intriguingly, studies with female hamsters revealed that AVP has the opposite
effect on competitive aggression with central AVP administration reducing and
V1aR antagonists stimulating aggression (Gutzler et al. 2010). In lactating rats,
intracerebroventricular administration reduced and a V1aR antagonist increased
aggression toward a male intruder (Albers 2012). The different activation of 5-HT
and AVP neurons in diverse brain regions in males and females supports the idea that
some behavioral outcomes (e.g., dominance and social recognition) can be acquired
through the activation of these systems in a sex-dependent manner.
The sex differences in the involvement of AVP and 5-HT in aggression suggests a
potential role for sex hormones and/or sex chromosomes. Castration reduces male
aggression in many species and administration of T can restore it (Nelson and
Trainor 2007). T, or its estrogenic metabolites, during the post-pubertal period,
can stimulate neural circuits organized perinatally making aggression-inducing
stimuli more salient (Nelson and Trainor 2007). Among rodents, T increases aggres-
sion in C57BL/6 J mice, whereas in the CF-1 strain, the conversion into E2 by the
aromatase enzyme is pivotal to increase aggression (Nelson and Trainor 2007).
Another T metabolite, DHT, can restore intermale aggression in castrated males of
some strains (e.g., Swiss-Webster, CD-1, Tuck TO) but not others (e.g., CFW, CF-1)
(Clipperton-Allen et al. 2010). Furthermore, male mice with targeted disruption of
the gene for ERα showed reduced aggressive interaction in different testing contexts
(Ogawa et al. 1997, 1998; Nelson and Trainor 2007). The number of ERα-positive
cells in the LS and BNST has been positively correlated to aggression in male mice,
whereas differences in ERβ expression were not related to aggressive behaviors
(Nelson and Trainor 2007; Aspesi and Choleris 2021). However, the subcutaneous
injection of the ERα agonist PPT, but not of the ERβ agonist DPN, reduced the
rejection rate of sexually vigorous male rates by ovariectomized females (Mazzucco
et al. 2008). This finding is consistent with previous literature in which ERβKO male
mice showed more aggressive behaviors than WT or ERαKO, suggesting that ERβ
may attenuate aggression in male mice (Nomura et al. 2002, 2006). Thus, both
androgens and estrogens are involved in the mediation of aggressive behaviors in
males of several strains.
Few studies investigated the role of sex hormones in female aggression. Virgin
ERα KO female mice showed increased aggressive behaviors toward ovariecto-
mized intruders, but not toward olfactory bulbectomized males, and estrogen
replacement after ovariectomy decreased aggression (Ogawa et al. 2004). However,
gonadally intact ERβ KO females showed no aggression toward ovariectomized
intruders, and T administration decreased aggression (Ogawa et al. 2004). These
findings suggest that the activation of ERα may inhibit, whereas that of ERβ may
facilitate, the expression of aggressive behaviors in adult female mice. Females
treated neonatally with estradiol benzoate display masculinized patterns of fighting
and urine marking in the presence of ovarian hormones production (Wu et al. 2009).
The administration of T in these female mice to mimic the normal circulating levels
of males increased the female aggressive behaviors to levels similar to those
observed in males. These results suggest a specific timing and role for sex hormones
in the two sexes: gonadal hormones in adult individuals of either sex can support a
male pattern of fighting after neonatal exposure to estrogens has masculinized the
underlying neural circuits. The use of selective ERα and ERβ agonists confirmed a
different role played by ERs in the modulation of aggressive behavior. The acute
activation of ERα through subcutaneous injections of the selective ERα agonist
1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) increased sex-typical ago-
nistic behavior in gonadectomized male and female mice (Clipperton-Allen et al.
2011). ERβ activation, on the other hand, had no effects in gonadectomized mice but
enhanced non-attack agonistic behavior in gonadally intact animals (Clipperton-
Allen et al. 2010). Moreover, a recent study showed that aromatase expressing
neurons in the MeA regulate both intermale and maternal aggression, revealing
that the same neural circuit underlie different forms of aggression (Unger et al.
2015). The enzyme aromatase, which converts T into E2, is expressed in <0.5% of
neurons in the brain of adult male mice, in regions involved in sexually different
behaviors (Unger et al. 2015; Wu et al. 2009). Even if female mice have low
circulating levels of T and high levels of E2 produced by the ovaries, the female
mouse brain expresses aromatase, though at lower levels than males (Wu et al.
2009). The involvement of aromatase in the same brain regions, such as the MeA, in
aggressive behaviors in both sexes suggests the possibility that a primordial neural
pathway underlying aggression may have been shared by males and females.
Fig. 6 Simplified (sagittal) diagram of the key brain regions involved in the aggressive behaviors
of male (inter-male) and female (maternal) mice. The highlighted brain areas showed greater
activation (cFos, pCREB, or citrulline) during aggressive behaviors. The dot pattern indicates
that the brain region has been shown to be involved in male aggression, while the vertical lines
indicate brain regions that have been shown to be involved in maternal aggression. The schematic
map clearly shows a large level of overlap between regions involved in sex-specific aggressive
behavior, although some areas of the brain are seen to be specifically involved in aggressive
behavior in one sex and not the other. Abbreviations: AMY amygdala, AVPV anteroventral
periventricular nucleus of hypothalamus, BNST bed nucleus of the stria terminalis, DRN dorsal
raphe nucleus, LHb lateral habenula, LS lateral septum, MPOA medial preoptic area; NAcc nucleus
accumbens, PAG periaqueductal gray, PFC prefrontal cortex, SCN suprachiasmatic nucleus, VMH
ventromedial hypothalamus, VTA ventral tegmental area
However, different contexts, functions, and evolutionary pressures led to divergent

modulation of aggression in the two sexes.
The processes of evolution and sexual selection drove different kinds of stimuli
activating diverse neurocircuitries in the sexes (see Fig. 6 for a schematic represen-
tation of the main brain regions involved in intermale and maternal aggression). It is
important to emphasize that agonistic interactions have different functional out-
comes. Fighting may be efficacious to establish territory for males, whereas
non-over aggression builds a dominance hierarchy and allows the sharing of the
same territory for females. To properly evaluate and understand the different forms
of aggression evolved in humans, and mimicked in some strains of rodents, it is
fundamental to question what each sex competes for and how lethal this competition
is. Specific ecological and evolutionary pressures shaped the emission of different
types and functions of aggressive behaviors in the sexes. For this reason, it is
important to adopt an ethological approach to the neuroendocrine investigation of
aggressive behaviors, not only when considering direct, overt attacks, but also
various other dominant and submissive behaviors displayed by males and females.
5 Conclusions and Relevance to Humans
The above-reviewed literature highlights the hormonal mechanisms underlying three

aspects of social behavior whose fine-tuned regulation is critical for normal group
living: social recognition, social learning, and aggression. As a social species, human
share not only the three social behaviors, but also the neural structures that in other
species are regulated by the sex steroid hormones and their interplay with other
neurochemicals. Thus, the literature described above can inform our understanding
of the human social brain and of disorders where its normal functioning is affected.
In humans, sex differences are observed from early childhood in the toys chosen
when given a free choice and the way children interact with others. For example,
girls are more likely to engage in prosocial behavior, maintain greater eye contact,
and obtain a greater social understanding than boys (Bono et al. 2018). Even just
2 days after birth, girls show preferences for social stimuli whereas boys show
preferences for mechanical stimuli (Bono et al. 2018). Generally, clear sex differ-
ences are observed in the type of play children engage in with boys spending more
time engaging in rough play, play in larger groups, and object play, whereas girls
typically prefer play parenting with dolls, even though this is influenced by parents
(Lonsdorf 2016). Notably, similar sex differences are observed in non-human
primates. For example, male vervet monkeys (Cercopithecus aethiops sabaeus)
preferred to play with “male-typical” toys whereas female vervets preferred
“female-typical” toys (Alexander and Hines 2002). These findings suggest that the
preference for sex-typical objects developed early within the evolution of primates.
Gender norms may also be learned socially. From a young age, children learn about
gender appropriate behavior through positive and negative reinforcement (Wharton
2009). For example, a young girl may be praised by her mother for playing with
dolls, whereas she may be ignored or not praised when playing with toy cars. Girls
may learn about femininity by observing their mother’s behaviors, whereas boys
may learn about masculinity by observing their father’s behaviors. While societal
norms likely drive some of these sex differences, a role for developmental sex
hormones in sociability, social play/interaction, and social learning in children has
also been suggested.
Interestingly, human females that were exposed to greater levels of T in utero
later spent more time playing with males, exhibited a greater preference for “male”
toys, and greater “male-typical” personality features (Bao and Swaab 2010). Thus, it
appears that sex hormone exposure during pregnancy may be important for behav-
ioral development during childhood. In a number of adult neurological diseases, sex
differences in prevalence are widely reported. Specifically, certain disorders with
neurodevelopmental and social cognitive disruption, such as autism spectrum dis-
order (ASD) and schizophrenia that involve deficits in social behavior, show greater
prevalence and/or severity in men than women.
ASD is a cognitive disorder characterized by deficits in social interactions and
patterned repetitive behaviors (Ferri et al. 2018). Individuals with ASD typically
experience deficits in communication, interpreting social cues, social reciprocity,
and initiation and maintenance of social exchange (Vivanti and Nuske 2017). In
comparison with age-matched controls, individuals with ASD do not show a pref-
erence toward social stimuli, display decreased social attention and engagement in
social and cooperative tasks, and show lower expressions of pleasure during social
activities (Vivanti and Nuske 2017). Thus, for people with ASD, there appears to be
a difference in the rewarding factors of sociability. ASD is significantly more
prevalent in males than females, suggesting a likely role of hormones.
ASD affects one in 68 children at a male to female ratio of 4.5:1 (Ferri et al.
2018). This male prevalence suggests that either male sex hormones increase
vulnerability to ASD, or female sex hormones protect against ASD. It is also
possible that both mechanisms are at play, or a completely sexually different system
is at play (Ferri et al. 2018). One theory suggests that high fetal T is linked to ASD.
Several, but not all, studies found that fetal T is correlated with greater ASD
diagnoses and autistic trait scores (Ferri et al. 2018). In females, this may include
individuals exposed to higher-than-normal levels of fetal T, such as females with
congenital adrenal hyperplasia, females with male twins, and females with mothers
that suffer from polycystic ovarian syndrome (Ferri et al. 2018). Within the hypoth-
esis that the sex difference in ASD is fetal T driven, the extreme male brain (EMB)
theory suggests that the brain of individuals with ASD is hyper-masculinized,
possibly due to high levels of fetal T (Ferri et al. 2018). These findings suggest
that perhaps male sex hormones increase vulnerability to the onset of ASD through
organizational action on the brain.
Schizophrenia is another thought disorder that is more prevalent in males than
females (Gogos et al. 2019). Among others, individuals with schizophrenia experi-
ence negative symptoms that include deficits in sociability (Gogos et al. 2019).
People with schizophrenia demonstrate decreased motivation to engage in social
interactive behaviors even before diagnosis (Catalano et al. 2018). Further, in
comparison with age-matched healthy controls, individuals with schizophrenia did
not show a preference for genuine smiles versus polite smiles suggesting a reduced
value for social reward (Catalano et al. 2018). Thus, decreased interest in social
engagement seen in individuals with schizophrenia may be due to decreased reward
of socialization.
Like ASD, there is reason to believe that the sex difference in the age of onset of
schizophrenia is driven by sex hormones. For example, the peak onset of schizo-
phrenia is between the ages of 18 and 25 for men and 22 and 26 for women,
suggesting a critical role for post-pubertal hormones (Galderisi et al. 2012). Inter-
estingly, unlike men, women typically experience a second wave of symptoms
around the age of 45–49, around the time of perimenopause (Hafner 2003; Gogos
et al. 2019). These findings suggest that the decline and/or change of circulating
estrogens in women during and before menopause contributes to the re-occurrence
of schizophrenic symptoms. Furthermore, in the brain, men with schizophrenia show
greater ventricular enlargement, greater abnormalities in white matter microstruc-
ture, and more severe frontal and temporal lobe atrophy than women with schizo-
phrenia (Gogos et al. 2019). Collectively, these findings suggest a critical role of sex
hormones in schizophrenia as well as concrete differences in brain regions that may

be involved in schizophrenia.
Overall, in this chapter we have described the animal research demonstrating a
role for the sex hormones in social recognition, social learning, and aggression. This
research has shown that along with the important role sex hormones play on these
behaviors, there are sex differences in how these behaviors are mediated, such as
vasopressin playing a larger role in social recognition in males than females or
serotonin decreasing aggression in males and increasing it in females. The findings
discussed here highlight the importance of investigating the mechanisms underlying
behavior in both sexes. The role the sex hormones have and their interactions with
other neurochemicals on these social behaviors may also inform further research into
the understanding of the neuroendocrine underpinnings of disorders with impaired
social cognition in humans.
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Sex Differences in Cognition Across Aging
Bonnie H. Lee, Jennifer E. Richard, Romina Garcia de Leon, Shunya Yagi,

and Liisa A. M. Galea
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
2 Sex Differences in Cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
2.1 Sex Differences in Cognitive Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
2.2 Sex Differences Across Cognitive Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
2.3 Sex Differences in Learning Strategies and Brain Networks . . . . . . . . . . . . . . . . . . . . . . . . 241
3 Cognition Throughout the Aging Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
3.1 Cognitive Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
3.2 The Neurobiology of Cognitive Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
3.3 Sex Differences in Cognitive Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4 Dementia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4.1 Cognitive Decline in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
5 Lifestyle Factors on Cognition, Cognitive Aging, and Dementia . . . . . . . . . . . . . . . . . . . . . . . . . . 252
5.1 Obesity and Cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
5.2 Diet and Cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
5.3 Body Weight, Diet Intake, and Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
B. H. Lee, R. G. de Leon, and S. Yagi

Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada
Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC,
Canada
J. E. Richard
Canada
L. A. M. Galea (*)
Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada
Canada
Department of Psychology, University of British Columbia, Vancouver, BC, Canada
e-mail: liisa.galea@ubc.ca
https://doi.org/10.1007/7854_2022_309
236 B. H. Lee et al.
Abstract Sex and gender differences are seen in cognitive disturbances in a variety
of neurological and psychiatry diseases. Men are more likely to have cognitive
symptoms in schizophrenia whereas women are more likely to have more severe
cognitive symptoms with major depressive disorder and Alzheimer’s disease. Thus,
it is important to understand sex and gender differences in underlying cognitive
abilities with and without disease. Sex differences are noted in performance across
various cognitive domains – with males typically outperforming females in spatial
tasks and females typically outperforming males in verbal tasks. Furthermore, there
are striking sex differences in brain networks that are activated during cognitive
tasks and in learning strategies. Although rarely studied, there are also sex differ-
ences in the trajectory of cognitive aging. It is important to pay attention to these sex
differences as they inform researchers of potential differences in resilience to
age-related cognitive decline and underlying mechanisms for both healthy and
pathological cognitive aging, depending on sex. We review literature on the pro-
gressive neurodegenerative disorder, Alzheimer’s disease, as an example of patho-
logical cognitive aging in which human females show greater lifetime risk,
neuropathology, and cognitive impairment, compared to human males. Not surpris-
ingly, the relationships between sex and cognition, cognitive aging, and Alzheimer’s
disease are nuanced and multifaceted. As such, this chapter will end with a discus-
sion of lifestyle factors, like education and diet, as modifiable factors that can alter
cognitive aging by sex. Understanding how cognition changes across age and
contributing factors, like sex differences, will be essential to improving care for
older adults.
Keywords Alzheimer’s disease · Diabetes · Diet · Females · Hippocampus · Males ·

Obesity
1 Introduction
Cognition allows animals to navigate their environments and learn from each
interaction, whether social or physical, granting organisms a degree of adaptation
to their environment (Shettleworth 2001). Cognitive deficits are common across a
wide variety of psychiatric and neurodegenerative diseases (Baiano et al. 2020; Rock
et al. 2014), and depending on the type of illness, sex differences in severity of
cognitive deficits with disease exist (Friedman et al. 2001). Thus, it is important to
consider sex as a factor when examining cognitive domains in both normal and
diseased states. Both sex (biological) and gender (psychosocial, identity) play a role
in these disparities and the intersectionality of race/ethnicity/income can further
exacerbate health inequities. We use the terms female/male to indicate biological
sex and use women/men to encompass sex/gender.
Research shows stark differences between sexes in cognitive aging trajectories,
from childhood to adulthood (McCarrey et al. 2016). Historically, there has been a
reluctance to investigate the contribution of sex in cognition due to the belief that this
Sex Differences in Cognition Across Aging 237
work contributes to the perception that one sex may be inferior to another. Indeed,
this field has been termed “neurosexism” (Eliot 2019), with the idea that sex
differences in cognition or brain volume imply inferiority in a particular sex. This
misconception restricts potentially important research endeavors in the field (Lisi
et al. 2002). Studying sex differences in cognition not only informs researchers of
differing patterns of behavior or underlying mechanisms in males and females, but it
also allows for rigorous investigation of cognitive manifestations in disease states.
Meta-analyses show that women with Alzheimer’s disease (AD) have a more rapid
cognitive decline compared to men with AD (Irvine et al. 2012; Sohn et al. 2018).
However, some have questioned the extent to which baseline sex differences in
cognitive performance may contribute to the rapid decline with AD in women. For
example, is it that human females are seemingly more resilient at earlier stages of the
disease due to the memory domains tested, which favor females, or are they more
susceptible to severe cognitive decline with AD (Seto et al. 2021)? More studies are
needed to elucidate possible sex differences in cognitive trajectories in normal aging
as well as in disease states and to identify if different tests and/or comparisons
against sex-based norms may be necessary. Moreover, to understand sex differences
involved in cognition-related neuropathology, it is critical to understand the
preexisting cognitive sex differences present at baseline as well as how this may
change with disease. This chapter will thus explore sex differences in cognition at
baseline, throughout the aging process, and in AD. This chapter will also consider
how lifestyle and sociodemographic variables like education and diet may contribute
to the nuanced relationship between sex and cognition.
2 Sex Differences in Cognition
In general, in humans, the most robust sex differences exist in spatial and verbal
cognition, with males performing better in spatial tasks than females and females
performing better in verbal tasks than males on average (Weiss et al. 2003). Men, on
average, excel in spatial perception, spatial memory, and mental rotation (Levine
et al. 2016), whereas, women, on average, excel at verbal tasks such as speed of
articulation, accuracy of speech production and fluency (Scheuringer et al. 2017),
and verbal memory (Asperholm et al. 2019a, b). A recent study found that the
consideration of sex differences in cognition can lead to more accurate diagnoses of
cognitive impairments such as amnesic mild cognitive impairment (aMCI)
(Sundermann et al. 2019). Females will score higher on verbal memory tests, and
as the cognitive tests to determine whether someone has cognitive impairment rely
on verbal memory, males will reach the threshold of impairment sooner than
females, based solely on their baseline performance (Sundermann et al. 2019).
More research analyzing sex in cognition will bring researchers closer to the
functional and biological underpinnings of human cognition, further allowing indi-
vidualized treatments for pathological cognitive aging. It is important to understand
that there are a number of cognitive domains, and within each, different neural
Fig. 1 A schematic summary of sex differences in the trajectory of cognitive aging across different
domains. In humans, women tend to have higher baseline scores on tasks that depend on global
cognition and executive functioning, but with advancing age, they experience a steeper decline in
performance, compared to men (Levine et al. 2021). In terms of verbal tasks, women consistently
outperform men (de Frias et al. 2006; Voyer et al. 2021). Lastly, men tend to have higher baseline
scores that depend on visuospatial abilities, but with advancing age, they experience a steeper
decline in performance, compared to women (McCarrey et al. 2016). See Sect. 3.3 for a more
in-depth account of the literature
networks are required. As such, it should not be surprising that sex differences are
not uniform across cognitive domains. Figure 1 illustrates putative sex differences
across three cognitive domains with aging. This following section of the chapter will
describe common tasks used to measure cognition, present data on sex differences
seen across cognitive domains, and discuss potential underlying neurobiological
mechanisms.
2.1 Sex Differences in Cognitive Domains
As noted above, the largest sex differences are seen favoring spatial ability in males
and verbal ability in human females. In humans, the mental rotation task is a well-
established cognitive task that evaluates the time participants take to mentally rotate
(i.e., imagine what something looks like when rotated) a 3-dimensional (3D) object
to match another (Shepard and Metzler 1971). This task has led to well-documented
sex differences, with males showing predominant activation of the parietal cortex
and females showing inferior frontal activation in humans (Hugdahl et al. 2006),
suggesting that males and females process visuospatial cognition differently. Indeed,
other studies show neural activation patterns are different between males and
females after undergoing mental rotation (Griksiene et al. 2019). Importantly, sex
differences in these tasks are generally not present in children under the age of
10 years (Fernández-Méndez et al. 2018), indicating that the pubertal transition
likely contributes to these sex differences. Other studies suggest that sex hormone
levels play a role in spatial rotation performance (Kimura and Hampson 1994).
Fluctuations of estrogens and progesterone during different menstrual cycle phases
affect activation of different brain areas during spatial tasks (Pletzer et al. 2019;
Schöning et al. 2007). For example, during the follicular phase, there is greater
activation in the left superior temporal gyrus and less activation in the middle
temporal gyrus and cingulate gyrus than during the midluteal phase when complet-
ing a mental rotation task (Schöning et al. 2007). Some studies find that women/
females demonstrate better performance in spatial rotation tasks during the follicular
phase (lower estradiol/progesterone) compared to the luteal phase (higher estradiol/
progesterone) (Hampson 1990; Noreika et al. 2014). But even when there is no
significant difference in performance across the menstrual cycle on spatial tasks,
brain activation differences persist with greater hippocampal activity during high
estradiol phases and greater frontal cortical and striatal activation during the
midluteal phase when progesterone is higher (Pletzer et al. 2019). These findings,
and others, indicate that ovarian hormones have modulatory effects on spatial
rotation performance and brain activation (Noreika et al. 2014).
Different cognitive domains are probed using different tasks and a widely used
verbal memory test in humans, the Rey Auditory Verbal Learning Test (RAVLT), is
a cognitive task that requires recall and recognition to test verbal memory abilities.
Females generally outperform males, with higher recall of words both immediately
and over time (Kramer et al. 1988). As RAVLT performance is heavily used in
clinical settings to diagnose mild cognitive impairment (MCI) and neurodegenera-
tive diseases such as AD, close attention to possible sex differences could spare
misdiagnoses, as females tend to perform better on this task, thus possibly obscuring
disease manifestation and preventing early AD diagnosis in human females
(Sundermann et al. 2017).
One of the most common spatial memory tasks in rodents is the Morris Water
Maze (MWM) test and the radial arm maze test, where males typically outperform
females – although this depends on a variety of factors (Hernandez et al. 2020;
Voyer et al. 2017). Similar to findings in humans, circulating gonadal hormones
influence both male and female rodents in terms of performance, strategy, and neural
activation – see below (Warren and Juraska 1997). Another common cognitive test
used is the Object Recognition (OR) task (Leger et al. 2013). Previous studies using
the OR task have identified sex differences, where female mice and rats tend to
perform better than male mice and rats (Sutcliffe et al. 2007). It is important to note
that using rodents to study sex differences in cognition may be influenced by strain
(Van Dam et al. 2006), age (Nicolle et al. 2003), and genotype (Zaharia et al. 1996).
Moreover, although the OR is a commonly used tool to study cognitive sex differ-
ences, protocol differences affect performance (Cohen and Stackman Jr. 2015), so
caution is urged when comparing across studies.
2.2 Sex Differences Across Cognitive Domains
Meta-analyses demonstrate that there is an overall male advantage in spatial refer-

ence and working memory in humans and in mice and rats (Jonasson 2005; Voyer
et al. 2017) and a female advantage in episodic memory and response inhibition in
humans (Asperholm et al. 2019a, b; Gaillard and Rossell 2021). However, there are a
number of studies failing to demonstrate significant sex differences or showing
opposing differences to those mentioned, and these equivocal findings likely depend
on a variety of factors, including age and type of cognitive task (reviewed in Grissom
et al. 2013; Yagi and Galea 2019).
Sex differences in working memory performance in humans, favoring males, are
affected by age of participants and types of working memory tasks (Shields et al.
2017; Voyer et al. 2017, 2021; Gaillard and Rossell 2021; Barel and Tzischinsky
2018). A meta-analysis demonstrates that in humans, males outperform females in
spatial working memory tasks across adulthood, whereas there is no sex difference
during childhood and adolescence (Voyer et al. 2017; see Table 1). In contrast,
females outperform males in verbal-related working memory tasks in all age groups,
though the magnitude of sex differences varies depending on the task (Voyer et al.
2021). During spatial working memory tasks in humans, males, compared to
females, show greater blood-oxygen-level-dependent (BOLD) response in various
brain regions including the prefrontal cortex, the hippocampus, verbal-related
regions, and visual-processing regions during adolescence, even when there is no
sex difference in their performance (Alarcón et al. 2014). During young adulthood,
males show greater activity in the hippocampus and visual-processing brain regions
Table 1 A summary of sex differences in visual-spatial and verbal working memory tasks in
humans
Sex differences
Type Task Age <13 13–17 18–29 30–49 50<
Visuospatial Corsi blocks Male N.S. N.S. Male Male Male
N-back Male – N.S. Male Male –
Memory for patterns Male Male Male Male Male Male
Memory for Female Female Female Female Female Female
locations
CANTAB spatial Male Male Male Male Male Male
working memory
Verbal Complex span Male N.S. N.S. Male N.S. N.S.
Cued task Female N.S. Female N.S. N.S. N.S.
Free recall Female Female N.S. Female Female Female
Serial recall N.S. N.S. N.S. N.S. N.S. N.S.
Simple span N.S. Female N.S. Male N.S. N.S.
There are task-dependent and age-dependent male advantages (Male), female advantages (Female)
or neither male nor female advantages (N.S.) in visuospatial (Voyer et al. 2017) and verbal (Voyer
et al. 2021) working memory tasks. – indicates no data, NS ¼ not significant,
CANTAB¼Cambridge Neuropsychological Test Automated Battery
during verbal working memory tasks, whereas females show greater activation in
verbal-related brain regions during spatial working memory tasks in humans
(Tschernegg et al. 2017). Furthermore, premenopausal women show greater inhibi-
tion of hippocampal activity during verbal working memory tasks compared to men
whereas hippocampal activity is comparable between older men and postmeno-
pausal women (Jacobs et al. 2017). In human males, higher levels of circulating
testosterone are associated with greater BOLD activation in the inferior lateral part of
the parietal lobe during adulthood, whereas testosterone does not associate with the
BOLD activation in the brain region during adolescence in response to visuospatial
working memory tasks (Schöning et al. 2007; Alarcón et al. 2014). These sex
differences in brain activation patterns suggest that males and females employ
different brain networks during working memory tasks, which is altered depending
on hormonal status and age.
In terms of sex differences in episodic memory in humans, there are variable
results depending on the types of tasks and hormone levels (Andreano and Cahill
2009; Andreano et al. 2013; Jacobs et al. 2016; Rentz et al. 2017; Asperholm et al.
2019a, b; Spets and Slotnick 2021). Meta-analyses reveal that in humans, females
outperform males in verbal-related episodic memory tasks, whereas males
outperform females in spatial related episodic memory tasks (Asperholm et al.
2019a, b; Spets and Slotnick 2021). Consistent with human studies, there is a male
advantage in standard spatial reference memory tasks in rats and mice (Jonasson
2005). Furthermore, a number of studies have reported a female advantage in
performance in emotional episodic memory tasks in humans (Naveh-benjamin
et al. 2004; Andreano and Cahill 2009; Gavazzeni et al. 2012) and in fear-
conditioning contextual discrimination tasks in rats and mice (Day et al. 2016;
Foilb et al. 2018). Interestingly, premenopausal and perimenopausal human females
outperform postmenopausal females in verbal-related episodic memory tasks, and
this is associated with circulating estradiol levels, where lower concentrations were
associated with more pronounced changes in hippocampal connectivity and poorer
memory performance (Jacobs et al. 2016; Rentz et al. 2017). Although
premenopausal and perimenopausal females are typically younger than postmeno-
pausal females, these studies found group differences that persisted even after
controlling for age effects.
2.3 Sex Differences in Learning Strategies and Brain

Networks
Several studies point to sex differences in strategy use during spatial memory tasks
(Merrill et al. 2016; Gagnon et al. 2018). In humans, verbal abilities and spatial
abilities in females are associated with route learning in a virtual environment,
whereas spatial abilities are associated with route learning in males (Galea and
Kimura 1993; Merrill et al. 2016), suggesting different strategies are employed by
males versus females. Furthermore, human females are more likely to remember
landmarks on routes and human males are more likely to remember more Euclidean
information (Galea and Kimura 1993). In addition, removal of directional cues
(geographical or geometric cues) impairs spatial navigation only in males, while
removal of landmark cues impairs spatial navigation in females, both in humans and
in rats (Sandstrom et al. 1998; Barkley and Gabriel 2007; Chai and Jacobs 2010;
Williams et al. 1990). In a pattern separation task, a male advantage was only seen in
those employing a spatial strategy and there was no sex difference in those
employing a response strategy in rats (Yagi et al. 2016). In addition, in rats, no
sex differences in performance were observed during a cue competition task in
which subjects can use both the place and response strategy to solve the task
(Yagi et al. 2017). These findings suggest that different strategies are employed
during route learning that indicate that males and females may recruit different brain
networks to solve the same task. Indeed, human males show greater activity in
visual-processing regions and the hippocampus compared to females during spatial
long-term memory tasks (Spets and Slotnick 2021). Furthermore, human females
show greater connectivity and activation between sensory regions and language
processing regions, and negative connectivity between the hippocampus and the
thalamus during spatial long-term memory tasks (Spets and Slotnick 2021). There-
fore, it is plausible that males and females process different types of information
during a spatial memory task so that the availability of favored memory cues
influences their performance during a given task.
Sex hormones also play a role in strategy use, as response versus place strategy
use varies in female rats across the estrous cycle, indicating that ovarian hormones
play a role in strategy use (Korol et al. 2004; Sava and Markus 2005). Adult male rats
and proestrous rats are more likely to use place strategies while estrous rats were
more likely to use response strategies (Hawley et al. 2012; Korol et al. 2004). This
shift from a response strategy during estrus to a place strategy during proestrus is
likely due to higher estradiol levels binding to estrogen receptors in the dorsal
hippocampus (Zurkovsky et al. 2007). Similar to the effects of estrogens in female
rats, the levels of androgens in male rats modulate learning strategies (Spritzer et al.
2013). Castrated adult male rats with a low dose of testosterone are more likely to
use response strategies whereas castrated adult male rats with a high dose of
testosterone are more likely to use place strategies (Spritzer et al. 2013). In humans,
estradiol and testosterone mediate resting-state functional connectivity in females
(Mueller et al. 2016; Pritschet et al. 2020). These studies suggest that gonadal
hormones mediate learning strategy use in male and female rats and brain connec-
tivity in human females.
The hippocampus plays a critical role in spatial cognition, episodic memory, and
response inhibition (Nadel et al. 1975; Olton and Werz 1978; Tulving and
Markowitsch 1998; Van Asselen et al. 2006). The hippocampus is highly plastic
and new neurons are continuously generated throughout the lifespan, including
adulthood, which is referred to as adult neurogenesis (Eriksson et al. 1998; Christie
and Cameron 2006; Neves et al. 2008). Although functional roles of adult
neurogenesis for cognition have not been fully understood, the last few decades of
research have demonstrated that adult neurogenesis is important for pattern separa-
tion in rats and mice (reviewed in Hvoslef-Eide and Oomen 2016). Pattern separa-
tion is the process during memory encoding that serves to separate and store
overlapping information as distinct from each other to reduce interference during
memory retrieval (Yassa and Stark 2011). Interestingly, male advantages in spatial
ability are more prominent during memory encoding compared to memory retrieval
(Driscoll et al. 2005; Piber et al. 2018; Subramaniapillai et al. 2019), suggesting a
possibility that there are sex differences in pattern separation. To the best of our
knowledge, no study has reported sex differences in the ability for pattern separation
in humans. This is partially due to lack of studies that analyze data with sex as a
variable in humans (Kumaran and Maguire 2006; Bakker et al. 2008; Chadwick et al.
2011; Lacy et al. 2010; Kyle et al. 2015; Favila et al. 2016; Ballard et al. 2019;
Rechlin et al. 2021). On the other hand, a few studies have reported sex differences
in the ability for pattern separation in rats and mice (Yagi et al. 2016; Kundey et al.
2019). Yagi et al. (2016) demonstrated a male advantage in the ability for spatial
pattern separation using the delayed non-match to place task in the radial arm maze
in rats. Importantly, this sex difference was observed only among rats that relied
more on allocentric spatial cues, suggesting that sex differences in spatial pattern
separation tasks emerge when requiring greater engagement of the hippocampus.
These findings in humans, rats, and mice imply that males and females use
different brain networks and learning strategies that depend on sex hormone status,
even when there are no sex differences in performance on the same cognitive task.
Therefore, it is important to consider sex hormone levels and the types of memory
cues available, as these may significantly influence the appearance and direction of
sex differences in cognitive performance. Furthermore, it is equally important to
consider age and hormone status of participants to gain a clearer picture of sex
differences in cognition.
3 Cognition Throughout the Aging Process
The world’s older population is growing at an unprecedented rate. It is projected that

the global population of those aged 65 or older will double over the next three
decades to 1.5 billion, and life expectancy will increase to 77 years by 2050 (United
Nations et al. 2020). It is important to consider sex in the question of what is normal
in cognitive aging and whether there are differential strategies needed to alleviate
detriments to cognitive health. Understanding these age-related cognitive changes
and their contributing factors, including possible sex differences in longevity and
mechanisms, will be critical to improving care for older adults and identifying
differences between healthy and disease states seen in aging. We will end this
chapter by exploring diet as a modifiable factor that can alter cognitive aging by sex.
3.1 Cognitive Aging
Cognitive aging is defined as the decline in certain cognitive abilities that occur as
people get older (reviewed in Buckner 2004). However, in all of the work described
in the next two paragraphs, neither sex nor gender were used as experimental
variables. Where we can, we indicate if sex differences were studied and noted.
Both cross-sectional and longitudinal research show robust declines across the adult
lifespan in processing speed, consolidation of newly learned information, and
episodic memory performance (reviewed in Hedden and Gabrieli 2004; reviewed
in Harada et al. 2013), however vocabulary increases with age (Salthouse 1996).
Executive function, defined as high-level cognitive processes that involve working
memory, flexible set shifting, and inhibition, is also impaired in normal aging
(Cepeda et al. 2001; reviewed in Diamond 2014; reviewed in Kirova et al. 2015;
Klencklen et al. 2017; McNab et al. 2015). A longitudinal study found declines in
working memory and inhibitory control started as early as 30–40 years old
(Ferguson et al. 2021). This supports previous literature showing age-related impair-
ments in executive functioning, verbal fluency and memory, and cognitive speed
tasks in healthy adults aged 64–81 (van Hooren et al. 2007). Overall, there is
heterogeneity in the trajectories of different cognitive domains impacted by aging
(reviewed Harada et al. 2013). Nevertheless, considerable empirical evidence shows
that executive function may be the first to deteriorate with age, and thus an important
mediator for age-related declines in other memory domains (Bugaiska et al. 2007;
Kim et al. 2013; reviewed in West 1996).
As noted above, some cognitive abilities remain stable or even improve with age.
Crystalized intelligence, which refers to skills and knowledge that are overlearned
and practiced – like vocabulary, recognition memory, temporal order memory, and
procedural memory – are relatively resilient to aging (reviewed in Craik and
Bialystok 2006; Horn and Cattell 1967). Semantic memory, which involves ideas,
concepts, and facts not drawn from personal experience and commonly regarded as
“general knowledge” is also preserved across most of the adult lifespan, with
declines occurring only very late in life (Nyberg et al. 1996; Rönnlund et al.
2005). As noted earlier, most of this literature has not examined how cognitive
aging may differ by sex. However, as highlighted in an earlier section of this chapter,
there are known sex-related differences in cognition, so including both sexes and
analyzing data by sex is crucial to understanding how cognition changes with
advancing age. Indeed, the trajectory of cognitive aging does differ by sex and this
has implications for diagnosis of disorders that result in cognitive impairments, and
this will be discussed in greater detail in a later section.
3.2 The Neurobiology of Cognitive Aging
To understand the neurobiology of cognitive aging, the prefrontal cortex and

hippocampus are two brain areas that are important to focus on because of their
roles in learning and memory (reviewed in Jobson et al. 2021; reviewed in Preston
and Eichenbaum 2013). Indeed, the prefrontal cortex is one of the initial brain areas
to lose integrity during normal aging in humans (Nyberg et al. 2010; Rajah and
D’Esposito 2005). With advancing age, large volumetric declines occur in the
prefrontal cortex, which subserves episodic encoding and executive processes
(Dotson et al. 2016; Meguro et al. 2001; Salat et al. 2004; Tisserand et al. 2002).
Combined with the loss of anterior white matter integrity and dopamine receptors in
the striatum and prefrontal cortex, these structural changes map onto the age-related
changes seen in executive functioning (Berry et al. 2016; reviewed in Caserta et al.
2009; Karrer et al. 2017; reviewed in Liu et al. 2017). Sex differences in age-related
white matter changes remain understudied, but recent studies show human females
with significantly faster decline in white and gray matter volume and increased white
matter hyperintensities with aging compared to human males (Than et al. 2021; Sang
et al. 2021). Network analyses of relationships between cognitive health and brain
health find that human females show greater links to brain aging with measures of
metabolic health (Foret et al. 2021), which will be discussed in more detail below,
and this may explain some of the equivocal findings in the literature. There are sex
differences in how changes in brain volume relate to cognitive performance in
humans. For instance, Gur and Gur (2002) show earlier brain aging, particularly in
frontotemporal regions associated with attention, inhibition, and memory, in males
than females. One recent study found that males had more cognitive functions that
were significantly correlated with gray and white matter volume declines, compared
to females (Sang et al. 2021). More research is needed to understand these under-
lying sex differences in the brain, as they may be closely related to the sex
differences seen in cognitive aging.
Many hippocampal processes decline with age, suggesting that the hippocampus
is susceptible to deterioration in the cognitive aging process (Papp et al. 2014;
Reuben et al. 2011). Indeed, decreased hippocampal volume is associated with
impairment in processing speed, working memory, episodic memory, and executive
function performance in 93 healthy humans (mean age 71.7 years, 60% female)
(O’Shea et al. 2016). Decreased hippocampal volume is also associated with impair-
ment in fluid intelligence (Reuben et al. 2011: sex not reported). Several studies have
shown significant hippocampal volume loss in aging (Jack et al. 2000; Nobis et al.
2019; Scahill et al. 2003; Schuff et al. 1999). Nobis et al. (2019) noted that the
acceleration in the rate of hippocampal volume decline in middle age was more
pronounced in human females than in human males (aged 45–80). Another study
found a later but faster decline in hippocampal volume in males (at around age 70)
(Wang et al. 2019). Wang et al. (2019) further reported that age-related hippocampal
volume decline was linear in females and quadratic in males in humans, indicating
sex differences in the trajectory of brain aging in the hippocampus. Still, other
studies have reported no significant sex differences in overall hippocampal volume
loss in aging (Sullivan et al. 1995). It is imperative to acknowledge that studies use
different methods of adjustment for brain size and the resulting measures vary
drastically depending on the correction factor used (O’Brien et al. 2017; Sanchis-
Segura et al. 2019). Indeed, Duarte-Guterman et al. (2021) have detected sex
differences in hippocampal volume in humans, but the direction was dependent on

the correction factor used. Regardless, given that the hippocampus is a heterogenous
structure with subfields which are histologically and functionally unique (the
subiculum, the four cornu ammonis sectors (CA1, CA2, CA3, and CA4), and the
dentate gyrus), examining hippocampal subfields separately may be more informa-
tive (Dimsdale-Zucker et al. 2018; Engvig et al. 2012; Reagh et al. 2014; reviewed in
Yassa and Stark 2011). Using a high-resolution MRI, the volume of the CA1 region
was found to decrease with advancing age in healthy individuals, regardless of sex,
while the rest of the subfields were spared (Mueller et al. 2007). Moreover, this
correlation was most pronounced in the seventh decade of life (Mueller et al. 2007).
These findings illustrate the importance of studying hippocampal subfields sepa-
rately to gain a better understanding of how they may be differentially altered in
aging.
Many studies focus on gray matter volume changes, yet changes to connectivity
and functional activation of the different hippocampal subfields with aging are also
present. Deficits in cognition with age are attributed to altered activity of the
hippocampus, including reduced activity in the CA1 along with hyperexcitation in
the CA3 and dentate gyrus (reviewed in Oh et al. 2016). Studies show that intrinsic
excitability in CA1 pyramidal neurons are reduced in aged rats, which would limit
their activity in the neural network during learning (reviewed in Disterhoft and Oh
2007; reviewed in Oh et al. 2016). Furthermore, studies in humans, mice, and rats
indicate increased excitability in CA3 neurons with age (reviewed in Oh et al. 2016).
fMRI studies indicate increased BOLD activity in the CA3/dentate gyrus region in
healthy male and female human adults (mean age 75, 80% females) who were
impaired on pattern separation tasks, in which performance is dependent on the
CA3 and dentate gyrus (Yassa and Stark 2011). Findings from animal studies agree
with this, as they show that CA3 neurons exhibit increased firing rates and network
activity in aged male rats (Wilson et al. 2005; Simkin et al. 2015). It is suggested that
this hyperactivity in the CA3 region leads to overgeneralization and impaired ability
to separate experiences or memories with similar features. A recent study using aged
(28–31-month-old) male rats formed two groups: one that retained cognitive perfor-
mance relative to younger adults and the other that was cognitively impaired
compared to younger adults on temporal and spatial working memory performance
(Buss et al. 2021). In the cognitively normal aged male rats, they found synaptic
architecture of the CA1 and CA3 was also maintained and similar to their young
adult counterparts, but in aged male rats that were cognitively impaired, there was a
redistribution in synaptic weights such that the influence from the dentate gyrus onto
CA3 pyramidal neurons weakened and the influence among CA3 pyramidal neurons
onto themselves to strengthened. In other words, it appears that age-related cognitive
deficits are associated with synaptic changes in CA3 pyramidal neurons in aged male
rats. Together, this body of research suggests that decreased excitability within the
CA1 and increased excitability and imbalanced synaptic weights within the CA3
contributes to age-related cognitive decline. These findings should be interpreted
with the awareness that the majority of the animal studies are conducted in males
only, and it remains unclear whether or how sex may influence the relationship
between age and hippocampal connectivity and functional activation.
3.3 Sex Differences in Cognitive Aging
Given the known sex differences in cognition, it is not surprising that the trajectory
of cognition throughout aging is different in males and females. Previous studies
have indicated that human females have higher baseline scores on most types of
cognitive tests, except those measuring visuospatial ability (de Frias et al. 2006;
Munro et al. 2012; McCarrey et al. 2016). Despite the higher baseline performance
in global cognition, executive function, and verbal memory in human females
compared to human males, females showed faster declines in global cognition and
executive function over a span of 20 years compared to males (Levine et al. 2021).
However, in verbal cognitive domains, human females maintained superior memory
performance compared to human males over a 10-year period (initially aged 35–80)
(de Frias et al. 2006). In this longitudinal study of cognitively healthy adults, human
females persistently outperformed human males on verbal memory, verbal recogni-
tion, and semantic fluency tasks whereas human males outperformed human females
on visuospatial ability tasks (de Frias et al. 2006). This finding is well replicated in
the literature (van Hooren et al. 2007; Proust-Lima et al. 2008). There are other
cognitive tasks in which steeper decline is observed in human males. For instance,
across the lifespan (12–69 years of age), although the decline on reasoning scores
began earlier in females, the decline was faster in males (Nichols et al. 2020). In
older human adults (baseline mean age 64.1–69 years), McCarrey et al. (2016) found
a steeper decline, across 3–9 years, in males, on measures of mental status,
perceptuomotor speed and integration, and visuospatial ability compared to females
(McCarrey et al. 2016). Together, this research illustrates that there are sex differ-
ences in cognitive aging and that human males and females differentially show either
different patterns of resilience or different aging patterns across brain structures that
contribute to sex differences in age-related decline depending on the cognitive
domain.
When trying to make sense of these sex differences in cognitive aging, sex
hormones are important to consider. Testosterone is a sex hormone primarily
secreted by the testes in males and, though to a much lesser extent, by the ovaries
in females. In human males, testosterone levels begin to diminish starting from age
30 and continue to decline throughout the lifespan (Feldman et al. 2002). Declining
levels of circulating testosterone are associated with declines in cognition and
alterations in brain functioning in older males (reviewed in Giagulli et al. 2016;
reviewed in Moffat 2005). Testosterone levels are associated with global cognition,
memory, executive functions, and spatial performance in human males (reviewed in
Holland et al. 2011). In human females, estrogens affect several cognitive domains
as well as cognitive aging (reviewed in Galea et al. 2017; reviewed in Russell et al.
2019). Of the three types of estrogens (estrone, estradiol, and estriol), estradiol
Fig. 2 A schematic summary of factors that contribute to differential cognitive aging outcomes,
including sex, hormones, education, APOEε4, diet, and obesity
(17β-estradiol) is the most potent and abundant during the reproductive period in
females (Nugent et al. 2012). During menopause, estradiol and estrone levels rapidly
decline (Nugent et al. 2012). As such, the neuroprotective effects of these estrogens
may diminish as well, exacerbating the effects of age on cognitive functions (Barha
and Galea 2010; Farrag et al. 2002; Maki et al. 2021; reviewed in Reuben et al.
2011). Indeed, cognitive decline is seen in surgically menopausal human females,
and those with a >50% drop in estrogen levels experienced more cognitive decline
(Farrag et al. 2002). Furthermore, surgical menopause is associated with faster
cognitive decline with age (Bove et al. 2014; Georgakis et al. 2019). Supporting
these findings, a recent study shows impaired verbal and working memory perfor-
mance in surgically menopausal human females, but among those who were receiv-
ing estradiol therapy, working memory ability was maintained (Gervais et al. 2020).
Importantly, the relationship between estrogens and cognitive aging is dependent on
lifetime estrogen exposure, reproductive experience, and hormone therapy (age at
initiation, length, type, and duration) (Barnes et al. 2003; Bove et al. 2014; Karim
et al. 2016; Kuh et al. 2018). Figure 2 contains a schematic of a summary of factors
that can contribute to cognitive aging. A more detailed account of the relevant
literature is beyond the scope of this chapter, and the reader is directed to other
reviews (Georgakis et al. 2019; Sherwin 2006).
4 Dementia
Undeniably, one of the biggest challenges associated with aging is dementia.

Although dementia typically affects older adults, it is not a normal part of aging.
The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) defines demen-
tia as the umbrella term for several neurological conditions of which the primary
symptom is cognitive decline that significantly interferes with independence in
everyday activities (American Psychiatric Association 2013). One or more of the
following cognitive domains may be impacted: complex attention, executive func-
tion, learning and memory, language, perceptual-motor function, and social cogni-
tion (American Psychiatric Association 2013). Although all types of dementia
deserve attention and research as they uniquely impact patients and their caregivers,
this part of our chapter will focus on AD, which accounts for 60–80% of dementia
cases (Alzheimer’s Association 2020). For detailed discussions about other types of
dementia, the reader is directed to other reviews (reviewed in Bang et al. 2015;
reviewed in Walker et al. 2015; reviewed in O’Brien and Thomas 2015).
AD is a neurodegenerative disorder characterized by progressive and irreversible
memory decline accompanied by pathological changes in the brain (Alzheimer’s
Association 2020). However, the reader is cautioned that not much work has
characterized possible differences in the disease by sex, which we describe below.
The neural hallmarks of this disease involve accumulation of the protein fragment
beta-amyloid (Aβ) (plaques) outside neurons, accumulation of an abnormal form of
the protein tau (tangles) inside neurons, and neurodegeneration (Jack et al. 2013).
Specifically, as the disease progresses, aggregated Aβ depositions and aggregated
hyperphosphorylated tau become increasingly evident around and inside neurons of
memory-related medial-temporal structures – particularly the entorhinal-
hippocampal circuit (Thal et al. 2000). Furthermore, prominent volume loss is
seen in the CA1 region of the hippocampus and entorhinal cortex early in AD
(Mueller et al. 2010). As the disease spreads, total hippocampal volume becomes
significantly reduced (Mueller et al. 2010). It is important to highlight that the
distribution of hippocampal volume loss in AD is distinct from that in healthy
cognitive aging, which as mentioned earlier is restricted to the CA1 region (Mueller
et al. 2007). Besides volume reduction, hippocampal structure and function are also
compromised in AD (Allen et al. 2007; Demars et al. 2010; reviewed in Setti et al.
2017). The AD-related neuropathology (AB, tau) and integrity loss in the hippo-
campus may underscore the decline in hippocampus-related cognitive functions seen
in the disease. As such, this will be discussed in greater detail in the next section.
The direct causes of sporadic late-onset AD, which represent more than 95% of
AD cases, remain unknown (reviewed in Tanzi 2012). However, there are known
modifiable risk factors, like obesity, diabetes, physical activity, sleep, and diet (see
Sect. 5.3 in this chapter; reviewed in Edwards III et al. 2019) and nonmodifiable risk
factors, like aging, Apolipoprotein E (APOE)ε4 genotype, and female sex (Holland
et al. 2013; reviewed in Mielke et al. 2014; Plassman et al. 2007; reviewed in Riedel
et al. 2016). In terms of genetics, the ε4 allele of the APOE gene is recognized as the
strongest genetic risk factor for sporadic AD (DiBattista et al. 2016; Sando et al.
2008). Although the worldwide APOEε4 frequency is only 14%, 40–50% of people
with AD carry at least one allele (Farrer et al. 1997; Ward et al. 2012). Indeed,
carrying one or two APOEε4 alleles confers a 3 or 25-fold increase, respectively, in
the odds ratio of developing AD (Saddiki et al. 2020). Controlling for APOEε4
status, human females show more severe AD symptoms and progression, greater
neuropathology, and faster cognitive decline compared to human males (Barnes
et al. 2005; reviewed in Ferretti et al. 2018; Holland et al. 2013). However, when
studied together, sex has been found to interact with APOEε4 to further modify AD
risk and manifestation (Altmann et al. 2014; Ghebremedhin et al. 2001). A large
meta-analysis found that human females aged 60–70 with one APOEε4 allele had a
fourfold increased risk for developing AD, whereas their male counterparts had no
increased risk for developing AD (Farrer et al. 1997). Even in those with both
APOEε4 alleles, AD risk peaked at 12-fold in human females compared to tenfold
in human males (Farrer et al. 1997). This interaction between sex and APOEε4 has
been replicated in several studies that followed (Bretsky et al. 1999; Neu et al. 2017).
In addition to increased risk, APOEε4 human females present with greater AD
pathology, measured by total tau and phosphorylated tau levels (Duarte-Guterman
et al. 2021) and tau-to-Aβ ratio (Altmann et al. 2014) compared to males. Clearly,
there are many sex differences seen in AD manifestation and progression, and
further examination of these sex differences may uncover critical information
about differential risk factors, pathogenesis, and treatment of the disease between
males and females.
4.1 Cognitive Decline in Alzheimer’s Disease
Although prodromal changes to cognition may occur earlier in some domains than
others, global cognitive function decline accelerates by 15-fold five years prior to
AD diagnosis (Wilson et al. 2011). Amnestic mild cognitive impairment (aMCI) is
considered an intermediary state between the cognitive changes seen in aging and
mild dementia (Davis et al. 2018). Indeed, the risk of AD is considerably higher in
people with aMCI compared to those who are cognitively healthy (Roberts et al.
2014; Bermejo-Pareja et al. 2016; Yaffe et al. 2006). Although many studies suggest
a higher prevalence (Petersen et al. 2010) and incidence (Brodaty et al. 2013;
Roberts et al. 2012) of aMCI in human males, others found no sex differences
(Au et al. 2017; Overton et al. 2019). The discrepancy in this literature may be due to
differences in study design, diagnostic criteria, MCI subtype, and age distribution of
the samples. Nonetheless, among people with aMCI, cognition decline has consis-
tently been found to be steeper in human females compared to males (Sohn et al.
2018; Lin et al. 2015; Wang et al. 2019). Moreover, there is a sex and APOEε4
interaction such that APOEε4 females show the steepest decline in a verbal learning
task compared to non-APOEε4 females and males (Wang et al. 2019). Supporting
the evidence of faster progression from aMCI to AD in human females relative to
human males, Skup et al. (2011) found that various gray matter regions in the brain
atrophied more rapidly in human female aMCI and AD patients compared to human
males. Together, these findings suggest that sex plays an important role in aMCI,
which has implications for AD risk and memory decline. More studies on this matter
are necessary to fully understand this relationship and its underlying mechanisms.
As mentioned earlier, human females have greater lifetime AD risk and carry a
higher burden of the disease. Females also experience greater cognitive impairment
than males as the disease progresses (Chapman et al. 2011; Irvine et al. 2012).
Among those with AD, females show worse performance in multiple cognitive
domains, including visuospatial, verbal processing, semantic, and episodic memory
than males (Irvine et al. 2012; reviewed in Laws et al. 2016). To understand
cognitive state across the different stages of normal aging, aMCI, and AD, Duarte-
Guterman et al. (2021) examined data from a database (ADNI). In the cognitive
normal and aMCI groups, human females had significantly better composite memory
scores than human males, but this sex difference disappeared in the AD group
(Duarte-Guterman et al. 2021). This finding is suggestive that, because of the female
advantage in verbal memory tasks, this advantage may delay diagnoses of MCI or
AD in females, as others have suggested (Sundermann et al. 2016a, b), and/or may
reflect the findings that females show faster decline in cognition in AD (Irvine et al.
2012).
There is an apparent interaction between sex and AD neuropathology in the
context of cognitive decline. Barnes et al. (2005) conducted a post-mortem exami-
nation of people who were diagnosed with AD and used the number of neuritic
plaques, diffuse plaques, and neurofibrillary tangles from four cortical brain regions
to measure AD neuropathology. They found that with each unit of AD neuropathol-
ogy, cognitive function scores for episodic memory, semantic memory, working
memory, perceptual speed, and visuospatial ability were significantly more reduced
in human females compared to human males (Barnes et al. 2005). Consistent with
this finding, Koran et al. (2017) showed that human females with high total tau levels
had faster hippocampal atrophy and executive functioning decline than human
males, and this sex difference was most pronounced among APOEε4 carriers
(Koran et al. 2017). In fact, APOEε4 alleles are additionally associated with greater
detrimental effects in hippocampal pathology, functional connectivity in the default
mode network, cortical thickness, and memory performance in human females than
human males across different stages of AD (Damoiseaux et al. 2012; Fleisher et al.
2005; Liu et al. 2010). The findings relating to sex differences in AD cognitive
decline are nuanced, yet until recently, there are few studies that have collected,
analyzed, and/or presented neurocognitive data separated by sex (reviewed in Laws
et al. 2016).
5 Lifestyle Factors on Cognition, Cognitive Aging,

and Dementia
The relationships between sex and cognition, cognitive aging, and dementia are
complex and because of their multifaceted nature, these relationships can change
when taking lifestyle and sociodemographic variables into account, like education
and diet – both of which will be explored in this chapter.
Not surprisingly, previous education plays a substantial role in how cognition
changes with age. Studies have demonstrated that a higher level of education is
protective against age-related cognitive decline (Beeri et al. 2006). For example, van
Hooren et al. (2007) showed that people with a “middle to high” level of education
(defined as intermediate secondary education to university education and scientific
education) performed better on cognitive tests than those with a lower level of
education, at older ages (van Hooren et al. 2007). In fact, low education (defined
as primary education and lower vocational secondary education) is related to
accelerated decline in processing speed, verbal memory performance, and general
cognitive scores in middle and old age (Bosma et al. 2003). One possible explanation
for this phenomenon is related to cognitive reserve capacity. Cognitive reserve refers
to preexisting cognitive processes or compensatory processes which help an indi-
vidual sustain threats to brain integrity and maintain function and can be obtained by
learning new skills, more education, exercise, and greater social interactions
(reviewed in Stern 2009). Therefore, cognitive symptoms may be more likely
to emerge after an insult to the brain in those with lower cognitive reserve compared
to those with greater cognitive reserve (reviewed in Stern 2009). It is also important
to be mindful that there are other factors that are related to education that may be at
play, including genetic factors as well as gendered factors like occupation and other
lifestyle habits (Bosma et al. 2003), though this discussion is outside the scope of this
review. The exact combination of mechanisms underlying the relationship between
education and cognitive aging remains unclear. Although this chapter only discusses
education and diet, it is important to keep in mind that other lifestyle factors like
exercise, stress, and social determinants of health will also affect cognitive health
across the lifespan.
5.1 Obesity and Cognition
Obesity is one of the greatest public health challenges of the twenty-first century,
affecting over 650 million adults and 124 million children (World Health Organi-
zation 2016; NCD Risk Factor Collaboration 2017). It is a complex disorder that
develops due to a range of biological, environmental, and social factors, and is
driven by an imbalance between energy intake and energy expenditure, resulting in
excessive fat accumulation. Energy intake has increased throughout many parts of
the world due to high energy foods being readily available at a low cost. There are
also rewarding aspects to food, especially in high energy foods, that drive excessive
consumption (Morales and Berridge 2020). Moreover, caloric expenditure from
physical activity has decreased historically, further accelerating body weight gain
and obesity (Booth et al. 2012). Although sometimes questioned as a viable measure
of body fat, body mass index (BMI) is one of the most commonly used indexes to
classify body weight status, calculated by dividing an individual’s weight in kilo-
grams (kg) by their height in meters (m) squared (weight/height2). In adults, the
World Health Organization (WHO) defines overweight as BMI 25 and obesity
30. Another measure of obesity, and more specifically abdominal obesity, is the
waist-to-hip ratio, calculated as the waist circumference divided by the hip circum-
ference, in which abdominal obesity is defined as having a waist–hip ratio above
0.90 for males and above 0.85 for females (World Health Organization 2008). The
latter method may be of more importance for addressing body fat level in relation to
disease risk, as abdominal (visceral) fat may be especially detrimental to metabolic
and cognitive health, which is more common in males, as discussed below.
Obesity can impact cognitive health and is negatively associated with cognitive
function and an increased risk of neurodegenerative diseases, such as AD (O’Brien
et al. 2017). In addition, obesity-related comorbidities, such as diabetes
(Papanikolaou et al. 2006; Vasquez et al. 2021), hypertension (Qiu et al. 2005),
and dyslipidemia (Panza et al. 2006), are all associated with cognitive impairment
and an increased risk of cognitive decline.
There is an imbalance in the prevalence of excess body weight between sexes,
with human females being overrepresented in overweight and obese weight groups
compared to human males (Organization 2016). These differences are especially
apparent after menopause, a timing at which human females’ risk of metabolic,
neurodegenerative, and cognitive disorders increase (Henderson 2014; Kozakowski
et al. 2017). In humans, females have a higher body fat percentage than males in
general, even at the normal weight range (Kyle et al. 2001; McCarthy et al. 2006;
Schorr et al. 2018), and males and females also have distinct patterns of fat
accumulation, with females primarily accumulating fat subcutaneously (hips and
thighs), while males accumulate fat viscerally (abdominally) (Karastergiou et al.
2012; Palmer and Clegg 2015). This effect is mainly driven by differences in sex
hormone levels (estrogens versus androgens) between sexes, and differences in
estrogen and androgen receptor expression in visceral and subcutaneous fat pads
(Brown and Clegg 2010). Although the main source of sex hormone production is
within the gonads, sex hormones are also produced directly within fat tissue (Labrie
1991), which becomes the main source of estrogens after menopause (Simpson
2000). Fat distribution patterns strongly impact health (Manolopoulos et al. 2010),
where subcutaneous fat accumulation, the main fat storage site in females, is
associated with reduced risk of metabolic disorders such as diabetes (Chan et al.
1994; Carey et al. 1997) and cardiovascular disease (Lapidus et al. 1984; Seidell
et al. 2001; Canoy et al. 2007; Despres 2007), whereas the contrary has been found
for excess visceral fat, the site of main fat storage in human males. In line with this
adipocentric view, studies suggest that obese human males, who have high levels of
visceral fat, are more susceptible to cognitive impairment than obese human females
(Elias et al. 2005) and have greater global gray matter loss than their obese female
counterparts (Taki et al. 2008; Ahonen et al. 2012). Further strengthening the
importance of the location of fat accumulation for cognition and dementia risk,
both normal and obese females with a high waist-to-hip ratio have increased risk of
cognitive impairment and probable dementia (Kerwin et al. 2011). Obesity is also
associated with reduced progesterone levels in males (Blanchette et al. 2006) and
premenopausal females (Jain et al. 2007). Progesterone has beneficial effects on
cognitive performance in male (Frye et al. 2021) and female (Si et al. 2013; Peterson
et al. 2012; Frye and Walf 2008a, b; Frye et al. 2013) mice and rats across several
different learning and memory tasks. Despite male fat accumulation patterns being
more detrimental to cognitive health, overall dementia risk is higher in human
females (Floud et al. 2020). As mentioned above females with AD are more
burdened by the disease and show greater neuropathology and cognitive decline
than males with AD (Baum 2005; Irvine et al. 2012). With almost 40% of adults
being overweight or obese, there is only a slight overrepresentation in human
females (Organization 2016), and obese males are more susceptible to cognitive
impairment than obese females. So what can be causing the overrepresentation of
females afflicted by dementia later in life? The answers lie, at least in part, in the
influence of sex hormone levels at different stages of life, and points to different
etiology of AD between males and females.
Although human females seem to be protected from the negative effects of
increased body weight on metabolic and cognitive health prior to menopause, this
shifts postmenopause due to the pronounced reduction of estrogens at this stage in
females’ lives (Zsido et al. 2019). The marked reduction in circulating estrogens can
be detrimental to cognition both directly and indirectly: As discussed above, estro-
gens promote neurogenesis and enhance cognition, albeit this depends on a number
of factors including the levels and the type of estrogens as well as the cognitive
domain tested (Barha and Galea 2010). Furthermore, removal of the main source of
estrogen production, the ovaries, results in impaired cognitive functions in humans,
mice, and rats (Farrag et al. 2002). The reduced levels of estrogens after menopause
also result in the redistribution of fat, from subcutaneous to visceral (Ley et al. 1992;
Svendsen et al. 1995), and increased fat mass (Chang et al. 2018), which can further
contribute to an increased risk of cognitive impairments and cognitive decline.
Furthermore, 17β-estradiol can indirectly reduce body weight by inhibiting food
intake, and the amount of food intake varies throughout the menstrual cycle in
humans and estrous cycle in rats (Drewett 1973; Gong et al. 1989; Lyons et al.
1989; Eckel et al. 2000). The vast reduction in 17β-estradiol after natural or surgical
menopause results in increased food intake and body weight, caused by an increase
in meal size and frequency, in addition to increased hedonic feeding (Rivera and
Stincic 2018). Reduced levels of estrogens and progesterone at menopause may also
result in a reduction in brown adipose tissue-mediated thermogenesis, as both
estrogens and progesterone stimulate brown adipose tissue-mediated thermogenesis,
a process which produces heat and increases body temperature by burning fat
(adipocyte lipolysis) (Rodríguez et al. 2002). Reduced brown fat activity therefore
results in a reduction in energy expenditure, which can contribute to postmenopausal
weight gain (Gonzalez-Garcia et al. 2017; Chang et al. 2018). Body weight gain after
menopause or ovariectomy can be blunted by administration of 17β-estradiol-based
hormone therapies (Gambacciani et al. 1997; Asarian and Geary 2002). In contrast,
progesterone, which also reduces postmenopause/ovariectomy, is not associated
with changes in food intake (Czaja 1978; Eck et al. 1997; Pelkman et al. 2001) or
food cravings (Michener et al. 1999) in female humans and primates.
Increased adiposity may play an especially important role for cognition at midlife,
as high BMI at this time is associated with reduced general cognitive ability and
faster cognitive decline later in life; an effect seen in studies that included both males
and females but did not always explicitly test for sex differences (Dahl et al. 2010;
Pedditzi et al. 2016). Importantly, detrimental effects of being overweight or obese
on cognition at midlife can be counteracted by weight loss. Weight loss mediated by
diet, exercise, or a combination led to improved attention, memory, executive
function, and memory in human females, and in a study that used both human
males and females combined [although the majority of subjects included in this
meta-analysis were female (78%)] (Veronese et al. 2017). The beneficial effects of
weight loss are especially apparent for individuals with higher BMI at baseline
(BMI 30) (Siervo et al. 2011). Although weight-loss effects on dementia and
AD are still unclear (Veronese et al. 2017), weight loss in obese individuals
improves memory, executive function, global cognition, and language in seniors
with mild cognitive impairment in a study conducted predominantly in women
(83.7%) (Horie et al. 2016). This association was strongest in younger seniors and
in APOEɛ4 carriers. However, in most of the studies cited above, sex was not used as
a discovery variable, so although both sexes were included, it is difficult to know
whether these associations were stronger in one sex versus the other.
In contrary to the detrimental effects of excessive body weight at midlife, the
opposite may be true at older ages, as some studies report that obesity later in life
(>65–70 years) may instead be protective against cognitive decline (Hughes et al.
2009; Kim et al. 2016; Pedditzi et al. 2016); this relationship was especially strong in
human females (Kim et al. 2016). In fact, several studies conducted in older human
males and females have found that obesity alters cognitive performance selectively
in males in a range of cognitive tests including orientation, concentration, language,
praxis, immediate and delayed memory, and logical memory (Elias et al. 2003;
Kanaya et al. 2009), further strengthening a non-pathological role for obesity in
females with advanced age. Thus, in addition to sex, detrimental versus protective
effects of excess body weight vary depending on age and the level of overweight/
obesity. In older adults, excessive BMI may be especially detrimental at greater ages
(>80 years), compared to between 65–79 years of age (Wu et al. 2021). Although
obese human males were the only group that demonstrated an increased risk of
cognitive impairment, sex was not a significant factor between the association of
BMI and cognitive impairment. The study also highlights the impact of the detri-
mental effect of being underweight on cognitive impairment (for 65–79, but not
>80 year old individuals), which poses a similar risk of cognitive impairment as
being overweight (Wu et al. 2021). This association was also found in a previous
study, with a stronger association in human females (Onadja et al. 2013). The
negative impact of being underweight on cognitive function in older human females

has previously been described (Ortega et al. 1997; Lee et al. 2001). In addition to
age, risk of cognitive impairment may also vary depending on level of excess body
fat, as a study recently demonstrated that, whereas being overweight was associated
with an increased risk in cognitive impairment in both human males and females,
obesity was protective in females and detrimental to cognitive health in males
(Wu et al. 2021).
Taken together, adverse effects of increased body weight in human females may
be especially driven by viscerally stored fat at midlife, and increased fat mass earlier
in life is not as detrimental to cognition if it is stored in other regions (e.g.,
subcutaneously), even after controlling for risk factors such as type 2 diabetes and
cardiovascular disease (Elias et al. 2005; Taki et al. 2008; Dahl et al. 2010; Kanaya
et al. 2009). Furthermore, at later stages of life (postmenopause), excess fat may
instead act in a protective manner against cognitive decline in human females,
especially with obesity. The protective properties of excess fat in older females
may, in part, be driven by the detrimental effects of malnutrition and underweight on
cognitive health in some studies, as individuals with low BMI at old age present with
reduced cognition, especially in older females (Ortega et al. 1997; Lee et al. 2001). It
is also possible that detrimental effects of fat accumulation at midlife in human
females is a result of the drastic loss of circulating estrogens, an effect which can
potentially be rescued long-term by increasing estrogen production locally within
subcutaneous adipose tissue. Adipose tissue is the main site of estrogen production
postmenopause (Labrie 1991; Simpson 2000), therefore, in the post-reproductive
years, the degree of a woman’s estrogenization is mainly determined by the extent of
her adiposity. In fact, postmenopause, obese females have higher levels of estrone,
estradiol, and estriol than non-obese females (Kozakowski et al. 2017). Females with
greater levels of body fat may, therefore, be able to compensate for the loss of
estrogens at midlife, and attenuate impairments in cognitive decline due to estrogen
loss, an effect which is not possible in females with lower adipose reserves. This may
also explain why higher levels of fat are required (obesity vs overweight) for
protective effects to be present in females. A protective role of high body weight
in females at old age has also been demonstrated in other disease states. For instance,
obese females at advanced age are relatively protected against osteoporosis (Melton
3rd 1997), and the incidence of AD is lower in postmenopausal women with greater
body weight than in their slimmer counterparts (VW Henderson, personal
communication).
5.2 Diet and Cognition
High energy diets, such as diets high in saturated fats (high-fat diet; HFD), refined
carbohydrates (high sugar diet; HSD), or both (often referred to as the western diet),
are well-known for their role in the development of obesity, and excessive con-
sumption has been linked to lower hippocampus-dependent cognition in both
humans and rodents, even in absence of obesity (Greenwood and Winocur 1996;
Granholm et al. 2008; Thirumangalakudi et al. 2008; Kanoski and Davidson 2010,
2011; Kosari et al. 2012; Francis et al. 2013; Kaczmarczyk et al. 2013; Cordner and
Tamashiro 2015; Morris et al. 2015; Moser and Pike 2017). High exposure of all
three high energy diets impairs hippocampus-dependent cognition, although diet
type, length of diet, and age at consumption play important roles in the magnitude of
impairment. Each of these diet types and sex differences in their effects on cognition
will be discussed in greater detail below.
5.2.1 Western Diet
Diets rich in fat and sugar are recognized as major contributors to obesity and type
2 diabetes, conditions that are strongly associated with impairments in cognition
(Morris et al. 2015). Females are less likely to report consuming a western diet and
are more likely to report consuming a healthy diet, than human males (Shakersain
et al. 2016). Several studies in males and females have reported negative effects on
cognition after chronic exposure to the western diet (Morris et al. 2004; Akbaraly
et al. 2009; Francis and Stevenson 2011; Brannigan et al. 2015; Shakersain et al.
2016). However, sex specific effects are seldom analyzed or reported, though there
are indications that sex differences in western diet exposure and cognition exist. In
older humans, although adherence to a western diet was associated with poorer
baseline cognition in males, but not in females, the western diet did not result in a
greater decline in global cognition over a 3-year time period [Mini-Mental State
Examination (3MS)], in either sex (D’Amico et al. 2020).
In mice and rats, the western diet is especially detrimental to learning and
memory tasks that require the hippocampus (i.e., spatial learning), including the
MWM and radial arm maze, at least in adult males, arising as early as 10 days after
diet introduction and after diet consumption up to 24 weeks (Kanoski et al. 2010;
Boitard et al. 2014; Takechi et al. 2017; Abbott et al. 2019). The contrary has been
demonstrated in a rat model of brain aging, where western diet exposure was not
sufficient to impair spatial learning and memory in males in the MWM further than
the impact of aging alone (Pancani et al. 2013). However, the diet did not result in a
significant increase in body weight during the 24-week diet exposure period.
Whether resistance to the diet is a result of the model used or age of diet introduction
is difficult to elucidate due to a lack of studies examining western diet on learning
and memory in older rats. Female data relating to the effect of western diet exposure
on spatial learning and memory are sparse, although two studies demonstrate a
negative effect of western diet on MWM performance in younger females
(3–4 months old) after 2 months of diet exposure (Molteni et al. 2002, 2004).
Furthermore, diets high in sugar and fat also impact neuropathology: a HFD coupled
with liquid sugar (mimicking soft drink consumption) for 14 weeks led to insulin
resistance and increased inflammation, in addition to displaying biochemical
changes associated with βA deposition and neurofibrillary tangle formation and
decreased synaptic plasticity in adult male mice (Kothari et al. 2017). Effects of
diet on these parameters in females have mainly been studied in models of AD,
where factors associated with amyloid pathology were more pronounced in female
mice, although the diet did not further exacerbate performance in the MWM after
3 months on the diet (Nam et al. 2017). However, in obese wildtype mice fed a HFD
for 28 weeks, only males display impaired hippocampal synaptic plasticity (long-
term potentiation) (Hwang et al. 2010). More studies in females are required to
investigate the impact of these diets on neural and electrophysiology mechanisms in
normal young and aging females. Future studies should, therefore, evaluate the role
of sex and hormonal status on the effects of western diet on learning and memory at
different ages, including at midlife and in older age groups.
5.2.2 High-Fat Diet (HFD)
Effects of diet on learning and memory are not restricted to combined high-fat and
sugar intake, as diets containing high amounts of either component alone also affect
cognition. For instance, midlife HFD is associated with a reduction in global
cognitive function and prospective memory and increases the risk of mild cognitive
impairment; an effect which was stronger in human females (Eskelinen et al. 2008;
Simpson 2000). HFD intake is also associated with impaired learning and memory in
younger human females (25–45 years old), as high intake of fat is associated with
impaired performance in tests measuring medial-temporal lobe function (Gibson
et al. 2013). The effect of high-fat intake on cognition is, however, strongly linked to
fat type, where high intakes of saturated and trans fat at midlife are detrimental to
cognitive health, and intake of polyunsaturated fatty acids (PUFAs) at midlife
reduces the risk of impaired cognitive function in studies including human males
and females (Kalmijn et al. 2004), and in females alone, later in life (Devore et al.
2009). However, intake of HFD at older ages is linked to impairments only in human
males (Elias et al. 2003; Naqvi et al. 2011; D’Amico et al. 2020), which is in line
with data on cognitive function in obesity by sex in humans, as reported above.
These findings are suggestive of a critical window for females, with HFD exposure
and increased body weight having a more detrimental effect at midlife, presumably
during the menopausal transition, but not later in life, significantly past menopause,
where increased adiposity and HFD may in fact be protective against cognitive
decline. In mice and rats, HFD has more detrimental metabolic effects in younger
males, with obesity and glucose dysregulation manifesting only in males in some
studies (Underwood and Thompson 2016). In aged mice, both sexes presented with
obesity after HFD exposure whereas only males exhibited increased ectopic fat
accumulation, hyperglycemia, and hyperinsulinemia (Barron et al. 2013), suggesting
again a possible protective component in females at advanced age. Similarly, long-
term HFD exposure (in rats aged 3 weeks to 9–12 months) led to obesity in both
males and females, but to larger degree in males, coupled with hyperinsulinemia and
hypercholesterolemia, hyperglycemia and hypertriglyceridemia that only manifested
in males (Hwang et al. 2010).
The majority of studies have found a negative impact of HFD on learning and
memory in males, using several tests of cognition and diet exposure lengths (for
reviews, see Kanoski and Davidson 2011; Cordner and Tamashiro 2015). However,
the effects of a HFD on cognition in females are unclear. For instance, HFD
exposure in mice led to poorer learning performance (hippocampus-dependent) in
males, but not in females, at middle age (up to 12 months), in the contextual fear-
conditioning and step-down passive avoidance tasks, compared to males and
females fed a control diet (Hwang et al. 2010). However, another study reported
an effect of HFD to be detrimental to performance in mice of both sexes using the
Y-maze (Barron et al. 2013). Furthermore, both age (18-month vs 4-month-old) and
diet (HFD vs standard diet) impaired cognition in the MWM independently in
female rats (Xu et al. 2019) but unfortunately, HFD exposure was not compared to
the intake of standard diet at old age, making it difficult to assess the effect of diet in
aged females. A study conducted in two different strains of middle age female mice
demonstrated differences in metabolic and cognitive impairments, where only Swiss
Webster mice, and not C57 mice displayed impairments in learning and memory in
the Barnes maze (Anderson et al. 2014), demonstrating the importance of strain
choice in study design. Cognitive impairment due to HFD intake may, however, be
rescued by a brief period of dietary fat reduction (1-month low-fat diet after 5 months
HFD exposure) in female mice (males not tested, Johnson et al. 2016). Furthermore,
data suggests that effects of HFD on cognitive impairments are accelerated after the
abrupt reduction in ovarian hormones following ovariectomy, including estrogens,
in rodents (Pratchayasakul et al. 2015). This suggests that a HFD may be particularly
detrimental to cognitive health in females at estropause. In summary, while HFD
may be more detrimental metabolically in males, the majority of the current literature
suggests that this diet affects hippocampus-dependent cognition in both sexes and
may be especially detrimental to cognitive health in females at midlife.
5.2.3 High Sugar Diet (HSD)
Diets high in sugars increase the risk of dementia (Stephan et al. 2010), potentially
by promoting insulin resistance (Yoshida et al. 2007; Bremer et al. 2010; Dekker
et al. 2010) and type 2 diabetes (Montonen et al. 2007; Malik et al. 2010), although
HSD may be detrimental to cognition even in absence of diabetes (Ye et al. 2011).
The risk of cognitive decline due to HSD intake may be especially high in females, at
least after middle age (Nanri et al. 2010; Eshak et al. 2013). Pre-menopause, females
have enhanced insulin sensitivity compared to age-matched males, and lower inci-
dence of type 2 diabetes (Yki-Jarvinen 1984; Park et al. 2003); a sex difference that
is shifted postmenopause (or after ovariectomy) (Sites et al. 2002; Carr 2003). In
rodents, ovariectomy results in insulin resistance (Campbell and Febbraio 2002), an
effect which can be counteracted by administration of 17β-estradiol (Stubbins et al.
2012), indicating that 17β-estradiol has a beneficial effect on glucose regulation.
Progesterone also increases blood glucose regulation by stimulating gluconeogene-
sis in female (Masuyama and Hiramatsu 2011) and male mice (Lee et al. 2020), and
may exacerbate hyperglycemia in diabetic subjects. However, progesterone also
stimulates insulin secretion in the pancreas (Costrini and Kalkhoff 1971; Ashby et al.
1978).
Estrogens alter the effects of HSD on memory, as sucrose exposure (for
14–28 days) impairs spatial memory in rats in both sexes, although in females this
effect was only present in the estrous phase, characterized by low circulating
estrogens (metestrus), but not in the proestrous phase, characterized by high circu-
lating estrogens (Abbott et al. 2016). Furthermore, rats of both sexes were impaired
in the MWM and passive avoidance task after 12 weeks on a diet containing 30%
fructose (Espinosa-Garcia et al. 2020). However, females demonstrated less cogni-
tive impairment in these tasks in response to diet compared to males. Whether this
difference is due to a general difference in diet-induced cognitive impairment
between sexes or due to combined analysis of females in various cycle phases is
unknown, as cycle phase was not examined in that study. However, together, these
data suggest that HSD’s effects on cognition are influenced by circulating estrogen
levels in females, with detrimental effects when estrogens are low.
In contrary to long-term effects of diets high in glucose, acute glucose exposure
improves memory and learning in both sexes in humans (Lapp 1981; Gonder-
Frederick et al. 1987; Scholey et al. 2009; Stollery and Christian 2015, 2016),
and in male rodents (Messier and White 1984; Gold 1986; Jurdak et al. 2008; Jurdak
and Kanarek 2009; Magnusson et al. 2015), in several reward-associated and
hippocampus-mediated tasks. However, intriguingly, these effects are more promi-
nent in older subjects (Hall et al. 1989; Manning et al. 1992; Parsons and Gold 1992;
Messier et al. 2003; Mantantzis et al. 2018). Memory was improved after peripheral
glucose injection in male rats, compared to saline, suggesting that post-ingestive
processes, and not necessarily the sweet taste of glucose, are required for memory
improvement (Messier and White 1987). This is further strengthened by data in male
rats showing that consumption of the non-nutritive sweetener saccharin, which
shares a similar sweet and pleasurable taste to glucose but does not result in a
subsequent rise in blood glucose post ingestion, does not improve memory (Messier
and White 1984). Effects of glucose on memory may, however, mostly be beneficial
if compared to individuals or rodent subjects who are fasted or who have reduced or
impaired glucoregulatory functions (Martin and Benton 1999; Messier et al. 2003).
To the best of our knowledge, there are no data on the beneficial effects of acute
sugar exposure on memory and learning in females at this time. In conclusion, sugar
can act to either improve or impair memory depending on the duration of exposure,
and a HSD is more detrimental to cognition in males than in females depending on
age and circulating estrogen levels.
5.3 Body Weight, Diet Intake, and Alzheimer’s Disease
In humans, there is a strong association between obesity, central adiposity, and AD

risk (Kivipelto et al. 2005; Whitmer et al. 2007; Luchsinger et al. 2012; Albanese
et al. 2017), especially in females (Gustafson et al. 2003; Hayden et al. 2006; Ma
et al. 2020). Females, but not males, who are overweight or obese show disruptions
to the blood brain barrier (Gustafson et al. 2007) and increased inflammation
(Ahonen et al. 2012), both of which are commonly described in AD. Certain types
of diet exposure can impact AD neuropathological markers even in absence of body
weight gain; for instance, in a transgenic mouse model of AD, although HFD-fed
females were spared from metabolic impairments that affected males, behavioral
performance was similarly impaired in both sexes and they demonstrated similar
levels of hippocampal β-amyloid protein accumulation of (Barron et al. 2013). As
discussed in an early section, in addition to advancing age and female sex, APOEɛ4
carrier status is one of the greatest genetic risk factors for sporadic late-onset AD
(Serrano-Pozo et al. 2021), and diet and sex modulate the risk of carriers of this
allele, effects of which will be discussed below.
Although APOEɛ4 allele possession increases AD risk in both sexes, the risk is
even greater in females (Koran et al. 2017). Furthermore, APOE allele carrier status
also impacts metabolic health. APOEɛ4 carriers have an increased risk of cardio-
vascular disease (El-Lebedy et al. 2016), which may not be a surprise due to the role
of the APOE protein in peripheral and central lipid metabolism. Although APOEɛ4
carriers have lower BMI in general, they are at greater risk of metabolic and
cognitive disturbances throughout aging, and in AD development (Volcik et al.
2006; Tejedor et al. 2014; Emrani et al. 2020). In addition, midlife carriers of one
or two APOEɛ4 alleles with a higher waist-to-hip ratio were associated with reduced
medial-temporal lobe and attention function (Zade et al. 2013), but unfortunately
sex-specific effects were not assessed. However, the findings are not consistent
across studies as others have found that obese or overweight APOEɛ4 carriers had
a lower risk of cognitive decline (Rajan et al. 2014). Differences between studies
may be due to the lack of consideration of both age and sex, as the effects of APOEɛ4
on metabolic parameters are not consistent across sexes. In male mice, rats, and
humans, APOEɛ4 carriers are more susceptible to diet-induced metabolic distur-
bances, such as increased visceral fat accumulation and glucose intolerance, than
APOEɛ3 males (Elosua et al. 2003; Arbones-Mainar et al. 2008) but this is not the
case in females, who are less metabolically affected by high-fat diets and exhibit
similar metabolic effects in response to diet between genotypes (APOEɛ3 vs ɛ4)
(Jones et al. 2019). Thus, it is important for future studies to consider sex in their
analyses of body fat content and AD risk, as sex matters for APOE genotype effects.
With APOE as a central player in lipid metabolism, it may be no surprise that
APOE genotype can influence plasma cholesterol levels, and these effects have been
demonstrated to be sex specific. In diabetic individuals, APOEɛ4 human males
display increased levels of low-density lipoprotein (LDL) cholesterol (Chaaba
et al. 2008), often termed the “bad” cholesterol, whereas females display higher
LDL in combination with lower high-density lipoprotein (HDL) cholesterol, known
as “good” cholesterol (Gomez-Coronado et al. 1999). Furthermore, postmenopausal
APOEɛ4 females demonstrate an increase in LDL cholesterol that exceeds the
increase in this lipoprotein of APOEɛ4 premenopausal females and males combined
(Schaefer et al. 1994), indicating that at least regarding cholesterol, APOEɛ4 may be
especially detrimental for females with an even greater risk after menopause. This
also highlights the importance of the consideration of age and sex in APOE effects as
more detrimental effects of diet may emerge after middle life in humans and rodents.
APOE also increases the risk of type 2 diabetes and sex differences have been
reported for brain glucose utilization based on genotype (Arbones-Mainar et al.
2008; Torres-Perez et al. 2016). Female APOEɛ4 carriers require higher central
glucose metabolism than female non-carriers, whereas male APOEɛ4 carriers
require lower levels of glucose compared to their same sex non-carrier counterparts
(Jiang et al. 2020). As insufficient central glucose metabolism has recently emerged
as a candidate, or consequence, of AD pathology, sex differences in central
glucometabolic efficiency between APOE genotypes may potentially contribute to
sex differences seen in AD lifetime risk.
Diet may also affect cognition dependent on APOE genotype. In female mice,
although both APOE genotypes fed a HFD displayed impaired spatial learning and
memory, these effects were exacerbated in APOEɛ4 females (Johnson et al. 2017,
2019). Curiously though, APOEɛ4 females demonstrated lower levels of β-amyloid
pathology after exposure to a western diet than APOEɛ3 females, although APOEɛ4
females had greater levels of β-amyloid overall (Christensen and Pike 2019).
However, in males, only APOEɛ4 mice demonstrated increases in amyloid deposits,
β-amyloid burden, and glial activation fed a western diet (Moser and Pike 2017).
Thus, while APOEɛ4 carriers may be at higher risk of metabolic impairments after
unhealthy diet exposure than APOEɛ3, whether these diets further exacerbate the
role of carrier status on cognition and AD pathology is not as clear and may depend
on sex and age. Further studies investigating the role of diet on cognition and AD
development in different APOE alleles, in addition to exploring differences in
mechanisms that result in impaired learning and memory, are needed to draw any
clear conclusions, in addition to uncovering potential sex differences in these effects.
Excess body weight and high energy diet intake, including western, HFD, and
HSD, impair cognition and increase the risk of cognitive decline and dementia in
males throughout adulthood. Although female data are limited, the impact of diet on
cognitive health seems to depend largely on age, site of fat accumulation, and
reproductive status, where levels of estrogens, like 17β-estradiol, play an important
role in protecting against cognitive impairment and dementia. Midlife and the time
of menopausal transition is likely an especially critical period for overweight,
obesity, and high energy diet intake on cognition later in life. Furthermore, the
menopausal transition and postmenopause are also time points in which females are
at higher risk of body weight gain, due to the strong reduction in 17β-estradiol’s
anorexigenic effects and energy expenditure, and a switch between subcutaneous
and visceral fat pad accumulation. Hormone therapy, initiated at the time of natural
or surgical menopause, can improve cognitive decline and reduce the risk of
dementia. Furthermore, hormone therapy containing 17β-estradiol blunts postmen-
opausal weight gain (Asarian and Geary 2013). Introduction of hormone therapy at
this time may therefore help protect females from cognitive impairments caused by
menopausal body weight gain. Body weight loss at midlife, through diet and
exercise, can also improve cognition, in both males and females. At advanced age,
excessive body fat may instead be protective against cognitive impairment, at least in
females, where being underweight is seen as a greater risk factor for dementia
compared to obesity. Further studies are required to assess the effects of high energy
diets on cognition and cognitive decline, especially in females, at different ages and
across the various phases of reproductive status. In addition, future studies should
investigate potential differences in the brain mechanisms behind cognitive effects
resulting from diet- or excessive body fat-induced cognitive impairments and AD
pathology.
6 Conclusions
Sex differences are seen in certain cognitive domains, and importantly, are accom-
panied by sex differences in brain activation patterns, suggesting that males and
females leverage different brain networks during the same cognitive tasks. Research
shows that sex hormone levels and age contribute to these relationships, with a
spotlight on menopause as a period in a female’s life at which risk of cognitive,
neurodegenerative, and metabolic disorders increase. More work in this area is
critical to furthering our understanding about sex differences in cognitive health.
Cognitive aging is the decline in some cognitive abilities, including processing
speed, episodic memory, and executive function, experienced by people as they age.
The trajectory of age-related cognitive decline varies by cognitive domain and sex.
For example, human females typically have higher baseline performance in global
cognition, and executive function, but show faster declines with age, compared to
human males. In verbal memory, human females maintained better memory perfor-
mance across aging, compared to human males. These sex differences have impli-
cations for the diagnosis, manifestation, and possibly treatment for disorders that
involve cognitive impairments, such as AD.
This chapter also explores education and diet as modifiable risk factors of
cognitive decline with aging. Not surprisingly, factors like age, reproductive status,
and hormonal levels play critical roles in the relationships between diet, cognitive
health, cognitive aging, and AD. Although not a focus in this chapter, other lifestyle
factors like exercise, stress, and social determinants of health also play significant
roles in cognition and cognitive aging.
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Part III
Diseases of the Brain and Relevance
of Sex
Sex Differences in the Long-Term
Consequences of Stroke
Courtney E. Stewart, Taylor E. Branyan, Dayalan Sampath,

and Farida Sohrabji
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
2 Post-Stroke Mobility and Muscle Weakness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
3 Post-Stroke Memory and Cognitive Deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
3.1 Stroke and Dementia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
4 Post-Stroke Mental Health and Mood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
4.1 Quality of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
4.2 Depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
4.3 Personality Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
4.4 Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
5 Preclinical Models of Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Abstract Stroke is the fifth leading cause of death and as healthcare intervention
improves, the number of stroke survivors has also increased. Furthermore, there
exists a subgroup of younger adults, who suffer stroke and survive. Given the overall
improved survival rate, bettering our understanding of long-term stroke outcomes is
critical. In this review we will explore the causes and challenges of known long-term
consequences of stroke and if present, their corresponding sex differences in both old
and young survivors. We have separated these long-term post-stroke consequences
into three categories: mobility and muscle weakness, memory and cognitive deficits,
and mental health and mood. Lastly, we discuss the potential of common preclinical
C. E. Stewart and D. Sampath

Women’s Health in Neuroscience Program, Neuroscience and Experimental Therapeutics,
Texas A&M Health Science Center College of Medicine, Bryan, TX, USA
T. E. Branyan and F. Sohrabji (*)
Women’s Health in Neuroscience Program, Neuroscience and Experimental Therapeutics,
Texas A&M Health Science Center College of Medicine, Bryan, TX, USA
Texas A&M Institute for Neuroscience, College Station, TX, USA
e-mail: f-sohrabji@tamu.edu
https://doi.org/10.1007/7854_2022_311
288 C. E. Stewart et al.
stroke models to contribute to our understanding of long-term outcomes following

stroke.
Keywords Post-stroke addiction · Post-stroke cognition · Post-stroke mental

health · Post-stroke mobility
1 Introduction
As of 2018, stroke is the fifth leading cause of death and accounts for 1 out of every
19 deaths in the USA (Virani et al. 2021). Annually, 60% of strokes occur in people
under 70 years of age, while 8% of all strokes occur in people under 44 years of age
(Lindsay et al. 2019). Fifty-two percent of all strokes occur in men whereas 48%
occur in women (Lindsay et al. 2019). Although the percentage of stroke occurrence
is similar between sexes, stroke-related comorbidity, etiology, incidence, preva-
lence, long-term functional outcomes, and overall quality of life have all been
shown to be sex-dependent (Branyan et al. for review (Branyan and Sohrabji
2020)). Additionally, the number of stroke deaths per year has increased by 10.2%
from 2008 to 2018 (Lindsay et al. 2019). This increase could be a result of worsened
comorbidities such as cardiovascular and metabolic syndrome both of which are sex
and/or age dependent and have become a global burden for all (Roth et al. 2020;
Saklayen 2018). For example, hypertension, which is defined as a blood pressure of
140/90 mmHg, is both age and sex dependent. Specifically, younger men exhibit
hypertension more often than age-matched women, which may partially account for
the heightened risk of stroke in this group. Contrarily, when women age the sex
difference in hypertension is inversed, with women between 65 and 74 years of age
presenting with hypertension more often than age-matched males; coincidentally,
women within this group also experience more strokes (Benjamin et al. 2017).
Approximately 80 million stroke patients are currently living (49% male and 51%
female) (Lindsay et al. 2019), though these survivors often experience significantly
worsened quality of life. These survivors experience physical changes, such as
muscle weakness (Carin-Levy et al. 2006; English et al. 2010; Pantano et al. 1995)
and balance issues (Hugues et al. 2019; Lund et al. 2018), sensory changes, such as
loss in vision (Pula and Yuen 2017), taste, or smell (Moo and Wityk 1999), multiple
changes in mood (Carota and Bogousslavsky 2012; Lees et al. 2012a), language
processing and communication (Li and Zhang 2017), and memory and cognition
deficits (Kathner-Schaffert et al. 2019; Khlif et al. 2021; Lammers et al. 2021) which
together can lead to prolonged social challenges and depression (Norlander et al.
2021; Sensenbrenner et al. 2020; Tse et al. 2020; Viscogliosi et al. 2011). The World
Stroke Organization reported that 63% and 18% of healthy life is lost due to death
and disability in people under the age of 70 and 44 years, respectively. When
analyzed by sex, 56% of men and 44% of women experience a reduction in quality
of life after stroke (Lindsay et al. 2019). Therefore, more work is needed to
Sex Differences in the Long-Term Consequences of Stroke 289
thoroughly understand the long-term consequences of stroke to improve their quality

of life.
This review will utilize data from studies which include patients who have
suffered an ischemic stroke, hemorrhagic stroke, “mini strokes” referred to here as
transient ischemic attack (TIA) and/or have cerebral amyloid angiopathy (CAA)
which is a vascular disorder of the leptomeningeal vessels resulting in the deposition
of the apolipoproteins, progressing to hemorrhage and associated clotting of in the
vessel walls (Yamada 2015). Ischemic stroke accounts for nearly 70% of all strokes
per year (Lindsay et al. 2019), 52% of which are men and 48% of which are female.
Hemorrhagic stroke accounts for 30% of all strokes per year, 53% of which are men
and 47% of which are female (Lindsay et al. 2019). As of 2017, 1.9/1,000 people
suffer transient ischemic attacks per year (Lioutas et al. 2021), and it has been
reported that men have a higher incidence (101 cases/100,000 population) than
women (70 cases/100,000 population) but more recent incidence reports have not
corroborated this sex difference (Lioutas et al. 2021; Bots et al. 1997). Furthermore,
29.5% of those with reported TIA have a major stroke (Lioutas et al. 2021). The
number of CAA cases per year is hard to define as it is obtained postmortem, but
based on a study examining 400 autopsies CAA occurred in 18.3% of men and 28%
of women between 40 and 90 years of age (Masuda et al. 1988). Additionally, CAA
accounts for 15% of all age-related intracranial hemorrhages (Revesz et al. 2002)
and, therefore, unless the literature discussed within this review specifically analyzes
hemorrhagic stroke and CAA separately, CAA will be included as part of the
hemorrhagic stroke subtype.
While patients under 44 years of age make up a comparatively small proportion
(~7%) of those who suffer strokes, this demographic is critical to consider when
assessing long-term outcomes of stroke, as they will live with these consequences
significantly longer than will older stroke survivors (Maaijwee et al. 2014). Second-
ary methods of stroke prevention are often prescribed for the remainder of the
patient’s life and are dependent on each patient’s lifestyle and known risks present
which likely contributed toward the initial stroke etiology. These preventatives often
include antiplatelet drugs, statins, and hypertension medications which act to
improve overall metabolic health and lower stroke risk (Maaijwee et al. 2014).
However even with secondary preventatives, young adults who have suffered an
ischemic stroke will exhibit an increase in all-cause mortality, with the 5-year
mortality rate being 9–11% (Putaala et al. 2009), the 10-year mortality rate increas-
ing to 12–17% (Kappelle et al. 1994), and the 20-year mortality rate being reported
as approximately 27% (Rutten-Jacobs et al. 2013), which is four times higher than
age- and sex-matched individuals (Maaijwee et al. 2014). The rate of stroke recur-
rence in the first year within this group was about 3.6%, which dropped to 1.7% in
the following years (Varona et al. 2004). The cumulative risk for stroke recurrence in
these patients is 17%, while the cumulative risk for other adverse cardiovascular
events is 28% (Rutten-Jacobs et al. 2013). Within the subgroup that survived 30 days
yet did not survive 5 years after their initial stroke, the most common cause of death
was vascular disease (not including stroke), recurrent stroke, and infections (36%,
21%, and 9% of deaths, respectively) (Putaala et al. 2009). Factors that predicted
incidence of 5-year mortality in these young stroke survivors included malignancy,

heart failure, heavy drinking, prior infection, type 1 diabetes, increasing age, and
large artery atherosclerosis responsible for the initial stroke (Putaala et al. 2009).
Among 30-day survivors, 20-year mortality was higher in men than in women
(33.7% vs. 19.8%) (Rutten-Jacobs et al. 2013).
This review will explore the causes and challenges of known long-term conse-
quences of stroke, separated into three categories: mobility and muscle weakness,
memory and cognitive deficits, and mental health and mood (“the 3 M’s,” Fig. 1). In
each category, sex differences in old and young survivors are discussed below and
summarized in Tables 1 and 2.
2 Post-Stroke Mobility and Muscle Weakness
Stroke survivors, if they can stand and walk, often report prolonged mobility
impairments such as slower gait, shorter step length, and wider step width when
walking (Chatterjee et al. 2018). Consequently, this reduction in mobility, specifi-
cally lower extremity impairments, can lead to poor or worsening cardiovascular
health with the increase in sedentary behavior which increases the risk of secondary
stroke. In fact, after stroke people are sedentary 10.9 h a day as compared to 8.2 h a
day in age- and sex-matched controls (Morton et al. 2019). Unfortunately, the
sedentary behavior persists up to one year after stroke, even if they have good
functional recovery (Morton et al. 2019). Sex differences in the correlation of
sedentary behavior and cardiovascular disease (CVD) mortality vary between stud-
ies and may vary with age. For example, men who are >60 years of age but of
normal weight, normotensive, and physically active (as per the 150-min minimum
weekly requirement suggested by the CDC) who reported prolonged periods of
combined sedentary activity (i.e., riding in a car) had a reduced risk of CVD
mortality (Warren et al. 2010). Alternatively, prolonged sedentary behavior in
women greater than an average of 78 years of age but normotensive resulted in
greater CVD mortality risk (Bellettiere et al. 2019).
Moreover, these mobility impairments and sedentary behaviors lead to muscle
weakness and muscle wasting, which may also be a sex-dependent event (Rosa-
Caldwell and Greene 2019). Sex hormones are critical for maintaining muscle
homeostasis and hormone therapy has proven to be effective in preventing
age-related muscle wasting (Anderson et al. 2017; Sipilä et al. 2013). However,
the association between stroke, muscle wasting and weakness, and the presence of
sex-dependent differences has not been explored.
Prolonged mobility impairment and reduced physical activity also correlate with
the overall quality of life. Specifically, overall quality of life improves more in those
who have greater levels of physical activity (Sheng et al. 2021). Exercise therapy is
often used in post-stroke recovery regimens for both men and women to improve
balance and overall mobility. However, these exercise programs also may improve
cognition in a sex-dependent manner (Barha et al. 2017). For instance, a group of
Fig. 1 Long-term stroke consequences

Table 1 Summary table of sex affect in stroke type and post-stroke outcome measures
Stroke types, post-stroke outcomes and factors affected by sex
Risk Is sex
factor difference
Outcome (sex) present? Reference(s)
Ischemic Sex Yes; more (Lindsay et al. 2019)
stroke prevalent in
males
Stroke Hemorrhagic Sex Yes; more (Lindsay et al. 2019)
subtype prevalent in
males
TIA Sexa Yesa; higher (Bots et al. 1997)
incidence in
males
CAA Sex Yes; more (Masuda et al. 1988)
prevalent in
women
Mortality Sex Yes; increased (Rutten-Jacobs et al. 2013)
in males
Mobility Sex Yes; more risk (Warren et al. 2010; Barha et al. 2017)
associated with
males
Muscle Sexa Yesa; women (Chatterjee et al. 2018; Anderson et al.
weakness/ possibly more 2017; Sipilä et al. 2013)
wasting at risk
Cognition/ Sex Yes; more (Barha et al. 2017; Scheyer et al. 2018;
dementia reported in Dufouil et al. 2014; Pendlebury and
females Rothwell 2009)
Mental Sex Yes; increased (Poynter et al. 2009; Andersen et al. 1995;
health/ incidence in Pompili et al. 2012; Stenager et al. 1998;
depression females Dong et al. 2020)
Mood Sex Not reported or –
well-studied
“Loss of Sex Yes; worse in (Lindsay et al. 2019)
quality life” males
a
Denotes outcomes with limited available data and/or contradictory findings
post-stroke men and women enrolled in aerobic and/or balance base exercise pro-
grams for 6 months underwent testing to analyze working memory, selective
attention and conflict resolution, and cognitive flexibility. Females showed improve-
ment in selective attention and conflict resolution tasks whereas the males did not
show a significant improvement (Khattab et al. 2020). Moreover, meta-analysis of
studies using aerobic training in rodents showed that the type of exercise and the
desire to complete said exercise had sex-dependent effects on cognition (Barha et al.
2017). For example, voluntary aerobic training improved non-spatial memory more
significantly in males than females; whereas forced aerobic training improved
hippocampal dependent learning and memory in females more than males (Barha
et al. 2017). Females had a greater increase in brain-derived neurotrophic factor
Table 2 Reference table organized by stroke outcome, general stroke information, sex differences,
and age effects in stroke
Organized by outcome, general info, sex differences and age effects
Stroke outcomes References
Incidence/mortality General (Virani et al. 2021; Ayerbe et al. 2014; Mortensen
et al. 2013, 2015)
Sex (Lindsay et al. 2019; Branyan and Sohrabji 2020;
difference Roth et al. 2020; Saklayen 2018; Benjamin et al.
2017)
Age (Lindsay et al. 2019; Branyan and Sohrabji 2020;
effects Roth et al. 2020; Saklayen 2018; Maaijwee et al.
2014; Putaala et al. 2009; Kappelle et al. 1994;
Rutten-Jacobs et al. 2013)
Post-stroke mobility and General (Carin-Levy et al. 2006; English et al. 2010;
muscle weakness Pantano et al. 1995; Hugues et al. 2019; Lund et al.
2018; Chatterjee et al. 2018; Sheng et al. 2021)
Sex (Morton et al. 2019; Warren et al. 2010; Bellettiere
difference et al. 2019; Sipilä et al. 2013)
Age (Morton et al. 2019; Sipilä et al. 2013)
effects
Post-stroke cognitive impair- General (Kathner-Schaffert et al. 2019; Khlif et al. 2021;
ment and dementia Lammers et al. 2021; Jacquin et al. 2014; Mellon
et al. 2015; Silva et al. 2015; Kokmen et al. 1996;
Khan et al. 2016; Pohjasvaara et al. 1998;
Madureira et al. 2001; Yang et al. 2006; Leys et al.
2005; Ali et al. 1996; Dewar and Dawson 1995;
Irving et al. 1997; Uchihara et al. 2004; Ishibashi
et al. 2006; Kiryk et al. 2011; Block and Schwarz
1997; Davis et al. 1986)
Sex (Khattab et al. 2020; Wang et al. 2014; Anstey et al.
difference 2021; Scheyer et al. 2018; Dufouil et al. 2014;
Pendlebury and Rothwell 2009; Podcasy and
Epperson 2016; Poynter et al. 2009; Gall et al.
2012; Bushnell and McCullough 2014; Bushnell
et al. 2014)
Age (Wang et al. 2014)
effects
Post-stroke mental health and General (Carota and Bogousslavsky 2012; Lees et al. 2012a;
mood/personality changes Turner et al. 2019; Naess et al. 2012; Das and
Rajanikant 2018; Gorelick et al. 2016; Hadidi et al.
2009; Lai et al. 2002; El Husseini et al. 2012; Gao
et al. 2017; Kim et al. 2017; Tsai et al. 2011;
Karaiskos et al. 2012; Niedermaier et al. 2004;
Paolucci 2017; Stone et al. 2004; Rabat et al. 2021;
Suñer-Soler et al. 2012, 2018; Huang et al. 2017;
Ebrahimi et al. 2018; Esse et al. 2011; Shah and
Cole 2010; Larsson et al. 2016; Osama et al. 2018;
Widar et al. 2002; Tang et al. 2013; Scuteri et al.
2020)
(continued)
Table 2 (continued)
Organized by outcome, general info, sex differences and age effects
Stroke outcomes References
Sex (Pompili et al. 2012; Stenager et al. 1998; Gaznick
difference et al. 2014; O’Donnell et al. 2013)
Age (Waje-Andreassen et al. 2013)
effects
CAA and TIA General (Yamada 2015; Revesz et al. 2002)
Sex (Masuda et al. 1988)
difference
Age (Masuda et al. 1988)
effects
Preclinical models (Shi et al. 1998; Ali et al. 1996; Ruan and Yao
2020; Panta et al. 2019; Kim et al. 2015; Chen et al.
2020; Kronenberg et al. 2014; Vahid-Ansari 2017;
Palmer et al. 2021)
(BNDF) after aerobic training when compared to males which could result in
increased neuroprotection (Barha et al. 2017; Alcantara et al. 2018). These studies
suggest that exercise programs could further be tailored based on sex (i.e., voluntary
or forced) to best improve individual cognitive benefit rather than assuming aerobic
exercise in general is enough. Moreover, anaerobic exercise could also be explored
to determine if there is greater benefit in this class which may shed light on the role of
oxygen intake in post-stroke recovery.
3 Post-Stroke Memory and Cognitive Deficits
More than half of stroke patients suffer from some level of post-stroke cognitive
impairment (PSCI) by 6 months after stroke (Jacquin et al. 2014; Mellon et al. 2015),
and ischemic stroke is a leading cause of vascular dementia and Alzheimer’s disease
(AD) (Silva et al. 2015). Moreover, patients who do not develop cognitive impair-
ment directly after stroke have a nine-fold increased risk of developing delayed
cognitive impairment (Kokmen et al. 1996).
Stroke survivors also report changes in short-term and long-term memory. Post-
stroke memory impairment is more commonly reported in older adults, women, and
African Americans (Wang et al. 2014). A recent study using immediate recall and
delayed recall memory tasks showed that memory decline is sex dependent. Specif-
ically, women 60–64 years of age had worsened memory decline when compared to
age-matched males (Anstey et al. 2021). This observed sex difference may be, in
part, explained by hypertension which was more negatively associated with memory
decline in women than men (Anstey et al. 2021). Interestingly, an increase in
memory decline in males was correlated with the presence of stroke further
supporting the idea that stroke may underlie some age-related dementias, such as
vascular dementia (VaD) and Alzheimer’s disease (AD).
Post-stroke memory deficits are not exclusive to older survivors. Cognitive
impairment affects many young stroke survivors. One year after the initial stroke,
60% of survivors are reported to have impaired cognitive performance, 50% of
survivals continue to suffer from this impairment 11 years after the initial stroke
(Maaijwee et al. 2014), and 30% develop dementia within a year (Khattab et al.
2020). Reduced cognitive performance can be devastating to a young patient’s
quality of life as this may affect their ability to return to work, remain independent,
or perform activities that they previously enjoyed. Cognitive impairment in young
stroke survivors often presents differently than in elderly stroke survivors. Elderly
patients often show impairment predominantly in frontal executive function, while
young stroke patients tend to exhibit deficits in multiple regions, including
visuoconstruction, delayed verbal memory, attention, and executive function.
Fatigue is reported in about 50% of young stroke survivors and is associated with
poor functional outcome and inability to resume pre-stroke activities (Maaijwee
et al. 2014). Only 53% of young stroke survivors were categorized as “independent”
and were able to return to work, though approximately 23% of these individuals
required adjustments to be made for them (Varona et al. 2004). This loss of
independence can lead to a change in social behaviors and mental health issues
which will be described in the next section.
3.1 Stroke and Dementia
In addition to physical disability, ischemic stroke also increases the risk for dementia
in the long term. Stroke is a major risk factor for dementia, with 25% of stroke
survivors developing dementia, and an even larger proportion that develop cognitive
impairment (Khan et al. 2016; Pohjasvaara et al. 1998; Madureira et al. 2001; Yang
et al. 2006). Relative to non-stroke patients, there is a four-fold relative risk of
dementia among stroke patients (Kokmen et al. 1996). Neurodegeneration in the
months and years following stroke may also contribute to dementia in this group
such that the incidence of new onset dementia after stroke increases from 7% after
1 year to 48% after 25 years (Leys et al. 2005). Furthermore, hippocampal dysfunc-
tion is one of the earliest hallmarks of dementia, and its direct connection to the
striatum (via nucleus accumbens projections) suggests a possible pathological path-
way for disease progression after stroke.
The two most common subtypes of dementia are Alzheimer’s disease (AD) and
vascular dementia (VaD). AD is characterized by the presence of β-amyloid plaques,
neurofibrillary (tau) tangles, and progressive neurodegeneration. VaD, however, is
directly linked to the occurrence of a stroke in a brain region critical for cognitive
function. Female sex is a non-modifiable risk factor for having a stroke and devel-
oping AD (Benjamin et al. 2017; Scheyer et al. 2018; Dufouil et al. 2014;
Pendlebury and Rothwell 2009); however, males are at higher risk for developing
VaD (Podcasy and Epperson 2016). Despite the higher lifetime risk of stroke in
women, worldwide stroke prevalence is greater in men, and strokes also tend to
occur at a younger age in men (Appelros et al. 2009), likely contributing to the
higher risk of VaD in men. Men are also more likely to have several significant risk
factors for stroke, such as atrial fibrillation and diabetes, though women seem to be
more at risk for developing dementia when they suffer these comorbidities (Dufouil
et al. 2014), possibly due to underdiagnosis or undertreatment of these conditions.
Moreover, a recent meta-analyses of 47 studies showed that women are more likely
than men to suffer from post-stroke depression (Poynter et al. 2009), which is
another significant risk factor for dementia. Finally, dementia prior to a stroke is
more common in women, and the occurrence of the stroke may worse existing
dementia pathology (Pendlebury and Rothwell 2009).
There is some evidence to suggest that stroke may directly influence the devel-
opment of AD pathology rather than the association being merely a correlation due
to shared risk factors and at-risk target demographics. Firstly, it has been shown that
ovariectomized rats exhibit an increase in the expression of amyloid precursor
protein mRNA after middle cerebral artery occlusion, and this effect is attenuated
by estrogen treatment (Shi et al. 1998). Moreover, astrocytic apolipoprotein E
mRNA, a gene localized to pathologic β-amyloid plaques and tau tangles, is
significantly upregulated in the CA1 region of the hippocampus at 7 days post-
ischemia (Ali et al. 1996), suggesting that stroke results in activated astrocytes that
directly contribute to AD pathology. Ischemia has also been shown to increase the
expression and activity of β-secretase, an enzyme critical to the conversion of
amyloid precursor protein to pathological β-amyloid (Wen et al. 2004). Tau protein
expression is increased in astrocytes, oligodendrocytes, and microglia following
ischemia (Dewar and Dawson 1995; Irving et al. 1997; Uchihara et al. 2004).
Increased deposition of amyloidogenic proteins and neuronal expression of apoli-
poprotein E is also increased after stroke in human brains (Jendroska et al. 1995; Qi
et al. 2007). Beyond pathology, significant behavioral changes associated with AD
have been observed after stroke. Ischemic stroke can lead to long-lasting spatial
memory impairment, deficits in reference and working memory, increased anxiety,
and locomotor hyperactivity, which are characteristic of AD (Ishibashi et al. 2006;
Kiryk et al. 2011; Block and Schwarz 1997; Davis et al. 1986). Overall, these studies
provide significant evidence that ischemic stroke may result in primary
neurodegeneration that triggers amyloidogenic and tau cascades responsible for
AD-related dementia.
4 Post-Stroke Mental Health and Mood
4.1 Quality of Life
Many people who have had a stroke or a TIA experience a persistent reduction in
quality of life and may have trouble returning to work or their usual daily activities
(Turner et al. 2019). Survivors may also struggle with family relationships and
normal social activities (Turner et al. 2019). Conditions that contribute to the
lower quality of life include post-stroke depression, fatigue, and chronic pain
(Naess et al. 2012). Lowered quality of life is a significant predictor of mortality in
stroke patients (Naess et al. 2012). Unfortunately, treating the causes of low quality
of life has proven to be extremely difficult, as traditional anti-depressants or anal-
gesics have not been proven to adequately treat these conditions.
4.2 Depression
Depression, anxiety, and problems regulating mood and emotions are commonly
reported long-term symptoms of stroke (Turner et al. 2019). Depression is the most
commonly reported post-stroke psychiatric disorder, with approximately one-third
of those who experience a stroke reporting depressive symptoms (Das and
Rajanikant 2018; Gorelick et al. 2016). Depression negatively affects functional
recovery and quality of life in those who have suffered a stroke. It is associated with
disability, sleep disorders, cognitive impairment, increased mortality, and social
withdrawal. Risk factors for developing post-stroke depression include a history of
previous stroke, a history of depression, female sex, living alone, and social distress
pre-stroke (Andersen et al. 1995). Women were at greater risk than men of dying due
to suicide after a stroke (Pompili et al. 2012; Stenager et al. 1998). Greater stroke
severity was associated with persistence of depressive symptoms beyond the acute
phase of stroke, which could be linked to the patient’s inability to perform basic
activities or daily living (BADL) and instrumental activities of daily living (IADL)
(Hadidi et al. 2009). Tasks that are considered BADL include walking, eating,
dressing and grooming oneself, using the restroom, bathing, and being able to
move from one body position to another. Tasks that are considered IADL involve
more complex thinking and organizational skill and include managing finances,
managing transportation, shopping and meal preparation, house maintenance and
cleaning, managing communication, and managing medications. Depressed stroke
patients were less able to accomplish BADL and IADL compared to non-depressed
stroke patients (Lai et al. 2002). Despite the major obstacles that post-stroke depres-
sion can pose to recovery, many clinical stroke studies do not use measures of
depression as a post-stroke outcome measure, instead favoring more physical symp-
toms such as long-term disability or mortality. An analysis of current stroke studies
showed that only 6% of studies that involve human stroke survivors contained a
cognitive or mood assessment scale (Lees et al. 2012a). Interestingly, various
aphasias can also contribute to depression (Kauhanen et al. 2000; Laures-Gore
et al. 2017, 2020) and the incidence of aphasia is reportedly sex-dependent
(Wallentin 2018). Specifically, males are more likely to express Wernicke’s aphasia
(i.e., difficulty in comprehension of speech) but females are more likely to express
Broca’s aphasia (i.e., difficulty in articulation (Kimura 1983, 1992; Sundet 2008).
However, sex alone does not explain the difference in rates of post-stroke aphasia
but appears to be caused by age and/or lateralization effects post-stroke (Wallentin

2018; Kimura 1992; Sundet 2008).
Many of those who suffer from post-stroke depression are not adequately treated
using anti-depressants. In patients that were are diagnosed with persistent depression
after a stroke or TIA, a small proportion (18.5% and 4.5%, respectively) were using
anti-depressants at 12 months after the cardiac event to treat these symptoms
(El Husseini et al. 2012). Several selective serotonin reuptake inhibitors (SSRIs)
have been evaluated in the context of post-stroke depression. A clinical trial dem-
onstrated that citalopram demonstrated higher efficacy compared to cognitive behav-
ioral therapy, both starting 6 months post-stroke, although these patients reported a
high incidence of adverse side effects such as increased fatigue, nausea/vomiting,
and palpitation/tachycardia during treatment. However, citalopram did not show
such improvement when the therapeutic intervention began at 9 months post-stroke
(Gao et al. 2017). Escitalopram has not shown efficacy in reducing moderate or
severe post-stroke depression in the acute phase in a randomized, multicenter,
placebo-controlled, double-blind trial (Kim et al. 2017). In addition to the meager
data that supports the use of SSRIs to treat post-stroke depression, there is some
controversy as to whether these drugs are safe to use in this context. One study
demonstrated that SSRI treatment was associated with greater mortality at 3 months
post-stroke (Ayerbe et al. 2014); however, another study produced opposing results,
as they showed that SSRI treatment reduced mortality 30 days post-stroke
(Mortensen et al. 2015). Moreover, a study with a mean follow-up of >3 years
showed that SSRI treatment reduced the risk of new cardiovascular events yet was
also associated with an increase in all-cause mortality, which was speculated to be
due to an increased risk of bleeding (Mortensen et al. 2013). A few studies evalu-
ating serotonin and noradrenergic reuptake inhibitors (SNRIs) have shown improve-
ment in treating post-stroke depression. Milnacipran treatment was shown to reduce
the incidence of depression through 1-year post-stroke (Tsai et al. 2011). Moreover,
patients treated with duloxetine showed greater and faster improvement on depres-
sive and anxiety symptoms compared to sertraline and citalopram (two commonly
prescribed SSRIs) (Karaiskos et al. 2012). While this data seems more promising
than trials involving SSRIs, the limited number of trials using SNRIs to treat post-
stroke depression means that the efficacy of these drugs in this context is not fully
known. Finally, mirtazapine, a tetracyclic antidepressant, showed promise in
preventing and also treating post-stroke depression, yet side effects of this treatment
include weight gain and sedation (Niedermaier et al. 2004; Paolucci 2017).
Furthermore, post-stroke depression is not exclusive to older stroke survivors.
Young stroke survivors often exhibit significantly greater levels of anxiety and
depression. Anxiety is reported in 19% of young stroke survivors 12 years after
the initial stroke (Waje-Andreassen et al. 2013). Additionally, symptoms of depres-
sion were present in 28–46% of young stroke survivors, assessed 6–12 years after
the initial stroke (Waje-Andreassen et al. 2013). These individuals were also at
increased risk of suicide and suicidal ideation, and these symptoms may be exacer-
bated by previous mood disorders (Stenager et al. 1998; Santos et al. 2012).
4.3 Personality Changes
Personality changes in stroke survivors are commonly reported by caretakers.

Caretakers most often report reduced patience, increased frustration, more dissatis-
faction, and reduced confidence in stroke survivors (Stone et al. 2004). Personality
changes may be associated with anxiety or depression in the patient or in an
emotional disorder in the caretaker (Stone et al. 2004). Frequently self-reported
mood problems include increased emotionalism, anger, mood swings, frustration,
irritation, lack of empathy, and loss of confidence (Turner et al. 2019). Addictive
behavior increase and complete cessation can also occur. For example, in a hospital-
based study examining substance use in “first-ever-non-severe” stroke patients with
post-stroke depression (PSD) showed that nearly 30% of patients had problematic
substance use (nicotine, alcohol, and/or cannabis) with approximately 9% of that
total being polydrug users. Moreover, within that group 51% reported they had PSD
and 30% would even describe the PSD as severe (Rabat et al. 2021). Interestingly,
addictive behavior changes may largely depend on the region damaged during the
stroke. Post-stroke damage to the insular cortex revealed how this region may play a
role in addiction. Approximately, 40% of those who suffer an insular cortical stroke
stopped smoking within a year (Suñer-Soler et al. 2012). Sex differences in cessation
rates were also observed if the insular stroke lesion occurred in the left hemisphere
with males having significantly higher cessation rates than females and if the lesion
was present within the right hemisphere but no sex difference was present (Gaznick
et al. 2014). This supports the hypothesis that the structural connectome of the brain
is sex-dependent (Hametner et al. 2015). Understanding the location of the stroke
lesion is being used to better tailor treatment of long-term outcomes but may be less
sex-dependent but more so dependent upon which hemisphere is affected (i.e.,
dominant vs non-dominant) which varies by person (Gao et al. 2021; Ito et al. 2008).
However, more recent long-term studies showed that after 6 years changes in
tobacco use or the absence of use was not influenced by the presence of an acute
insular lesion indicating the substance use changes may be transiently present after
stroke (Suñer-Soler et al. 2018). In addition to nicotine use, opium consumption and
overall route of opium entry was also shown to change in response to insular strokes
and this observation was also shown to be more likely in younger patients
(Yousefzadeh-Fard et al. 2013). Alcohol use post stroke is largely understudied
but may also depend on the location of the stroke. For example, dorsolateral striatum
stroke in male rats was shown to increase alcohol preference as early as 5 days post
stroke (Huang et al. 2017). Overall, more work is needed to better understand
substance dependence patterns post stroke as many of these can increase stroke
risk (Ebrahimi et al. 2018; Esse et al. 2011; Shah and Cole 2010; Larsson et al.
2016).
4.4 Pain
Chronic pain may affect up to 50% of stroke survivors (Naess et al. 2012), and this
pain is often not treated appropriately. Post-stroke pain can present as central,
musculoskeletal, spasticity-related pain and headache. In a 434 stroke-survivor
study, overall prevalence of post-stroke pain was 29.56%. Time course analysis
revealed that approximately 14% occurred in the acute phase, 43% occurred in the
subacute phase, and 32% occurred in the chronic phase (Osama et al. 2018). One
study showed that two-thirds of those suffering with chronic pain post-stroke did not
receive adequate pain management with one-third of the patients not receiving any
prescription for pain management at all (Widar et al. 2002). Patients suffering from
post-stroke pain are more likely to experience greater cognitive and functional
impairment (O’Donnell et al. 2013) and may also be at an increased risk of suicide
(Tang et al. 2013). Women are at a higher risk of developing post-stroke pain, as are
those with a history of depression, type 2 diabetes, and hyperlipidemia (O’Donnell
et al. 2013). Anti-depressants, such as amitriptyline and duloxetine, and α2γ calcium
channel blockers, such as gabapentin and pregabalin, are recommended first-line
therapeutics for post-stroke pain, even though there is limited evidence on the
efficacy of these drugs in treating central neuropathic pain (Mulla et al. 2015).
Opioids are extremely effective analgesics; however, a meta-analysis evaluating
the effect of morphine and opioid antagonist naloxone demonstrated that neither of
these drugs results in the reduction of post-stroke pain (Scuteri et al. 2020). More
work is needed to better understand the mechanisms of post-stroke pain to improve
upon and expand upon the available therapeutic options.
5 Preclinical Models of Stroke
Although this review outlined the causes and challenges of known long-term
consequences of stroke and the existence of sex differences, if known, within each
of the outcomes, it also shed light on the scarcity of preclinical studies that focus on
long-term outcomes following stroke. Preclinical studies have contributed toward
our understanding of stroke pathophysiology, but as human stroke is heterogeneous,
it can be difficult to generate a perfect stroke model. Moreover, neurobehavioral
outcomes post stroke can be difficult to assess in preclinical stroke models which are
a large component of human based long-term assessments following stroke
(Maaijwee et al. 2014; Varona et al. 2004). For example, sensorimotor tests and
cognitive function post stroke are often assessed in rodent models (Ruan and Yao
2020) but other common stroke deficits like aphasia, visual impairment, and depres-
sion are harder to reliably examine. However, many labs have begun to assess and
treat long-term depressive-like behaviors in these preclinical models (Panta et al.
2019; Kim et al. 2015; Chen et al. 2020; Kronenberg et al. 2014; Vahid-Ansari
2017). Motor deficits are the easiest and most studied outcome in rodent models, but
more work is needed to standardize and optimize assays which address memory,
cognition, and mood in the context of stroke. As in human stroke studies, neurolog-
ical scores in rodents are assessed using the Bederson and Garcia score systems
(Garcia et al. 1995; Kleinschnitz et al. 2009). Mobility and motor function is
assessed with the pole test, cylinder test, corner test, adhesive removal test, rotarod
test, and gait analysis (Ruan and Yao 2020; Lubjuhn et al. 2009). Mood is assessed
with open field or elevated-plus maze as a measure of anxiety, and sucrose prefer-
ence as a measure of depression. Cognition/memory is often assessed with Morris
water maze, y-maze (only used in hemorrhagic stroke models), novel object recog-
nition tests, and radial-arm tests (Ruan and Yao 2020; Schaar et al. 2010). However,
if locomotion is severely impaired after acute stroke the assessment may not be of
value. Long-term studies conducted after sensorimotor function returns could benefit
from a comprehensive behavioral assessment. A standardized scoring system for
these locomotor dependent assays could be developed for the changes observed at
various times throughout stroke recovery to best use these assays in both acute and
long-term analysis. Alternatively, cognitive tests that are based on touchscreen
platforms may be less dependent on proper forelimb function and readily used in
preclinical stroke models (Palmer et al. 2021).
6 Conclusion
The need for a more thorough understanding of stroke pathology and post-stroke
therapy approaches and interventions is increasing as the amount of stroke survivors
and those who suffer stroke at a younger age rise. This need may also increase as
novel COVID19 related strokes are being reported in an atypically younger age
group. Moreover, a better understanding of long-term consequences of stroke in
each sex is critical as many long-term recovery obstacles are sex-dependent. During
our literature search we found that many studies examine individual long-term
consequences of stroke, but we wrote this review with the intent of generating a
single resource for fellow researchers to use and add to the field for preclinical and
clinical researchers alike.
We explored common post-stroke issues in the known stroke subtypes and noted
if sex differences were present in the following: post-stroke mobility, muscle
weakness, memory and cognitive deficits, dementia, mental health, depression,
mood and personality changes, pain, and overall quality of life (See Table 1 for
quick reference access). We found sex differences in post-stroke mobility which
worsened mortality risk (Gall et al. 2012) and that mobility interventions should be
tailored based on sex as not all exercise post-stroke benefits each sex equally (Barha
et al. 2017; Alcantara et al. 2018). Post-stroke memory changes, dementia, and
depression were also sex-dependent and in some cases, age/sex-dependent events
with women exhibiting a poorer prognosis (Dong et al. 2020). However, these
studies do not always mention the type of memory assessment used on subjects.
Furthermore, very few studies incorporate this post-stroke outcome at all. One study
determined that as little as 6% of stroke studies use cognitive and mood assessments
as a post-stroke outcome measure (Lees et al. 2012b). More work is needed in post-
stroke memory, cognition, and mood fields and this topic needs to be discussed
openly so more researchers are inclined to include these post-stroke outcomes and
push to develop a new field standard. Furthermore, these assessments are also
needed in preclinical studies, where long-term stroke effects are often poorly studied.
Experimental studies, where the type of stroke and other factors can be controlled,
will be better able to assess whether sex is a contributing variable for long-term
consequences.
Lastly although we do not explore the context of region and cultural affect
interactions on long-term outcomes of stroke, there is some literature that suggests
culture and region do play a role in modifiable risk factors preceding the stroke
(O’Donnell et al. 2016) and stroke symptom awareness (Yuan et al. 2021). Addi-
tionally, there are regional disparities in the quality of stroke care (Seabury et al.
2017), stroke rehab outcome (Reistetter et al. 2014), and 30-day stroke mortality
(Thompson et al. 2017). Surprisingly, we found that many studies do not explicitly
state which geographically population is being studied. Furthermore, if one uses
laboratory or senior author location, we find that subtopics of interest (i.e., mobility
and muscle weakness, memory and cognitive deficits, or mental health and mood)
are studied in many locations which infers that many long-term consequences are
regionally conserved but this should be confirmed possibly by comparing meta-
studies currently available.
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Sex Differences in Dementia
Eef Hogervorst, Sophie Temple, and Emma O’Donnell
Contents
1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
1.1 The Dementias, Increasing Prevalence and Costs Worldwide . . . . . . . . . . . . . . . . . . . . . . . 310
1.2 Non Modifiable Demographic Risk Factors for Alzheimer’s Disease and Other
Dementias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2 Possible Reasons for Sex Differences in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
2.1 Loss of Sex Steroid Hormones in Middle-Aged Women as a Modifiable Risk Factor 313
2.2 Cardiovascular Age at Mortality and Sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
2.3 Cardiovascular Disease and Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
2.4 Lifestyle-Related Protective Factors: Exercise and Other Activities . . . . . . . . . . . . . . . . 322
3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Abstract
Background
Women in many cohorts have a higher risk for Alzheimer’s disease (AD), the
most common form of dementia. Sex is a biological construct whereby differences in
disease manifestation and prevalence are rooted in genetic differences between XX
and XY combinations of chromosomes. This chapter focuses specifically on
sex-driven differences in dementia, as opposed to differences driven by gender – a
social construct referring to the societal norms that influence people’s roles, relation-
ships, and positional power throughout their lifetime.
Methods
Using a narrative review, this chapter explored the characteristics and risk factors
for the dementias, alongside a discussion of sex differences including loss of sex
steroid hormones in middle-aged women, differences in the prevalence of cardio-
vascular diseases and engagement in lifestyle protective factors for dementia.
E. Hogervorst (✉), S. Temple, and E. O’Donnell

National Centre for Sports and Exercise Medicine (NCSEM), School of Sports Exercise and
Health Sciences (SSEHS), Loughborough University, Loughborough, UK
e-mail: e.hogervorst@lboro.ac.uk
https://doi.org/10.1007/7854_2022_408
Published Online: 26 January 2023
310 E. Hogervorst et al.
Results
The sex difference in AD prevalence may exist because of systematic and historic
differences in risk and protective factors for dementia, including level of education
obtained and socioeconomic status differences, which can impact on health and
dementia risk.
Levels of sex steroids decline significantly after menopause in women, whereas
this is more gradual in men with age. Animal and cell culture studies show strong
biological plausibility for sex steroids to protect the ageing brain against dementia.
Sex steroid hormone replacement therapy has in some observational studies shown
to protect against AD, but treatment studies in humans have mainly shown disap-
pointing results. Cardiovascular disease (CVD) shares midlife medical risk
(e.g. hypertension, hyperlipidaemia, obesity etc.) factors with AD and other forms
of dementia, but also with related lifestyle risk – and protective factors (e.g. exercise,
not smoking etc.). Men tend to die earlier of CVD, so fewer survive to develop AD at
an older age. Those who do survive may have healthier lifestyles and fewer risk
factors for both CVD and AD. An earlier age at menopause also confers great risk for
both without hormone treatment.
Discussion
It could be the case that the decline in sex steroids around the menopause make
women more susceptible to lifestyle-related risk factors associated with dementia
and CVD, but this remains to be further investigated. Combining hormone treatment
with lifestyle changes in midlife (e.g. exercise) could be an important preventative
treatment for dementia and CVD in later life, but this also requires further research.
Keywords Age · Cardiovascular · Dementia · Risk/protective factors · Sex · Sex

steroids
1 Background
1.1 The Dementias, Increasing Prevalence and Costs
Worldwide
People living with dementia (PWD) show significant acquired and usually progres-
sive cognitive-behavioural impairments (e.g. in thinking, learning, problem solving,
and decision making), as well as personality and emotional changes. These changes
impact on their ability to live independently and perform activities of daily life,
including social interactions and occupational functions (APA 1994; ICD-10-CM
2022). There are many types of dementia (ICD-10-CM 2022), which are
characterised by brain pathology identified in life using brain and/or cerebrospinal
fluid scans to identify biomarkers of the pathology and through neurological,
psychiatric and neuropsychological examination of the clinical presentation of
symptoms. The most common types of dementia are: Alzheimer’s disease (AD),
Sex Differences in Dementia 311
with A-beta amyloid plaques and neurofibrillary tangle pathology build-up and a
slow progressive clinically relevant cognitive decline in multiple domains, often
starting with episodic memory dysfunction (APA 1994). Dementia with Lewy
Bodies (DLB) clinically presents with fluctuating levels of attention and alertness,
progressive decline in attention, visuospatial, and executive function, visual hallu-
cinations, REM sleep disorders and Parkinsonism features, which do not occur
before the cognitive disorders become apparent (McKeith et al. 2017). Vascular
cognitive impairment (VCID) is caused by strategic or widespread cerebrovascular
lesions (APA 1994) and is clinically usually accompanied by stepwise cognitive
decline and focal signs and symptoms. Like DLB, VCID also often does not present
with episodic memory disorders initially (Hogervorst et al. 2002). The dementia
diagnosis is confirmed as ‘definite’ using post-mortem pathological evidence, but
various brain pathology (e.g. finding vascular – with AD pathology) is usually found
at that stage (Hogervorst et al. 2003). The cognitive, behavioural, and psychosocial
changes seen in dementia should not be better explained by other morbidity
(e.g. major clinical depression, nutritional deficiencies, thyroid disease etc.),
although such morbidity can cause secondary dementia, which can sometimes
(partly) reverse with treatment of the morbidity. This type of morbidity can also
co-occur independently alongside dementia pathology (see McKhann et al. 1984), or
can be a risk factor for dementia at a later stage (e.g. subclinical hyperthyroidism for
later life AD, Tang et al. 2021).
Because of high care needs and loss of dependency, dementia has huge economic
and social costs, which were calculated to be more than $1 trillion worldwide in
2018 (ADI 2017, 2018). The World Alzheimer Report (ADI 2018) estimated that
there were 50 million people worldwide living with dementia in 2018, and with
10 million incident or newly diagnosed cases every year, this could result in
82 million cases in 2030, with costs estimated to increase to $2 trillion worldwide.
Absolute numbers of dementia cases are estimated to almost double again to
152 million cases worldwide by 2050. Developing countries are estimated to be
burdened with most of the dementia cases, and their worldwide share is thought to
further increase, from 66 to 72% of all dementia cases by 2050 (ADI 2018).
However, developing countries have few resources to deal with this onslaught in
new cases presenting with dementia, in terms of finances, insurance, medical
specialists, care centres, and facilities, especially in rural areas (Hogervorst et al.
2021). There is no current long-term medical cure for dementia and prevention is the
main strategy to stem the number of new dementia cases predicted to occur over the
next decades (ADI 2018).
1.2 Non Modifiable Demographic Risk Factors

for Alzheimer’s Disease and Other Dementias
Population aging is at least partly held responsible for an increase in numbers of

people living with dementia (ADI 2018). Although the risk for dementia increases
with age, it is distinct from healthy aging due to significant brain pathology. Brain
pathology (e.g. amyloid plaques and deep white matter disease) is associated with
cognitive decline, but presence of pathology substrates does not always equate
cognitive dysfunction. For instance, some cases – with substantial post-mortem
confirmed Alzheimer’s disease pathology – were found not to have been diagnosed
with dementia in life (Hogervorst et al. 2003).
This may be related to cognitive reserve, which refers to the ability to adapt
flexibly (e.g. by using alternative strategies to cope with cognitive dysfunction, such
as searching for other synonyms from vocabulary, when confronted with word
finding difficulties and memory loss) to the loss of physiological brain reserve
capacity (brain volume, loss of neuronal connections and synapses, etc.) in the
presence of pathology. Having obtained higher levels of education in childhood
and beyond is associated with a lower dementia risk (Livingston et al. 2020). This is
possibly related to having increased brain reserve capacity, with increased neuron
interconnectivity, adaptability, and flexibility of neuronal networks, but is also likely
related to cognitive reserve, with a wider range of available and alternative coping
skills and vocabulary to overcome brain reserve loss (Stern 2012; Stern et al. 2019,
2020).
In addition, related to having better childhood education, is people often being
then able to obtain a higher socioeconomic status (SES) in later life (Hogervorst and
Clifford 2013). SES and engagement in other activities, including reading and social
interactions also later in life contribute to higher cognitive reserve (Stern et al. 2019)
and possibly brain maintenance and/or physical/psychological resilience (Stern et al.
2019, 2020). Higher SES usually means better financially rewarded occupations,
with more leisure time to engage in stress-reducing activities including sports,
holidays abroad with vitamin D exposure in winter months; lifelong learning;
knowledge of lifestyle choices to promote healthy aging and the financial ability to
pay for heat, medical care and good and varied nutrition, while living in less polluted
and safer areas, which factors can all reduce dementia risk (Hogervorst and Clifford
2013; Stern et al. 2019, 2020; Livingston et al. 2020). Higher obtained education
also includes a higher likelihood of having been born to affluent and educated
parents, who are more likely to have provided more conducive preconception –
and child developmental – environments to support optimal brain development
resilient to later life pathological impacts.
Early childhood cognitive performance and intelligence have also been found to
be related to lowered dementia risk in later life and a reduced risk of some of the
preventable AD risk factors, such as midlife hypertension (Hogervorst and Clifford
2013; Hogervorst et al. 2019, 2021) and the risk of undergoing surgical menopause
(Kuh et al. 2018). Independent of demographic factors is sex. Being of the female
sex is associated with an almost doubled risk of AD in some cohorts, particularly in

people older than 80 years of age (Launer et al. 1999). It was estimated that 2/3 of
people living with dementia are female (Subramaniapillai et al. 2021). This review
also pointed out that historical educational and occupational differences between
men and women could have systematically biased health outcomes.
Using data from various cohorts, we (Hogervorst et al. 2012) found interactions
of age, education, and health by sex on verbal episodic verbal memory performance
(free word recall), one of the first functions to decline in AD (Hogervorst et al. 2002).
In older healthy cohorts where women had obtained more education or were very
well matched, women outperformed men on verbal episodic memory function after
age 70. When men had obtained more or similar education in healthy cohorts, older
women had similar performance. In rural cohorts characterised by poor overall
health, where older women had obtained less education, their performance was
worse than that of older men after age 68/70. Even if education and health are
statistically controlled for, systematic differences between the sexes and their dif-
ferent life history and health status can still bias results. It was suggested
(Subramaniapillai et al. 2021) that cognitive reserve may have different brain system
pathways by sex. In addition, reviewing the data suggested that carrying the genetic
risk factor for AD – the APOE e4 allele – was reported to be worse for women than
men. Some studies in this review showed that having obtained less education was a
worse risk factor for AD in women, but not men. This could suggest that women’s
aging brains are more susceptible to adverse risk factors for dementia, such as having
the APOE e4 genotype or having obtained little education.
2 Possible Reasons for Sex Differences in Alzheimer’s

Disease
2.1 Loss of Sex Steroid Hormones in Middle-Aged Women

as a Modifiable Risk Factor
Apart from systematic differences in education and SES in some cohorts between
sexes, it could be the case that women lose physiological brain reserve capacity at a
faster rate than men, because after menopause women have much lower levels of sex
steroids than older men (Hogervorst 2012b). Sex steroids are known to affect almost
any of the mechanisms thought to be involved in age-related cognitive decline and
dementia and the biological plausibility for this theory substantiated by cell culture
and animal studies is very strong (Hogervorst 2012b, 2014; Barrett-Connor and
Goodman-Gruen 1999).
2.1.1 Cognitive Change Over the Menopause and Taking Hormone

Replacement Therapy
Our recent review (Hogervorst et al. 2022) found that around half of women
complain that going through menopause affects their jobs, with loss of concentration
and memory, brain fog and mood swings. Studies showing that there are some
objective and measurable changes in brain function – but mainly in the perimeno-
pausal (transitioning) stage – and found these changes negatively affect episodic
memory in around 23% of women. However, for most women, the cognitive and
mood issues reversed after the menopause (Hogervorst et al. 2022). This transient
change in brain function in some women may occur because 60–72% of women
report hot flushes and night sweats in the (peri)menopausal transition, which can
affect sleep, which can in turn affect brain function. In addition, with multiple
described effects on neuronal and systemic function, it is likely that the brain may
need to adapt to the change in endocrinological status.
Taking hormone replacement therapy (HRT) in this phase, when symptoms
significantly affect quality of life could be considered, but most women or GPs are
not sufficiently informed (Hogervorst et al. 2022). There is a small (but
overperceived) increased risk of breast cancer with combined – but not estrogen
only – treatment. This risk is comparable to drinking two or more units of alcohol per
day (Women’s Health Concern 2015). Studies suggest that with HRT this risk is
mainly seen when using combination hormone therapy for longer than 5–7 years. As
such, most guidelines suggest that HRT can be used safely for up to 5–7 years after
the menopause, if there are no contraindications, such as risk for blood clotting or
familial risk for breast – or reproductive organ cancers (Hogervorst et al. 2022).
2.1.2 Should Women Take HRT in Midlife to Reduce Risk for Dementia
in Later Life?
Although previous observational studies showed a halved risk of dementia with sex
hormone treatment (Hogervorst et al. 2000), this was not reflected in randomised
controlled treatment trials (Lethaby et al. 2008). Benefits on cognition-if found of
HRT – did not exceed more than 2–4 months of treatment and were seen to be
reversed to baseline after 1 year of treatment (Hogervorst et al. 2022). Some
researchers suggested that giving sex hormones closer to age at menopause would
benefit brain function, the so-called ‘Critical window’ or ‘Window of Opportunity’
theory (Resnick and Henderson 2002). However, several large randomised con-
trolled trials using sex steroid hormone treatment showed no long-term improve-
ments (>1 year) in cognitive function in recently menopausal women (see also
cardiovascular Sect. 2.3). In addition, the possibly transient benefit of HRT on
cognitive function was seen in small studies of highly symptomatic women with
early menopause, but also in women with dementia, again also only for 2–4 months,
which data contradict the Critical Window theory (Hogervorst et al. 2009, 2022). In
contrast, some treatment studies showed that dementia risk was increased with
combination treatment in older women (>65 years of age) and that dementia
symptoms worsened with longer term combination treatment (>1 year). This rever-
sal of benefit with longer term treatment (>1 year) was already mentioned in the 50s
(Hogervorst 2013; Hogervorst et al. 2022). These data contrast with earlier and some
recent observational studies.
2.1.3 Why Is There a Difference Between Animal, Observational

and Treatment Studies?
Given estradiol’s strong biological plausibility and data from observational studies,
it is unclear why there would be a discrepancy with randomised controlled trials,
which show very little if any effect. It may be that recall bias plays a role. One study
found that people with dementia were half as likely to accurately recall whether they
had used hormones when comparing medical prescription data with personal recall
of use (Petitti et al. 2002). Contraindication may also create bias in observational
studies because women who already have dementia are less likely to receive HRT
due to compliance issues and potential interactions between existing dementia
medications and the HRTs (Peters et al. 2021; Leblanc et al. 2001).
Another possibility explaining the discrepancy between observational and treat-
ment studies is the healthy user bias, with hormone users also engaging in other
healthy lifestyle behaviours which prevent dementia. This was shown in a follow-up
study where women 8 years before they would choose to take hormones already had
fewer risk factors for AD, including having higher levels of education, but also being
more physically active, with lower average body weight, lower systolic blood
pressure, less insulin resistance and better blood lipid profiles (Matthews et al.
1996). This healthy user mechanism may play a role as outlined in three large recent
observational studies, which had medical registry data and were not affected by
recall bias. These studies will be discussed in more detail below.
A recent US retrospective observational study (Kim et al. 2021) using insurance
claims records of 379,352 women with an average age of 67.5 years, showed that
those who had used sex hormones after menopause had a halved risk of dementia,
with natural bio-identical estradiol and progesterone having the largest beneficial
effect. However, synthetic estrogens such as Premarin and Prempro (combination)
treatment also showed benefits in reducing risk. This is important, as the synthetic
estrogen Premarin with a synthetic progesterone showed the increased risk for
dementia and a worsening of symptoms in longer term (>1 year) treatment studies
of older women (>65 years) as mentioned above. Oral use and longer duration of use
were seen to have most benefit. There was no risk reduction in women who were
between 60 and 64 years of age (but dementia prevalence is low in that age range),
but the older the women were, the more the authors suggested they seemed to benefit
from HRT. Although education was not controlled for, data suggested that
co-morbidities (which could affect dementia risk, such as diabetes, CVD etc.)
were much more prevalent in non-users, although this apparently was controlled
for using propensity score matching. However, women had been followed up for
HRT use for a maximum of 10 years, with an average of 5 years in this study. Only
5% had used HRT for 6 or more years, with the majority only using HRT for a year
This study had also included only women who had been followed from age 45, so
not those with early or premature menopause.
A Finnish nationwide registry of medical prescriptions study including 84,739
women and showed an increased risk of AD in women over 60 years of age with any
HRT and any duration >3 years. However, in women who had started HRT when
younger than 60 years of age, increased risk was only seen when using estrogen by
itself for over 10 years (with a non-significant reduced risk for shorter durations of
treatment). For combination HRT, this risk increase was seen to occur after 5 years
of use (Savolainen-Peltonen et al. 2019). Women with AD had been matched for age
and district. The authors stated (using other studies) that education and SES were not
different for Finnish HRT users vs. non-users, as were cardiovascular disease (CVD)
risk factors (hypertension, hypercholesterolaemia, and smoking). However, CVD
(but not AD) mortality was reduced in Finnish HRT users.
Vinogradova et al.’s (2021) menopausal hormone therapy and risk of dementia
study of 16,291 cases and 68,726 age – and practice – matched controls using two
primary care databases did control for the CVD risk factors (and also included body
mass index or BMI), but also lifestyle (smoking, alcohol) and mental health (anxiety
and depression) variables. They reported no overall effect of HRT on dementia in
analyses adjusted for these confounds. In adjusted analyses, however, there was
8–12% dementia risk reduction in women taking only estradiol (the natural most
potent estrogen) for 1–5 years. These would have likely been women undergoing
surgical menopause with hysterectomy. In this large UK study, progesterone (which
is needed for women with a womb who underwent non-surgical or natural meno-
pause to prevent endometrial cancer) either mitigated the beneficial effect or
increased the risk of dementia slightly when given with estrogens for more than
5 years by 11% and when given for more than 10 years by 19%, similar to the
treatment data of older women mentioned above. Some of the synthetic progesto-
gens seemed to have a worse risk profile than others. 1 So, similar to breast cancer
risk, combination treatment up to 5 years and estradiol alone for 10 years could be
safe for negating potential negative effects on brain function and possibly help some
(e.g. highly symptomatic) women with cognitive impairments for a limited amount
of time, especially women undergoing surgical menopause who are treated with
estradiol only.
In the UK study, HRT users in general were younger and more likely to live in
more affluent areas, but also more likely to report mental health issues and to be
prescribed benzodiazepines. Combination HRT users were generally healthier, with
lower rates of CVD, stroke, diabetes, and hypertension than never users or estrogen
only users, but these factors were controlled for using multivariate regression
1
Surprisingly only dydrogesterone (a synthetic progesterone) showed a trend to decreased dementia
risk of 12% when given with an oestrogen together and taken between 1 and 11 years.
analyses. These typical CVD risk factors are found to confer increased risk for AD
and other dementias. The observed combination of deciding to use hormones during
and after menopause with long-term other healthy lifestyle behaviours and having
had better education makes these associations difficult to disentangle, even when
these are controlled for statistically.
Price et al.’s (2021) Canadian study on aging showed that women undergoing
surgical menopause had obtained less education and were more likely to be obese
and to smoke compared to women who underwent natural menopause (Price et al.
2021). This would already put them at increased risk for dementia (Livingston et al.
2020; Kivipelto et al. 2005), regardless of undergoing surgical menopause. A UK
study followed women from midlife to an average age of 69. In women undergoing
natural menopause, better verbal memory function was associated with an older age
at menopause. However, this association was not seen in women undergoing
surgical menopause, which was explained by education, among other covariates
(Kuh et al. 2018). This suggests that education or SES and its association with
surgical menopause could be a major source of bias in these studies, but it remains to
be investigated. In many countries such as the USA, HRT needs to be paid for, which
could explain why women who are better off are less affected by surgical menopause
because of their SES and associated reduced risk (see Sect. 1.2) rather than HRT use
by itself.
It may also be the case that the loss of sex steroids affects brain maintenance to
such a degree that other risk factors then have a magnified effect on reducing brain
reserve capacity. The ‘healthy cell bias’ theory (Brinton-Diaz 2005) suggests that
oestrogen will benefit healthy neurons and help with brain maintenance. However,
when neurons are undergoing pathological changes in mitochondria or in the
calcium channels, estrogens in in vitro studies were shown to accelerate neuronal
decline (Brinton-Diaz 2005). As this type of pathological change is more common in
older women and/or those with risk factors for pathology development (such as
women who have CVD risk factors), it could explain the data of older women having
a slight increased risk of dementia with prolonged hormone treatment. Progesterone
seems to worsen that effect, with possible differences with different preparations.
Earlier reviews (Hogervorst et al. 2022, see also ESHRE 2016) described several
previous prospective studies reporting that women who undergo menopause at a
younger age, especially surgical menopause which occurs after removal of the
ovaries, can have severe menopausal symptoms and significant objective memory
loss. Treatment with estradiol was shown to reverse these effects in small short-term
studies (2–4 months). Larger observational studies also showed an increased risk for
dementia in these women when undergoing early (<45 years of age) or premature
(<40 years of age) surgical menopause without HRT. For these women, EU
guidelines suggested to prescribe HRT up to the age of natural menopause (around
age 50) and to engage in healthy lifestyles to prevent increased risk for neurological
disorders, including dementia (ESHRE 2016). In general, the earlier the age at
menopause, the worse the risk of accelerated cognitive decline and dementia.
Conversely, a later age at natural menopause was seen to protect against dementia
in UK cohorts (Kuh et al. 2018). This could be related to not smoking (smokers have
an earlier age at menopause) or longer-term exposure to estrogens over the lifespan,

but these data are inconclusive. Whether cognitive ability in childhood and in later
life is a function of education, higher SES of parents, and/or subsequent better
environments or lifestyles for optimal health, both brain and ovarian reserve remain
to be untangled. Further complicating matters is the overlap between sex, CVD, and
dementia risk factors which will be discussed in more detail in the next paragraph.
2.2 Cardiovascular Age at Mortality and Sex
According to the World Health Organisation, while cardiovascular disease remains

the most common cause of mortality worldwide, there is a relative decrease by 15%
in European countries (Townsend et al. 2016). AD and other forms of dementia are
now among the top ten causes of death, reaching third place in the Americas and
Europe in 2019. Women are disproportionally affected: globally: 65% of deaths
from Alzheimer’s and other forms of dementia are women. Despite a lower increase
in numbers of dementia cases over the last decades than initially estimated, dementia
was the leading cause of death for women (and the second cause of death for men) in
2019 in the UK (ONS 2017a).2
In 2019, men were more likely to die of ischemic heart disease at an earlier age.
The gap in sex difference in heart disease mortality is reducing, however, with
relatively more women dying of heart disease, but at a later age. A closer look at
UK data shows that more men die of heart disease in midlife and before age 80, after
which mortality rates for women and men for this morbidity are almost equal and
then increase for women. Risk of dying of cerebrovascular disease, such as stroke,
has overall decreased over the years, e.g. Feigin et al. (2015). This could potentially
be due to quicker interventions with better public recognition of early symptoms.
However, for women risk of dying of stroke was higher than that for men, but again
only in women who were over 80 years of age (Appelros et al. 2009; Löfmark and
Hammarström 2007; Alabas et al. 2017). This could be the case because men are no
longer part of these statistics (because they died earlier of other causes) and/or
because men who have survived to this age are less likely to develop dementia,
heart disease or have a stroke (healthy survivor bias).
Cerebral small vessel disease (SVD) is one of the most common pathological
underpinnings for cognitive impairment and VCD. SVD is observed on T2-weighted
MRI scans as focal lacunar infarcts, micro-haemorrhages and diffuse white matter
hyperintensities (WMH). One hypothesised mechanism for the affect of SVD on
VCD is that SVD pathologies increase discrete lacunar infarcts, diffuse regions of
leukoaraiosis, and cerebral microbleeds (CMB), which in turn, can disrupt global
2
This was overtaken by COVID-19 in 2020 (ONS 2017b). However, excess mortality (because of
COVID-19, but also because of non COVID-19 related deaths) during lockdown in the UK was
most common in people diagnosed with dementia.
cognitive function, processing speed, executive functions and memory. Although

there are sex differences in CBF such as women showing an 11% higher global flow
and men showing asymmetry in frontal brain regions frontal brain regions (with
higher values on the right), it is unclear by which mechanism this leads to sex
differences in SVD (Binnie et al. 2022).
2.3 Cardiovascular Disease and Estrogens
Despite dementia being the most common cause of death for women on death
certificates, CVD remains one of the leading causes of death for women (ONS
2017a) During reproductive years, women demonstrate lower CVD risk compared
with age-matched men. After menopause, CVD risk increases markedly, such that
by the seventh decade of life CVD risk surpasses that observed in age-matched men
(Mosca et al. 2011). Not only do cardiovascular disease risk factors show overlap
with dementia, having been diagnosed with cardiovascular disease, such as atrial
fibrillation, myocardial infarct, and cardiac insufficiency are risk factors for cogni-
tive impairment and dementia, possibly because of hypoxia and/or other mecha-
nisms such as chronically elevated pro-inflammatory cytokines, which damage brain
integrity and function.
Loss of the cardioprotective effects of estrogen is thought to play a role in the
increased risk of heart disease in women. Estrogen exerts multifactorial effects on
the cardiovascular system, including metabolic, neurohumoral, and anti-
inflammatory effects that contribute to favourable cardiometabolic health, vascular
function, and blood pressure regulation. Accordingly, estrogen deficiency due to
menopause is associated with increased risk of CVD in association with the evolu-
tion of a pro-atherogenic lipid profile, metabolic dysregulation, hypertension, and
the development and progression of coronary artery disease (Mauvais-Jarvis et al.
2013; Mendelsohn and Karas 2005; Reckelhoff 2005; Sack et al. 1994; Saleh and
Connell 1999).
Observational studies showed that hormone replacement therapy (HRT)
improves vascular function (Gerhard et al. 1998; Lieberman et al. 1994), decreases
blood pressure, favourably modulates the lipid profile, and reduces CVD incidence
and all-cause mortality (Grodstein et al. 1997; Stampfer et al. 1985, 1991;
Savolainen-Peltonen et al. 2019). However, evidence from these and other observa-
tional studies was called into question in 2002 when the initial clinical trial from the
Women’s Health Initiative (WHI) reported that cardiovascular events in women
receiving HRT (conjugated equine estrogen with medroxy progesterone acetate or
MPA) were increased compared with non-users (Rossouw et al. 2002) and that there
was a greater risk of dementia (Shumaker et al. 2004). This finding precipitated a
rapid worldwide decline in HRT use of ~80% (Santen et al. 2014).
Subsequent sub-analysis of WHI findings (Rossouw et al. 2013), and other
interventional trials (Harman et al. 2014; Hodis et al. 2016; Schierbeck et al.
2012) supported the concept of a ‘Critical Window of Time’ or ‘timing hypothesis’
(Clarkson et al. 1994) which posits – similar to the effects on brain function – that the
timing of initiation of HRT is also important for the cardiovascular system. Specif-
ically, similar to data on brain function, neutral or beneficial cardiovascular effects of
HRT are observed when initiated <60 years of age or within 10 years of menopause
(Harman et al. 2014; Hodis et al. 2016; Schierbeck et al. 2012; Clarkson et al. 1994),
but negative effects may be seen when treatment is initiated in older women, such as
those of the WHI.
The optimal type of treatment, route of administration and dosage of HRT to elicit
cardiovascular benefits remains to be elucidated. According to a recent review
(Shufelt and Manson 2021), oral unopposed estrogens were shown to have a
favourable effect on lipoprotein levels, glycemia, insulin, and CVD risk. However,
the addition of progestogens (especially of MPA, with least negative effect seen of
micronised progesterone) blunted the beneficial lipid-related effects, similar to that
seen for brain function. Oral estradiol was associated with lower risk for stroke
compared to oral conjugated equine estrogens or CEE (Hale and Shufelt 2015). A
cross-sectional study (Wroolie et al. 2011) showed that 68 healthy postmenopausal
women (aged 49–68 years) who had used oral estradiol had better verbal memory
than women using oral CEE controlled for age, IQ, years of education, risk factors
for AD, duration of endogenous and exogenous estrogen exposure, concurrent
progesterone use, or natural versus surgical menopause status. Transdermal estro-
gens had less negative effect on coagulation, inflammation, and lipids than oral
estrogens and observational studies suggested that these routes of administration
posed a lower risk of venous thromboembolism and stroke than oral estrogens
administration (Shufelt and Manson 2021). Local administration of vaginal estradiol
(gel, pessary, vaginal ring) has been associated with significantly decreased risk of
stroke and coronary artery disease (CHD) compared with non-hormone users in a
large observational Finnish study (Mikkola et al. 2016).
The Kronos Early Estrogen Prevention Study (KEEPS, Harman et al. 2014;
Miller et al. 2019) treated 727 recently menopausal women (>6 months and
<3 years from natural menopause, aged 42–58 years) without subclinical vascular
disease (or untreated cardiovascular risk factors including body mass index or
BMI > 35, unfavourable untreated lipid profiles, hypertension, or diabetes, smoking
>10 cigarettes) (Miller et al. 2019), Women were treated with either oral conjugated
equine estrogens [o-CEE] or transdermal 17β-estradiol [t-E2]) with for both
micronised progesterone added for 12 days of the cycle or placebo to investigate
whether HRT would slow the progression of atherosclerosis as measured by changes
in carotid artery intima-media thickness (CIMT). In this randomised controlled trial
(RCT), there was no effect of either treatment on rate of increase in CIMT after
4 years. There was a trend for reduced accumulation of coronary artery calcium with
o-CEE. Importantly, there were no severe adverse effects, including that of venous
thrombosis or cognitive decline. Several ancillary studies demonstrated a positive
effect on mood with o-CEE, and reduced hot flashes, improved sleep, and mainte-
nance of bone mineral density with both treatments. Sexual function improved with
t-E2. There were no significant effects of either treatment on cognition, breast pain,
or skin (Miller et al. 2019).
The other large RCT called Early vs. Late Intervention Trial with Estradiol
(ELITE) tested the timing hypothesis mentioned earlier (Hodis et al. 2016). In this
study, 643 healthy postmenopausal women were divided into two groups according
to time since menopause (<6 years [early postmenopause] or ≥10 years [late
postmenopause]) and were randomly assigned to receive either oral 17β-estradiol,
plus progesterone vaginal gel administered sequentially for women with a uterus) or
placebo. The main outcome was the rate of change in CIMT, which was measured
every 6 months. Secondary outcomes included an assessment of coronary athero-
sclerosis by cardiac computed tomography (CT) and cognition. In this study after
5 years, oral estradiol therapy was associated with less progression of subclinical
atherosclerosis (measured as CIMT) as compared to placebo when therapy was
initiated within 6 years after menopause, but not after 10 or more years after
menopause. Estradiol had no significant effect on cardiac CT measures of athero-
sclerosis or on cognition (Hodis et al. 2016).
Data combined suggest that HRT can be indicated for the management of
menopausal symptoms in women within 10 years postmenopause, in whom HRT
appears not to increase CVD or dementia risk. However, most guidelines do not
advocate use of HRT to prevent these morbidities.
As stated, apart from sex steroid exposure after menopause, the dementias
(including AD) share other risk factors with cerebrovascular and cardiovascular
disease, such as obesity, high blood pressure, smoking, insulin resistance, and
high total cholesterol levels, but also depression (Barnes and Yaffe 2011; Kim
et al. 2018; Kivipelto et al. 2005; Livingston et al. 2020; Norton et al. 2014). Each
of these factors individually doubles dementia risk, as well as increasing risk
cumulatively when occurring together (Kivipelto et al. 2005). Addressing these
factors could reduce the number of dementia cases by up to 40% (Barnes and
Yaffe 2011; Norton et al. 2014; Livingston et al. 2020). These risk factors can be
addressed through lifestyle changes, such as healthier diets, smoking cessation and
exercise (Clifford et al. 2013; Barnes and Yaffe 2011; Norton et al. 2014).
2.3.1 Sex Differences in Engaging in Protective/Risk Factors Associated

with Mortality
In countries that have actively promoted the benefit of healthy lifestyles in public
health messages used to prevent – but also recognise – early stage cardiovascular
disease, the number of projected cases with dementia is lower than expected
(Baumgart et al. 2015). Moreover, mortality due to chronic diseases, such as diabetes
mellitus and ischemic heart disease, has shown a decline over the last decade
(Bennett 2018). However, as prevalence of these diseases is going up, more people
are living with chronic morbidity for longer which increases their risk of dementia in
later life.
Are women more likely to engage in healthy behaviours than men which allows
them to live longer with morbidity? Women are more likely to visit the GP for earlier
detection of morbidity and are more likely to adhere to medication prescribed for this
(Galdas et al. 2005; Maleki et al. 2016). There is a systematic bias, meaning that
comparing risk cross-sectionally between cohorts (using age as a proxy of aging) is
difficult. For instance, women in the older cohorts assessed in their 80s, could many
decades ago (when dementia plaques are thought to start building up, e.g. before WO
II) be less likely to smoke and drink alcohol than men, cook meals ‘from scratch’,
with fresh local vegetables and would have been more physically active doing
housework with fewer appliances. More people worldwide now eat processed
foods, live in polluted environments, are stressed, lonely and depressed and are
less physically active, which are all possible important risk factors for both heart
disease and dementia (Durazzo et al. 2014; Lafortune et al. 2016; Livingston et al.
2020; Maleki et al. 2016). Less physically demanding housework, for instance, and
increased sedentary activities including computer work and watching TV could
increase risk for both heart disease and dementia disproportionally in future gener-
ations (Chau et al. 2015; Falck et al. 2017). Sedentary behaviour was found to
increase cognitive impairment and dementia risk, independent of being physically
active (Falck et al. 2017). About half of the UK population does not meet guidelines
for physical activity and healthy diets (Scholes 2017).
Not all cohorts show the trends discussed; of increased absolute numbers of
dementia due to an aging population or of the sex difference. In the UK original
estimates of growth and absolute numbers of people living with dementia have
consistently been downscaled, up to a 20% deviation from the original estimate
(Ahmadi-Abhari et al. 2017). On the other hand, numbers of dementia cases may be
worse than originally estimated in developing countries, such as Indonesia
(Hogervorst et al. 2017). The lessening or even disappearance of sex differences is
also mainly found in Western affluent societies (Qiu et al. 2009), such as the
Framingham cohort (Elias et al. 2000).
This could also be the result of more women engaging in less healthy lifestyles
including smoking, sedentary behaviour, and unhealthy diets.
2.4 Lifestyle-Related Protective Factors: Exercise and Other

Activities
Observational studies generally show a reduced risk of dementia in people who

exercise (Guure et al. 2017; Hogervorst 2017). The risk reduction is anywhere from
none to 16%, and even to a halved risk reduction (Guure et al. 2017; Hogervorst
2017). Frequency and intensity of activity engaged in may explain some of the
discrepancies in these numbers. For instance, in a 10-year follow-up study of people
who did not have dementia at baseline, we found that engaging in moderate exercise
reduced dementia risk by a third, but that vigorous exercise could reduce risk by half
(Hogervorst 2017; Soni et al. 2019). People who had developed dementia during the
10-year follow-up study mentioned above, but who kept being physically active also
showed reduced decline in memory functions (Hogervorst 2017), a major hallmark

of dementia risk and progression.
There is sufficient biological plausibility to suggest that this association is causal.
Exercise can improve cardiovascular, immune and lung function, and acts on many
other factors also implicated in dementia risk (Hogervorst et al. 2019; Trigiani and
Hamel 2017). Exercise can also directly affect brain function by altering neurotrans-
mitter levels and promoting dendritic outgrowth in areas implicated in memory, such
as the hippocampus (Hogervorst 2017, 2019). Reviews of randomised controlled
trials have often (but not always) showed beneficial effects of exercise programmes
on global cognition in people with dementia (Bauman et al. 2016; Erickson et al.
2015; Farina et al. 2014; Hogervorst et al. 2019; McDermott et al. 2019; Rao et al.
2014). In contrast, a Cochrane meta-analysis reported no effect of exercise on
cognition in 17 trials including people with dementia, but there was an overall
beneficial effect on activities of daily life (Forbes et al. 2015). All reviews mentioned
heterogeneity and poor methodology of studies investigated. Two large trials
(Oxford and Swedish based) with people with dementia compared a high-intensity
exercise programme to a seated control activity and found no benefit of the exercise
on global cognition after 4 months of participation (Lamb et al. 2018; Toots et al.
2017). Another recent trial suggested a benefit of participating in high-intensity
exercise for 60 min three times per week, but this was not really substantiated in their
data analyses where none of the dependent outcome variables reached significance
(Hoffmann et al. 2016).
It may be that some forms of exercise are more beneficial than others for people
with and without dementia. The studies mentioned that found no difference of
exercise in people with dementia used high-intensity programmes which might be
responsible for not finding effects. In contrast, seated strength exercises for
20–30 min 2–3 times a week were seen to improve memory acutely and after
6 and 12 weeks in sedentary older people with – and at risk for – dementia (Clifford
et al. 2011). In addition, our reviews show that overall effects of exercise are much
stronger in middle-aged and older women compared to men, which may explain why
some studies, having included more women reported effects (Clifford et al. 2011;
Stock et al. 2012).
Our review of reviews (McDermott et al. 2019) and a meta-analysis of
10 randomised control trials (Karssemeijer et al. 2017) suggested that combined
cognitive and physical activity for people with dementia improved global cognitive
function most, with a small to medium effect size, while effects on activities of daily
living were moderate to large in people with dementia or mild cognitive impairment
(MCI, a risk factor for dementia). Other controlled trials combining treatments
(e.g. FINGER, MAX, see Hogervorst et al. 2019) also showed beneficial effects
on cognition, where more time spend on any physical and/or mental activities was
associated with better cognition in people who had not developed dementia yet, but
who were at risk. However, again effect sizes in general were small to medium only.
Previous reviews suggested that many of the nil effects found in exercise studies
could be explained by poor uptake and adherence to the exercise protocols, which is
perhaps more likely to be the case in more strenuous, high-intensity exercise
programmes. Our earlier work (Hogervorst 2012a) found a 55% adherence in people
over 80. In comparison, adherence to the same protocol was 85–88% in middle-aged
sedentary participants. Drop-out due to illness was common in the older group.
To our knowledge there are only three observational studies investigating the
interaction of HRT use and benefits of exercise. Although one study showed an
additive effect of combining both, the other two did not. This area remains to be
investigated further.
3 Discussion
This chapter discussed how demographic factors such as sex, age, education, and
socioeconomic status can interact in uptake of healthy lifestyles including use of
HRT and subsequent risk of morbidity and mortality related to heart disease and
dementia. Differences between cohorts (young vs. old) and cultures limit future
projections of risk, as risk and protective factors may interact with one another,
through off-setting (e.g. exercising while smoking and eating poor diets) or by acting
cumulatively. Efforts are made to combine these factors in risk algorithms, but
limitations due to systematic differences between past cohorts and future lifestyle
behaviours hamper reliable projections of future numbers of people with dementia.
Exercise and regular physical activity combined with psychosocially stimulating
activities may seem the most promising approach for all levels of prevention
including after people have developed dementia. A lifespan approach is advocated
where prenatal advice on healthy diets, not smoking and keeping active may be the
most cost-effective approach (Hogervorst et al. 2019; Shah 2013). However, this
requires concerted policy efforts, e.g. through public health messages, good and
affordable primary health care and education, to implement healthy life styles from
an early age and to future parents, to be most effective in the long term. Whether
short-term HRT can play a role in further reducing dementia risk needs to be
investigated. Longer term treatment trials have generally shown disappointing
results for cognitive improvement. It may be that brain scan data show more
promising benefits but without clinical improvement, its benefits may either be
limited or lie further in the future in terms of reducing dementia risk. With socio-
economic status influencing the uptake of healthy lifestyles (including the ability to
take HRT and undergoing surgical menopause), with growing gaps between rich and
poor, significant efforts need to be made to reduce these systematic prognostic risks
to reduce further burdens to healthcare costs.
Disclosure EH and EoD both worked as consultants on a UKRI grant to test an instrument for a
medical technology client to detect and reduce menopausal hot flushes. EH is an elected committee
member as dementia expert for NICE to update Guidelines for Hormone Replacement Therapy.
However, none of the material described in this chapter is related to both disclosures at this stage.
Both have given many public and academic lectures on this topic but received no honorarium
for this.
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Immune Cell Contributors to the Female
Sex Bias in Multiple Sclerosis
and Experimental Autoimmune
Encephalomyelitis
Nuria Alvarez-Sanchez and Shannon E. Dunn
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
2 RR-MS is a Female-Biased Disease that Manifests in the Reproductive Years . . . . . . . . . . . 337
3 Relapses and Inflammatory Lesion Formation are more Frequent in Females
in Early MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
4 Pregnancy in MS Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
5 EAE Models that Recapitulate the Female Sex Bias in CNS Autoimmunity . . . . . . . . . . . . . . 339
6 Role of Sex Hormones and Chromosomes in MS and EAE Onset . . . . . . . . . . . . . . . . . . . . . . . . 340
7 Sex Differences in Immune Mechanisms that Contribute to the Sex Disparity in CNS
Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
7.1 Primer on the Autoimmune Mechanisms in MS and EAE . . . . . . . . . . . . . . . . . . . . . . . . . . 343
7.2 Myelin-Specific Th1 Cells Expand More in Females Post-Vaccination . . . . . . . . . . . . . 345
7.3 CD4+ T Cells are more Abundant in Females and Exhibit Intrinsic Differences
in Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
7.4 Myelin-Specific T Cells Isolated from Male TCR Transgenic Mice Are More Able
to Cause EAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
7.5 Sex Differences in DC Function and MHC-II Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
7.6 T Effector Responses are Skewed Towards Th1 in Females . . . . . . . . . . . . . . . . . . . . . . . . 349
7.7 T Effector Responses Are Skewed Towards Th2 in Males . . . . . . . . . . . . . . . . . . . . . . . . . . 350
7.8 Sex Differences in Th17 Responses in EAE/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
7.9 Sex Differences in CD4+ Treg Cell Numbers and Activity . . . . . . . . . . . . . . . . . . . . . . . . . . 352
7.10 Sex Differences in T Cell IL-10 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
7.11 Sex Differences in CD8+ T Cells in MS Risk and MS Progression . . . . . . . . . . . . . . . . . 353
7.12 Sex Differences in NK Cells as Contributors to the Increased Tissue Damage
in Males with MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
7.13 Sex Differences in B Cells: Humoral Responses Are More Robust in Females . . . . 355
N. Alvarez-Sanchez
Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada
Department of Immunology, University of Toronto, Toronto, ON, Canada
S. E. Dunn (*)
Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada
Department of Immunology, University of Toronto, Toronto, ON, Canada
Women’s College Research Institute, Toronto, ON, Canada
e-mail: Shannon.Dunn@unityhealth.to
https://doi.org/10.1007/7854_2022_324
334 N. Alvarez-Sanchez and S. E. Dunn
7.14 Sex Differences in the Trafficking of T Cells Across the BBB . . . . . . . . . . . . . . . . . . . . . . 355
7.15 Sex Differences in Microglia Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Abstract Multiple sclerosis (MS) is a chronic, autoimmune, demyelinating disease

of the central nervous system (CNS) that leads to axonal damage and accumulation
of disability. Relapsing-remitting MS (RR-MS) is the most frequent presentation of
MS and this form of MS is three times more prevalent in females than in males. This
female bias in MS is apparent only after puberty, suggesting a role for sex hormones
in this regulation; however, very little is known of the biological mechanisms that
underpin the sex difference in MS onset. Experimental autoimmune encephalomy-
elitis (EAE) is an animal model of RR-MS that presents more severely in females in
certain mouse strains and thus has been useful to study sex differences in CNS
autoimmunity. Here, we overview the immunopathogenesis of MS and EAE and
how immune mechanisms in these diseases differ between a male and female. We
further describe how females exhibit more robust myelin-specific T helper (Th) 1
immunity in MS and EAE and how this sex bias in Th cells is conveyed by sex
hormone effects on the T cells, antigen presenting cells, regulatory T cells, and
innate lymphoid cell populations.
Keywords Autoimmunity · Experimental autoimmune encephalomyelitis ·

Multiple sclerosis · Sex chromosomes · Sex differences · Sex hormones · T helper
cell
Abbreviations
Aire Autoimmune regulator

APC Antigen presenting cell
BBB Blood-brain barrier
cDC Classical dendritic cell
CFA Complete Freund’s adjuvant
CNS Central nervous system
CSF Cerebrospinal fluid
DC Dendritic cell
DC1 Type 1 classical dendritic cell
DC2 Type 2 classical dendritic cell
E2 17-beta-estradiol
EAE Experimental autoimmune encephalomyelitis
Gd Gadolinium
GM-CSF Granulocyte-macrophage colony-stimulating factor
GWAS Genome-wide susceptibility study
IFN Interferon
Immune Cell Contributors to the Female Sex Bias in Multiple Sclerosis. . . 335
Ig Immunoglobulin
IL Interleukin
ILC2 Type 2 innate lymphoid cell
LPS Lipopolysaccharide
MBP Myelin basic protein
MHC Major histocompatibility complex
MHC-II Major histocompatibility complex class II
MMP Matrix metalloproteinase
MOG Myelin oligodendrocyte glycoprotein
MRI Magnetic resonance imaging
MS Multiple sclerosis
NK Natural killer cell
NKT Natural killer T cell
PLP Myelin proteolipid protein
PPAR Peroxisome proliferator-activated receptor
PP-MS Primary progressive multiple sclerosis
PTX Pertussis toxin
RR-MS Relapsing-remitting multiple sclerosis
S1PR Sphingosine-1-phosphate receptor
SP-MS Secondary progressive multiple sclerosis
TCR T cell receptor
TEC Thymic epithelial cell
Th T helperspiepr146 cell
TLR Toll-like receptor
TMEV Theiler’s murine encephalomyelitis virus
TNF Tumour necrosis factor
Tr1 Type I regulatory cell
Treg T regulatory cell
TSPO-PET 18-kDa translocator protein positron emission tomography
1 Introduction
MS is a chronic, autoimmune-mediated, CNS demyelinating disease that affects 2.8

million people worldwide (MSIF 2020). The aetiology of MS is multifactorial with
both genes and environmental factors contributing to disease risk (Reich et al. 2018).
People with MS are initially diagnosed as having RR-MS or primary progressive
(PP-) MS (Reich et al. 2018). RR-MS and PP-MS are not distinguishable according
to the major MS risk genes (Cree 2014), suggesting that these two subtypes are on
the spectrum of the same disease.
RR-MS is characterized by the occurrence of relapses followed by intervening
periods of recovery (Reich et al. 2018) (Fig. 1). This presentation of MS is the most
common (~85% of MS cases) and exhibits a strong female preponderance with a
Fig. 1 Sex differences in clinical features of relapsing-onset MS. The onset of clinical MS is
thought to be preceded by a long period of subclinical autoimmune activity initiating sometime in
adolescence. This subclinical autoimmunity does not surpass the clinical threshold of neurological
dysfunction until adulthood (dotted line). The first clinical attack of MS is termed clinically-isolated
syndrome (CIS). Preclinical CNS autoimmunity (indicated in grey) could represent an expansion of
autoreactive T cells in the peripheral lymphoid organs or the formation of small lesions in the CNS
that do not result in neurological dysfunction. Once a person has two CNS attacks in space or time,
followed by total or partial recovery, they are diagnosed with relapsing-remitting (RR-MS). RR-MS
may transition to secondary progressive MS (SP-MS), which is characterized by the steady
accumulation of disability, with few to no relapses. In terms of the underlying disease mechanisms,
early attacks in MS are driven by peripheral-mediated autoimmune mechanisms (grey), while the
progression of the disease is driven by CNS-compartmentalized inflammation and
neurodegeneration (aqua). The MS disease course in females is characterized by a slightly earlier
onset and more frequent T cell-mediated inflammation, whereas males have a higher risk of
developing SP-MS and exhibit more severe brain atrophy, axonal injury, and neurological dys-
function. The Y-axis in the figure represents the severity of the indicated measure
female:male ratio of 3:1. RR-MS also presents in the reproductive years with a mean
age of onset of 30 years (Cossburn et al. 2012). In comparison, PP-MS is charac-
terized by steady neurological progression from onset with few to no intervening
relapses (Reich et al. 2018). This type of MS tends to present in middle age (mean
age of onset of 45) and affects males and females more equally (Kalincik et al. 2013;
Miclea et al. 2019; Kampman et al. 2013; Zhou et al. 2020). PP-MS is considered to
be an “older” presentation of MS (Tutuncu et al. 2013). Without treatment, a
significant proportion of RR-MS patients transition to having a progressive course,
called secondary progressive MS (SP-MS) (Fig. 1) (Brown et al. 2019). Interest-
ingly, males are more likely to convert to SP-MS and exhibit more extensive axonal
loss and brain atrophy as the disease progresses (Voskuhl 2020). The biological
factors that contribute to MS onset and relapse activity versus disease progression
are thought to be distinct, with peripheral immune-mediated mechanisms driving the
former and CNS-localized inflammatory and neurodegenerative mechanisms driving
the latter (Fig. 1).
This chapter focuses on the female preponderance of RR-MS and overviews the
evidence that the peripheral-mediated autoimmune mechanisms that drive the onset
of this disease are initially more robust in females. It also describes how autoimmune
mechanisms differ between males and females to potentially explain this sex differ-
ence in CNS autoimmunity.
2 RR-MS is a Female-Biased Disease that Manifests

in the Reproductive Years
RR-MS is 3 times more prevalent in females than males (Miclea et al. 2019;
Kampman et al. 2013; Briggs and Hill 2020; Kearns et al. 2019; Bostrom et al.
2013; Trojano et al. 2012). The female:male ratio of RR-MS also varies according to
a person’s age, being close to parity in children with MS (0.8:1 to 1.8:1), sharply
increasing in adolescent-onset MS (2.2:1 to 4:1), remaining high in adult-onset MS
(1.6:1 to 4.71:1), and decreasing in late-onset onset MS (>50 years) (0.33:1 to 1.3:1)
((Cossburn et al. 2012; Achiron and Gurevich 2009; Chitnis et al. 2009), reviewed in
Dunn et al. (2015a)). The increased incidence of RR-MS in females after puberty,
together with the waning of relapses in middle age implicates gonadal hormones as
regulators of the sex bias in MS onset.
Most studies with a few exceptions (Bostrom et al. 2013; Rotstein et al. 2018)
have reported that the female:male ratio of RR-MS has increased over the last half of
the twentieth century due to a rising incidence in females; the incidence of RR-MS in
males remained stable over this same period ((Rojas et al. 2017; Westerlind et al.
2014), reviewed in Dunn et al. (2015b)). This finding suggests that environmental
factors are interacting with sex to increase RR-MS, particularly in females. Some
environmental factors implicated in these effects include lowered vitamin D levels
(Ascherio et al. 2014; Kragt et al. 2009), reduced sunlight exposure (Tremlett et al.
2018), adolescent obesity (Munger et al. 2013), and early onset of puberty
(Ramagopalan et al. 2009). This chapter overviews how autoimmune mechanisms
are different between males and females to regulate CNS autoimmunity, but does not
discuss how environmental factors interact with sex to alter these immune
mechanisms.
3 Relapses and Inflammatory Lesion Formation are more

Frequent in Females in Early MS
In addition to the female bias in MS, there is evidence that relapses are more frequent
in females early in this disease. Females are more likely than males to convert to
having RR-MS after experiencing a first demyelinating event (Achiron and Gurevich
2009; Dobson et al. 2012). Females with RR-MS also have higher annualized
relapse rates than males (Kalincik et al. 2013; Held et al. 2005; Tremlett et al.
2008; Magyari et al. 2014), even when treated with disease modifying therapies
(Magyari et al. 2014). Accordingly, the female:male ratio is higher in those
experiencing high relapse rates in the first five years post disease onset (Kalincik
et al. 2013).
Inflammatory-demyelinating lesions, which are the substrate of relapses (Held
et al. 2005; Bruck et al. 1997), are visualized on T1-weighted magnetic resonance
imaging (MRI) scans of the brain and spinal cord after injection with gadolinium
(Gd) contrast agent (Held et al. 2005). Gd leaks into the CNS in areas associated with
blood-brain-barrier (BBB) breakdown (Bruck 2005). Once inflammation subsides,
residual inflammation or tissue damage is seen as areas of hyperintensities on
T2-weighted scans (called T2 lesions) and as hypointensities on T1-weighted
scans (called T1 black holes). Although no consistent sex differences have been
reported for the number or volume of T1- or T2-weighted lesions (Antulov et al.
2009; Dolezal et al. 2013; Schoonheim et al. 2012; Pozzilli et al. 2003; Shin et al.
2019; Li et al. 2001; van Walderveen et al. 2001; Li et al. 2006; Datta et al. 2017), a
number of studies have found that Gd-enhancing lesions are more prevalent in
women compared to men with MS (Pozzilli et al. 2003; Tomassini et al. 2005;
Weatherby et al. 2000). One study reported that the number of Gd-enhancing lesions
was highest in people with MS who had low levels of testosterone (Tomassini et al.
2005) or a high oestrogen/progesterone ratio (Bansil et al. 1999), suggesting that
hormonal mechanisms may underlie this sex difference in CNS inflammatory
disease activity.
Post-mortem studies of the brain and spinal cord have shed light on the patho-
logical mechanisms in established MS, but have not been optimal in understanding
the pathogenic mechanisms involved in the onset of MS; this is because the majority
of brain specimens studied are from people who have lived with MS for a long
period of time. Nonetheless, a few of these studies scored the proportion of active
(contain abundant leukocytes), inactive (have no immune infiltrates), and chronic-
active or mixed-active lesions (have an inactive demyelinated centre with residual
CD8+ T cell and microglia inflammation along the lesion edge) in white matter in
male and female MS brain specimens (Frischer et al. 2015; Kuhlmann et al. 2009).
These studies observed no sex difference in the number of active white matter
lesions in the brain, but did find that males had a higher proportion of chronic-
active lesions at the expense of inactive lesions (Frischer et al. 2015; Kuhlmann et al.
2009). This finding suggested the presence of residual T cell inflammation in the
white matter of males with MS as they aged. Taken together, these MRI and
pathological findings suggest that peripheral immune mechanisms that underlie
BBB breakdown and white matter lesion formation in early RR-MS are initially
more robust in females, but that this sex difference dissipates with time and is
replaced by persistent, smouldering white matter inflammation in males.
4 Pregnancy in MS Risk
When trying to understand the underlying basis for sex differences in MS onset it is
important to consider the effects of pregnancy, a biological state that is unique to
women. Pregnancy has well-established effects in decreasing relapse rates in MS,
which later re-bound post-partum (Confavreux et al. 1998; Vukusic et al. 2004). It
remains controversial, however, whether pregnancy protects against MS onset in
women (Nguyen et al. 2019). Early studies on large prospective cohorts of women
reported no effect of parity (which is the number of live or still-born births) on MS

risk (Villard-Mackintosh and Vessey 1993; Hernan et al. 2000). More recently, a
number of case-control studies have reported a protective effect of increased parity
in MS (Nielsen et al. 2011; Hedstrom et al. 2014; Ponsonby et al. 2012; Magyari
et al. 2013); however, some of these studies found that this protection occurred in
both men and women (Nielsen et al. 2011; Hedstrom et al. 2014). This finding is
suggestive of reverse causality. Those men and women with yet undiagnosed MS
were less likely to start a family in the years leading up to their MS diagnosis
(Hedstrom et al. 2014). Therefore, there is no strong evidence supporting a protec-
tive effect of pregnancy on MS risk.
5 EAE Models that Recapitulate the Female Sex Bias

in CNS Autoimmunity
EAE is a Th cell-mediated autoimmune disease that targets CNS myelin that has
been an important model to study the importance of MS risk factors including female
sex on the development of autoimmunity. EAE is most commonly induced in
rodents by immunization with myelin antigens emulsified with complete Freund’s
adjuvant (CFA) containing heat-killed Mycobacterium tuberculosis (Stromnes and
Goverman 2006a). In some mouse strains, an additional adjuvant called pertussis
toxin (PTX) is administered to increase disease incidence (Stromnes and Goverman
2006a). EAE can also be induced by isolating and expanding myelin-specific T cells
from EAE donor mice and transferring these cells into healthy mice (this is called
passive or adoptive transfer EAE) (Stromnes and Goverman 2006b). Alternatively,
EAE can develop spontaneously in transgenic mice that over-express myelin-spe-
cific T cell receptors (TCRs) (reviewed in Lassmann and Bradl (2017)).
Similar to RR-MS, a female bias is seen in specific models of EAE. For example,
the incidence and severity of active EAE is higher in female compared to male mice
of ASW, NZW/LACJ, and SJL strains (Papenfuss et al. 2004). For SJL mice, the
female bias in EAE is evident regardless of whether disease is induced by myelin
proteolipid protein (PLP)139–155, myelin basic protein (MBP), or myelin oligoden-
drocyte glycoprotein (MOG)92–106 or whether PTX is administered (Papenfuss et al.
2004; Russi et al. 2018; Bebo et al. 1996; Cua et al. 1995; Butterfield et al. 1999;
Massilamany et al. 2011). Sex differences in the course of EAE are also seen in SJL
mice with females being more prone to relapses (Bebo et al. 1996; Butterfield et al.
1999). Female MOG-specific TCR transgenic mice on the SJL background also
exhibit a higher incidence of spontaneous EAE compared to male counterparts
(Pollinger et al. 2009). These findings have lead to SJL/J being the mouse strain of
choice for studying sex differences in the development of CNS autoimmunity.
By varying the sex of the donor or recipient mice in passive transfer studies in SJL
mice, researchers have been able to discriminate whether female sex enhances EAE
through effects on T helper cell priming (which is distinguished by an effect of sex in

the donor T cells), versus downstream mechanisms that constitute the so-called
effector phase of EAE, that include BBB permeability, interaction with antigen
presenting cells (APCs) in the CNS, and glial cell activity (which is distinguished
by an effect of sex in the recipient mice). It has been found that myelin-specific T
cells isolated from female immunized SJL mice are more able to transfer disease
compared to those isolated from male mice (Cua et al. 1995; Massilamany et al.
2011; Ding et al. 1997; Bebo et al. 1998; Kim and Voskuhl 1999; Voskuhl et al.
1996; Bebo et al. 1999), whereas the sex of host of the transferred T cells does not
influence the severity of passive EAE in a consistent way (Massilamany et al. 2011;
Ding et al. 1997; Bebo et al. 1998; Voskuhl et al. 1996). Therefore, female sex
impacts the induction phase of EAE.
Notably, not all mouse strains exhibit a sex bias in EAE development (e.g., NOD,
PL/J, or B10.PL mice) (Papenfuss et al. 2004). It is also controversial whether sex
differences are apparent in the popular MOG35–55-induced EAE model in C57BL6/J
mice. In this regard, some groups report more severe disease in females (Murphy
et al. 2020; Zhu et al. 2016; Wiedrick et al. 2021), or alternatively no sex difference
(Papenfuss et al. 2004; Rahn et al. 2014; Tassoni et al. 2019; Catuneanu et al. 2019)
or increased disease susceptibility in the males (Bearoff et al. 2015). In our hands, we
have observed that a female bias in disease in C57BL6/J mice becomes apparent at
lower doses of PTX (Fig. 2a, b), suggesting that a sex difference in the response to
the PTX adjuvant may be a confounding factor in this model. Nonetheless, even in
instances where sex differences are not observed in the clinical scores in MOG35–55-
induced EAE, female C57BL6/J mice exhibit more severe spinal cord and optic
nerve inflammation (Wiedrick et al. 2021; Tassoni et al. 2019; Doroshenko et al.
2021) (Fig. 2c, d). In addition, female MOG-specific TCR transgenic mice on the
C57BL6/J background are more susceptible to developing spontaneous EAE than
male counterparts (Cahill et al. 2019). MOG35–55-specific Th cells isolated and
expanded from female C57BL6/J mice are also more able to transfer EAE as
compared to male cells (Fig. 2e, f). Together, these data suggest that female sex is
associated with heightened T helper cell-mediated autoimmunity in EAE in com-
monly studied mouse strains including SJL and C57BL/6.
6 Role of Sex Hormones and Chromosomes in MS

and EAE Onset
Sex differences in CNS autoimmunity can arise because of sex differences in

gonadal hormone production or sex differences in the expression of X- and
Y-encoded genes as a result of differences in X- or Y-gene dosage or parental
imprinting effects on the X-chromosome (Golden et al. 2019). In MS, the finding
Immune Cell Contributors to the Female Sex Bias in Multiple Sclerosis. . .
Fig. 2 T helper cell-mediated autoimmunity and CNS inflammation in the spinal cord is more robust in female compared to male C57BL6/J mice during EAE.
(a–b) Clinical scores and cumulative scores of male and female C57BL6/J mice immunized with MOG35–55/CFA and injected i.p. with either 200 ng (a) or 75 ng
(b) of pertussis toxin (PTX) on the day of, or 2 days following immunization [n ¼ 6–7 mice/group for (a); n ¼ 17–18 animals/group for (b)]. (c–d) Histological
scoring of spinal cord parenchymal inflammation (c) and perivascular infiltration (d) [n ¼ 5 animals/group] for representative mice in the experiment in (a). The
presence of parenchymal inflammation or perivascular cuffs was examined in each quadrant of the spinal cord, in 10–12 spinal cross-sections per mouse. (e–f)
Clinical scores of sex-matched recipient B6 mice after transfer of female and male MOG-reactive T cell lines. The MOG p35–55-specific T cells were expanded
341
from the draining lymph nodes of male or female donor C57BL6/J mice for 10 days with MOG p35–55 and IL-2. Fresh media containing IL-2 was added on day
that the female bias in RR-MS manifests post-puberty and wanes with menopause
(reviewed in Dunn et al. (2015a)) strongly implicates gonadal hormones as the major
regulator of sex differences in MS onset. Differential expression of genes off the X
and Y chromosome could be an additional, more minor factor regulating the sex
differences in MS onset. A large transcriptional profiling study of immune cells in
healthy men and women reported that only 7% of genes differentially expressed
between the sexes were X- or Y-encoded (Schmiedel et al. 2018). The most recent
genome-wide susceptibility (GWAS) study in MS identified that of the >200 MS
risk variants identified, only one was encoded on a sex chromosome (International
Multiple Sclerosis Genetics Consortium 2019).
The influence of sex hormones and sex chromosomes on the development of CNS
autoimmunity has been more extensively explored in the EAE model, particularly in
SJL mice. To parse out the influence of sex hormones, researchers have removed the
gonads of mice or supplemented mice with exogenous hormones. Such studies have
demonstrated that castrating SJL males enhances EAE severity, whereas supple-
mental androgen treatment is protective against EAE in SJL and C57BL/6 mice
(Palaszynski et al. 2004; Voskuhl and Palaszynski 2001). Ovariectomy has less of an
effect on disease, but can be protective in SJL mice if performed prior to pubertal
onset (Palaszynski et al. 2004; Voskuhl and Palaszynski 2001; Ahn et al. 2015).
Therefore, gonadal hormones are major regulators of the sex difference in EAE.
To parse out the influence of sex chromosomes in EAE, researchers have
employed the “four-core” genotype mouse model. In this model, mice are generated
that have the testes-determining, Y-encoded gene Sry either knocked out on the Y
chromosome or expressed on an autosome. Crossing these strains of mice with XX
females allows for the generation of gonadal males with XY and XX-Sry+ chromo-
some complements and gonadal females with XX and XY-Sry/ chromosome
complements (Arnold 2009). When the four groups of mice are gonadectomized,
the influence of the sex chromosomes can be studied apart from the influence of
gonadal hormones. Studies performed using these mice on the SJL background
demonstrated that the XX chromosome complement conferred more severe EAE
than the XY chromosome complement that correlated with a Th2 bias in the immune
response in males (also see Sect. 7.7). This effect of sex chromosome complement
was not seen in C57BL6/J mice (Smith-Bouvier et al. 2008). These data in EAE
thereby align with the epidemiological findings in MS and suggest that the sex
difference in EAE onset is largely specified by sex differences in gonadal hormone
production with sex chromosome complement contributing in a more minor way.
The following sections will overview the immunopathogenesis of MS and EAE, will
describe the immune mechanisms that contribute to sex differences in CNS
Fig. 2 (continued) 2 of culture. Cells were then re-primed with anti-CD3 and anti-CD28 (5 μg/mL
each) in the presence of IL-23 for 24 h prior to transfer into n ¼ 5 mice/sex. For (e), recipients were
treated with 50 ng PTX and injected with 4 106 male or female cells. In (f), recipients received
sub-lethal irradiation and were treated with 50 ng PTX prior to being transferred with 5 106 cells
(f). *p 0.05, **p 0.01
autoimmunity, and will discuss the modulatory role of sex hormones and sex
chromosomes on these mechanisms.
7 Sex Differences in Immune Mechanisms that Contribute

to the Sex Disparity in CNS Autoimmunity
7.1 Primer on the Autoimmune Mechanisms in MS and EAE
7.1.1 Autoimmune Mechanisms in MS
Since MS risk factors have an impact on disease risk in adolescence, a decade or

more prior to the average age of clinical MS onset, it has been difficult to study the
immune system in MS at the time when autoimmunity is most likely to be initiated.
However, genetic studies in MS have provided hints to what cells may be involved in
disease initiation (Canto and Oksenberg 2018). The strongest genetic association for
MS maps to the major histocompatibility class (MHC) region on chromosome 6p21.
In Caucasians, this associates with the HLA-DR15 haplotype (Canto and Oksenberg
2018). HLA-DR15 consists of two MHC class II (MHC-II) alleles (HLA-DRB1*15:
01 and HLA-DRB5*01:01) that encode MHC-II beta chains that are almost always
co-inherited. These beta chains pair with an alpha chain (HLA-DRQA1*01:01) to
form DR2b and DR2a MHC-II molecules (Martin et al. 2021). The involvement of
MHC-II alleles in MS onset strongly implicates a role for Th cells in this disease.
Indeed, recent research has found that DR2a/DR2b-restricted Th cells recognize self-
MHC peptides as well as epitopes on MBP (amino acids 83–99) and antigens
encoded by Epstein Barr Virus (Wang et al. 2020) and microbiota species such as
Akkermansia muciniphila (Martin et al. 2021; Wang et al. 2020). It is speculated that
the recognition of self-MHC peptides permits the survival of the DR2a/2b-restricted
Th cells during thymocyte selection. It is further speculated that these cells can
become activated and expand in response to a variety of microbes that leads to a
reduction in their threshold of activation and eventual activation by self-antigens
including MBP (Martin et al. 2021). Consistent with this model, although individ-
uals with MS and healthy people both have MBP-specific T cells, those in MS
patients have a more antigen-experienced phenotype (Cao et al. 2015).
Immunological studies have also furthered the understanding of Th cells in
MS. Th cells can differentiate along a variety of Th lineages including T helper
1 (Th1), Th2, Th17, T regulatory (Treg), and T follicular helper. Of these Th cell
types, Th1 (interferon γ [IFN-γ]-producing CD4+ cells) and Th17 cells (interleukin-
[IL-]17-producing CD4+ cells) have been implicated in MS. Both of these Th cell
types are detected in MS lesions (Lock et al. 2002; Kebir et al. 2007) and are
increased in the blood or cerebral spinal fluid (CSF) just prior to or coincident
with relapse (Bielekova et al. 2000; Beck et al. 1988; Brucklacher-Waldert et al.
2009). MS is also associated with defective T regulatory mechanisms. Tregs in those
affected by MS are functionally impaired in their ability to suppress T effector cells
(Carbone et al. 2014; Viglietta et al. 2004) and single nucleotide polymorphisms in
the high-affinity IL-2R alpha chain (IL2RA), which is important for Treg cell
function, confer MS risk (International Multiple Sclerosis Genetics Consortium
et al. 2011). In addition to Treg dysfunction, Th cells isolated from individuals
with MS do not differentiate into IL-10-producing type I regulatory T cells (Tr1)
cells as readily as those from healthy people (Astier et al. 2006). Consistent with
there being an imbalance in T effector and T regulatory mechanisms in MS, myelin-
specific T cell clones expanded from the blood of individuals with MS have been
shown to produce higher levels of IFN-γ, IL-17, and granulocyte-macrophage
colony-stimulating factor (GM-CSF), and less IL-10 compared to those expanded
from healthy controls (Cao et al. 2015).
Notably, half of MS-affected individuals are negative for HLA-DRB15, impli-
cating the involvement of other genes in MS. In this regard, GWAS in MS have
identified more than 200 independent risk variants outside of the MHC region
(International Multiple Sclerosis Genetics Consortium 2019). These risk variants
contribute in a much smaller way to MS risk and include genes expressed by CD4+ T
and other immune cell types including CD8+ T cells, B cells, natural killer
(NK) cells, monocytes, dendritic cells (DCs), and microglia, further supporting the
concept that MS initiates as an autoimmune-mediated disease (International Multiple
Sclerosis Genetics Consortium 2019).
7.1.2 Autoimmune Mechanisms in EAE
In contrast to MS, where the steps leading to autoimmunity are still speculative, the
sequence of events leading to the development of EAE is well understood (Robinson
et al. 2014). As in humans, Th cells with weak to moderate reactivity against myelin
antigens are present in the peripheral T cell pool in mice. These Th cells though
usually ignorant of self-antigen become activated in the context of the myelin/CFA
immunization. Toll-like receptor (TLR) ligands in the CFA are thought to trigger the
upregulation of MHC-II and co-stimulatory molecules (e.g., CD80/CD86) on clas-
sical DCs (cDCs) and B cells, arming these APCs with an ability to prime myelin-
reactive Th cells in peripheral lymphoid organs (Jager et al. 2009; Dendrou et al.
2015). Activated APCs also produce cytokines (e.g., IL-12, IL-23, IL-6) that pro-
mote the differentiation of Th cells into Th1 or Th17 lineage cells. These activated
myelin-specific Th cells then leave the peripheral lymphoid organs and traffic
through the blood and lung prior to entering the CNS through the blood-brain and
blood-meningeal barriers (Odoardi et al. 2012; Vajkoczy et al. 2001; Dusi et al.
2019). Once in the CNS, myelin-specific CD4+ T cells become reactivated by local
CD11c+ dendritic cells presenting myelin antigen (Giles et al. 2018; Waisman and
Johann 2018; Juedes and Ruddle 2001; Bartholomaus et al. 2009), which leads to the
local production of cytokines (IFN-γ, IL-17, and GM-CSF) and chemokines (CCL2,
CXCL10, etc.). This inflammation leads to activation and proliferation of microglia
(Boehm et al. 1997; Kawanokuchi et al. 2008), upregulation of adhesion molecules
on the BBB (Kebir et al. 2007, 2009), and the migration of additional immune cells
including monocytes into the CNS (Kebir et al. 2007; Griffin et al. 2012; Kroenke
et al. 2008). The CCR2+ inflammatory monocytes that accumulate in EAE lesions
(Matyszak and Perry 1996; Almolda et al. 2009) differentiate into macrophages that
can strip myelin away from axons (Yamasaki et al. 2014) or become inducible nitric
oxide synthase-expressing inflammatory DCs (Giles et al. 2018). GM-CSF produced
by the CNS-infiltrating Th1 or Th17 cells is crucial to the upregulation of these
pro-inflammatory pathways in monocytes and for mediating myelin damage in EAE
(Becher et al. 2016; Codarri et al. 2011; Croxford et al. 2015).
Knowing the cascade of events that occur in EAE, sex differences in inflamma-
tory lesion formation in EAE and MS may occur as a result of sex differences in:
(1) the priming of myelin-specific Th cells by APCs, (2) the expansion or differen-
tiation of myelin-specific Th cells, (3) the trafficking of myelin-specific Th cells into
the CNS, (4) regulatory T cell function, (5) Th cell interactions with brain APCs,
and/or (6) the functioning of other immune cell subsets (CD8+ T cells, B cells,
monocytes, NK cells). Here we will overview how sex impacts each of these
immune mechanisms.
7.2 Myelin-Specific Th1 Cells Expand More in Females

Post-Vaccination
A number of studies have reported that when female and male SJL mice are
immunized with myelin antigen and CFA and the lymph node and spleen cells are
later harvested and activated with the same myelin antigens, that the recall prolifer-
ative response of myelin-specific Th cells is higher in females compared to males
(Kim and Voskuhl 1999; Luna et al. 2010; Wilcoxen et al. 2000; Zhang et al. 2012;
Palaszynski et al. 2005). This sex difference in the response to vaccination is not
specific to myelin antigens or to the SJL strain (Kim and Voskuhl 1999; Dunn et al.
2007; Weinstein et al. 1984). This biology may be conserved in humans, since
studies that evaluated myelin-specific T cell responses in the blood using either [3H]-
thymidine incorporation or IFN-γ ELISPOT assays detected higher T cell reactivity
to certain myelin epitopes in females compared to males (Greer et al. 2004; Pelfrey
et al. 2002).
The four-core genotype mouse model has been used as a tool to parse out the
influence of sex hormones versus sex chromosomes on the proliferation and IFN-γ
production by MBP-specific T cells after EAE induction in SJL mice (Palaszynski
et al. 2005). In these experiments, mice were vaccinated with MBP/CFA and the
recall proliferation and MBP-specific IFN-γ production was assessed in the draining
lymph nodes. It was observed that MBP-specific T cell proliferative responses and
IFN-γ production were higher in gonadal females compared to gonadal males and
that these sex differences were reversed by castrating the males, but not by
ovariectomizing the females, and were not impacted by the sex chromosome com-
plement (Palaszynski et al. 2005). These findings suggest that sex differences in
androgen levels are the major driver of the enhanced expansion of myelin-specific
Th1 cells in EAE. The following sections will discuss potential cellular mechanisms
that contribute to the increased expansion of myelin-reactive Th1 cells in females
including sex differences in the starting CD4+ T cell repertoire, sex differences in
APC function, Th1/Th2/Th17 cytokine balance and T and NK cell regulatory
mechanisms. When available, information regarding the influence of hormones
versus sex chromosome effects on these immune features will be described.
7.3 CD4+ T Cells are more Abundant in Females and Exhibit

Intrinsic Differences in Activation
When compared with males, females have higher CD4+ T cell counts and exhibit a
higher CD4+ to CD8+ ratio in peripheral blood (Abdullah et al. 2012; Lee et al. 1996;
Amadori et al. 1995). This phenotype may be a result of higher thymic output in
females (Pido-Lopez et al. 2001). T cells in healthy human females at baseline also
appear to be in higher state of activation, expressing higher levels of HLA-DR,
CD69, and certain proliferation markers (Abdullah et al. 2012; Sankaran-Walters
et al. 2013). Therefore, one reason why MS may initiate more readily in a female is
that female CD4+ T cells are more abundant and in a higher basal state of activation
compared to male CD4+ T cells.
Sex differences in T cell activation could relate to sex differences in Autoimmune
regulator (Aire) expression and related effects on the T cell repertoire. Aire is a gene
that is expressed in medullary thymic epithelial cells (TECs) that plays a role in the
negative selection of autoreactive thymocytes (Proekt et al. 2017). Interestingly, a
number of reports have found that Aire is expressed at higher levels in males than
females and is under the control of sex hormones, being induced by androgens and
repressed by oestradiol (Zhu et al. 2016; Dragin et al. 2016). This higher expression
of Aire in the male TECs might be anticipated to induce more extensive negative
selection of myelin-specific T cells in males. In support of a role for Aire in
regulating sex differences in EAE, it was reported that the female bias in
MOG35–55-induced EAE seen in C57BL/6 mice and the suppressive effect of
androgens on this disease were not observed in Aire/ mice (Zhu et al. 2016).
However, Aire/ mice also exhibit defective Treg development (Malchow et al.
2016) and develop more severe EAE (Nalawade et al. 2018), making these results
difficult to interpret.
Insights into sex differences in the T cell repertoire have been provided by studies
that examined the ability of PLP139–151-reactive T cells in SJL mice to recognize
other microbe-encoded epitopes. One peptide encoded by Acanthamoeba castellanii
(ACA83–95) was found to be capable of cross-activating PLP139–151-reactive T cells
in SJL mice. Further studies revealed that male and female PLP139–151-reactive T
cells proliferated equivalently to ACA83–95 and that expanded male and female
PLP139–151- or ACA83–95-reactive T cells exhibited similar TCR Vβ usage
(Massilamany et al. 2011). Similarly, TCR Vβ usage by CNS-infiltrating

PLP139–151-reactive T cells has been examined in male and female SJL mice with
EAE and was found not to differ between the sexes (Bebo et al. 1996; Massilamany
et al. 2011). However, since the TCR hypervariable regions of the PLP139–151-
reactive Th cells were not sequenced, these data do not rule out a sex difference in
the myelin-specific T cell repertoire in SJL mice.
7.4 Myelin-Specific T Cells Isolated from Male TCR

Transgenic Mice Are More Able to Cause EAE
Recent studies have shown across a variety of models and mouse strains that when
male and female MOG-reactive TCR transgenic CD4+ T cells are cultured with
MOG antigen under Th1 or Th17-polarizing conditions and are used to induce
adoptive transfer EAE, male T cells induce a more severe EAE disease (Dhaeze
et al. 2019; Doss et al. 2021; Williams et al. 2011). In the TCR1640 model of passive
EAE, the female MOG-specific TCR1640 cells induced a relapsing form of EAE,
whereas male TCR1640 cells induced a more severe and chronic form of this disease
(Dhaeze et al. 2019). In this model, the sex of the host in the passive transfer studies
did not influence the EAE phenotype (Dhaeze et al. 2019). Regarding the mecha-
nisms of this sex difference, it was found that while the cytokine profile of the
transferred cells did not differ, the female CNS T cells expressed higher levels of
genes involved in Treg function or differentiation (Ctla4, Gpr15, Foxp3) (Dhaeze
et al. 2019).
In another model, transfer of male Th17-polarized MOG-specific TCR1C6 CD4+
T cells was more likely to produce a severe progressive EAE phenotype compared to
female counterparts when transferred into NOD.Scid recipient mice (Doss et al.
2021); however, in this case, the phenotype tracked with lowered expression of an
X-chromosome encoded-histone H3K4 demethylase called Jarid1c in the male T
cells and an increased T cell plasticity for cytokine production. In yet another TCR T
cell transfer model, the higher potential of Th1-polarized male MOG-specific
TCR2D2 cells to transfer EAE associated with a higher memory effector phenotype
of the transferred male TCR2D2 T cells (Williams et al. 2011). Together, these studies
suggest that on a cell-per-cell basis male myelin-specific TCR transgenic Th1 or
Th17 effector cells are more capable of causing EAE than female counterparts. This
finding that male, but not female TCR transgenic Th cells are more pathogenic in
EAE would not explain why MS or EAE is more likely to occur in females, but could
go further in explaining why males with MS are more likely to show neurological
progression with age. Furthermore, this finding that the male T cells are more
pathogenic potentially contradicts the idea that Aire mediates increased negative
selection of autoreactive T cells in males.
7.5 Sex Differences in DC Function and MHC-II Expression
CD11bCD11c+ conventional dendritic cells (cDCs) or DC1 cells are the most
efficient at priming CD8+ T cells in the draining lymph nodes and myelin-reactive
Th cells at the CNS site (Giles et al. 2018) whereas CD11b+CD11c+ cDCs or DC2
cells are the main DC involved in priming naive Th cells in peripheral lymphoid
organs. No sex differences have been reported in the frequency or activation state of
DC1, DC2, or plasmacytoid DCs in the peripheral blood of individuals with MS
(Tillack et al. 2013). Because of the difficulty of accessing lymphoid and CNS tissue
in humans, the activation state of DCs at the likely sites of Th cell priming or
re-activation has not yet been examined in MS.
As discussed in Sect. 7.1.1, carriage of the HLA-DRB15 haplotype is the major
genetic risk factor for MS. In this respect, a number of case-control studies have
reported that HLA-DRB15+ status is a MS risk factor for females, but not males
(Olsson et al. 2017; Irizar et al. 2012; Hensiek et al. 2002; Celius et al. 2000;
Weatherby et al. 2001). The HLA-DRB15 haplotype also exhibits parent-of-origin
effects, being more frequently transmitted from MS mothers to offspring than from
MS fathers to offspring, particularly if the offspring are female (Ramagopalan et al.
2008; Chao et al. 2010). Therefore, some of the sex difference in MS may relate to
how HLA-DRB15 functions in a male versus a female.
Studies in mice have also explored sex differences in the functionality of DC and
other APC populations in EAE. Such studies found that splenic DCs or macrophages
isolated from SJL female mice are more efficient at priming naïve myelin-specific
CD4+ T cells in vitro compared to male APCs (Wilcoxen et al. 2000; Zhang et al.
2012). The sex of the APC is not as critical of a factor when APCs are used to
activate myelin-specific Th effector cells that had been previously activated in vivo
(Bebo et al. 1998), suggesting that sex impacts APC function at the time of initial Th
cell priming in EAE.
One reason why female DC may be more able to prime CD4+ T cells is that
differentiation and maturation state of DC is under the control of 17-beta oestradiol
(E2) and the oestrogen receptor-α (reviewed in Kovats (2012)). Bone marrow pro-
genitors isolated from C57BL6/J mice differentiate into CD11c+CD11bint DC more
efficiently in response to GM-CSF when the cultures are also supplemented with E2
(King et al. 2009). These E2-generated DCs exhibit higher MHC-II and
co-stimulatory marker expression and higher IL-12p40 secretion (Siracusa et al.
2008; Delpy et al. 2005) and have a greater capacity to prime naïve CD4+ T cells
in vitro (Siracusa et al. 2008; Paharkova-Vatchkova et al. 2004) compared to those
cultured without E2. Similarly, E2 addition also generates more functionally com-
petent DCs when bone-marrow precursors are differentiated with Flt3 ligand
(Kovats 2012).
Consistent with female cDCs being more functionally competent, when DCs are
isolated from spleens of female SJL mice and are co-cultured with myelin-specific
Th cells, responding T cells produce higher levels of IFN-γ that correlates with
higher production of IL-12p40 in the co-cultures (Zhang et al. 2012). Similarly,
peritoneal macrophages isolated from female SJL mice produce higher levels of
IL-12p40 and lower levels of IL-10 compared to male macrophages (Kim and
Voskuhl 1999; Wilcoxen et al. 2000; Hoghooghi et al. 2020). IL-12p40 is a cytokine
that can be produced as a monomer, a homodimer, or a heterodimer with IL-12p35 to
form the Th1-promoting cytokine IL-12p70 or as a heterodimer with IL-23p19 to
form the Th17 promoting cytokine IL-23 (Drohomyrecky et al. 2019; Gee et al.
2009). Consistent with IL-12p40 having a sex difference in function in CNS
autoimmunity, this cytokine is detected at higher levels in the draining lymph
nodes of female SJL mice during EAE (Kim and Voskuhl 1999) and in the serum
of females with MS (Miteva et al. 2019) compared to male counterparts. Therefore,
sex differences in IL-12p40 production are a conserved feature between mice and
humans.
Further exploration of the mechanisms of the sex difference in IL-12p40 produc-
tion by SJL macrophages revealed that the lowered IL-12p40 production by male
macrophages in vitro was accompanied by increased IL-10 production and was
reversed by neutralizing IL-10 in the culture or by castrating the male SJL donors
(Wilcoxen et al. 2000; Hussain and Stohlman 2012). In this study, the higher
IL-12p40 correlated with increased T cell IFN-γ production. These findings go
further in explaining why myelin-specific Th1 cells may expand more in a female
mouse during EAE.
Although sex differences in monocyte-derived DC populations have not been
described for EAE or MS, monocyte signaling and myeloid cell phenotype have
been reported to differ between males and females. For example, experiments that
knocked out p38 MAPK selectively in cells of the myeloid lineage in C57BL/6 mice
demonstrated that p38 MAPK signalling functioned only in the female myeloid cells
to promote Th1/Th17 inflammation in the CNS during EAE (Krementsov et al.
2014). In addition, in vivo treatment of C57BL/6 mice with E2 at levels that are seen
in a normal cycling female enhanced the potential of isolated macrophages to
produce IL-12p40, IL-6, tumour necrosis factor (TNF), and IL-1β in response to
TLR4 stimulation (Calippe et al. 2010), and increased the production of type I
interferon production by plasmacytoid DCs in response to TLR7 or TLR9 stimula-
tion (Seillet et al. 2012). Transcripts encoding pro-inflammatory cytokines (IL6,
TNF, and CXCL10) are also reported to be expressed at higher levels in monocytes
of female humans (Schmiedel et al. 2018). Taken together, these findings suggest
that both sex and gonadal hormones can regulate aspects of DC and monocyte
differentiation function that have the potential to contribute to the female bias in
CNS autoimmunity in EAE and MS.
7.6 T Effector Responses are Skewed Towards Th1

in Females
It is well established that myelin-specific Th1 responses are enhanced more in female
SJL mice during EAE (Russi et al. 2018; Bebo et al. 1996; Cua et al. 1995; Bebo
et al. 1999; Luna et al. 2010; Zhang et al. 2012) and Th1 cells are more frequent in
peripheral blood of female humans living with MS (Pelfrey et al. 2002; Moldovan
et al. 2008; Eikelenboom et al. 2005). The increased number of myelin-specific Th1
cells in females could relate to the higher IL-12 production by female APC (Sect.
7.5) or increased Th cell expansion in females (Sect. 7.3); however, there is also
evidence that female Th cells are intrinsically geared to produce higher levels of
IFN-γ compared to male T cells. For example, female murine CD4+, CD8+, and
natural killer T (NKT) cells secrete higher levels of IFN-γ in vitro upon stimulation
with anti-CD3 and anti-CD28 or PMA/Ionomycin (Schmiedel et al. 2018; Zhang
et al. 2015). In humans, T cells from females also produce higher levels of IFN-γ
than male T cells when activated in vitro with anti-CD3 and anti-CD28 or
PMA/Ionomycin (Zhang et al. 2012; Hewagama et al. 2009).
It has become clear that the sex difference in IFN-γ production by T cells is
controlled by the balance in gonadal sex hormones, with androgens having repres-
sive, and nM levels of E2 having enhancing effects on IFN-γ expression. For
example, testosterone levels are inversely correlated with myelin-specific production
of Th1 cytokines in EAE SJL mice (Foster et al. 2003) and castration results in
increased myelin-specific Th1 responses in SJL male mice in EAE (Zhang et al.
2012). Low levels of supplemental E2 also promote Th1 responses in ovariecto-
mized C57BL/6 and BALB/c mice (Maret et al. 2003). Though some of these effects
of sex hormones on IFN-γ may be directed through APC expression of IL-12, there
is also evidence of direct hormone-dependent regulation of Ifng expression. Treat-
ment with E2 in vitro increases Ifng promoter activity through an oestrogen receptor
response element (Fox et al. 1991). In addition, androgens repress and oestrogens
enhance IL-12-induced STAT4 phosphorylation in T cells (Bao et al. 2002; Kissick
et al. 2014). Androgens also induce the expression of peroxisome proliferator-
activated receptor (PPAR)-α in male T cells, which functions as a transcriptional
repressor at the human and mouse Ifng loci (Zhang et al. 2012; Dunn et al. 2007).
Importantly, there is evidence that this female Th1 bias in the immune response
can contribute to enhanced CNS inflammation in mice with EAE and in humans with
MS. In mice, the enhanced Th1 immunity that accompanies deletion of PPARα
increases the severity of EAE in SV.129 mice (Dunn et al. 2007) and the incidence of
spontaneous EAE in male 2D2 TCR Tg mice (Cahill et al. 2019). Similarly, several
polymorphisms in the IFNG gene, including one that leads to increased expression
of IFNG, associate with increased MS in males (Kantarci et al. 2008; Kantarci et al.
2005; Goris et al. 2002). Therefore, heightened Th1 cytokine production is likely
one contributing factor to the sex difference in MS and EAE.
7.7 T Effector Responses Are Skewed Towards Th2 in Males
While Th responses are biased towards Th1 in females, most MS studies and EAE
studies in SJL mice, with some exceptions (Kim and Voskuhl 1999; Zhang et al.
2012; Tillack et al. 2013), report that males exhibit a Th2 bias in the immune
response (Russi et al. 2018; Bebo et al. 1996; Pelfrey et al. 2002; Moldovan et al.
2008). There is evidence from MS genetic (Akkad et al. 2007) and EAE (Falcone
et al. 1998) studies that the Th2 cytokine IL-4 protects against autoimmunity. The
higher Th2 cytokine production in males may occur in part as a by-product of
reduced Th1 immunity. Th1 and Th2 lineages are mutually exclusive and cross-
regulatory mechanisms exist to ensure that the Th cells that differentiate towards Th1
do not differentiate towards Th2 (Zhu et al. 2010). However, a recent study in SJL
mice demonstrated that higher type 2 innate lymphoid cells (ILC2) also drive
increased Th2 responses in males during EAE (Russi et al. 2018). It was shown
that androgens induce IL-33 production by mast cells. IL-33 induces the accumula-
tion of rare, but potent ILC2 cells in the draining lymph nodes that promote Th2
immunity (Russi et al. 2015, 2018). In addition to sex hormone influences, in the
four-core genotype model in SJL mice, the protective effect of the XY chromosome
complement mapped to higher production of Th2 cytokines and expression of the
IL-13 receptor α2 in APC (Smith-Bouvier et al. 2008). Therefore, both androgens
and the XY chromosome complement may promote Th2 immunity in male
SJL mice.
Though the expression of the Th2 cytokine IL-4 is clearly protective in EAE
(Falcone et al. 1998), Th2 cells and other Th2 cytokines also have the potential to be
pathogenic in EAE and MS. For example, expression of the Th2 cytokine IL-13 is
associated with increased EAE susceptibility in female, but not male C57BL/6 mice
(Sinha et al. 2008). This effect of IL-13 was mediated through increased expression
of MHC-II on the female APCs (Sinha et al. 2008). MBP-specific Th2 cell lines can
also induce EAE in immunocompromised mice, albeit with lower efficiency than
Th1-polarized MBP-specific cells (Lafaille et al. 1997). In addition, Th cells and
cytotoxic T cell clones expanded from fresh autopsy samples from the brains of
individuals with MS are reported to exhibit a Th2 or a mixed Th1/Th2 cytokine
profile (Planas et al. 2015). Therefore, while IL-4 cytokine production is more
prominent in males, it remains to be clarified whether other Th2 cytokines or
Th2-associated mechanisms are protective or detrimental in EAE and MS.
7.8 Sex Differences in Th17 Responses in EAE/MS
Whether sex differences exist in the Th17 responses in MS/EAE is controversial.

Some groups have reported that male SJL mice exhibit enhanced myelin-specific
IL-17 production in the spleen during EAE; however, these same reports also noted
IL-17 to be lowered in males in the draining lymph nodes, tracking with reduced
myelin-specific Th cell expansion (Massilamany et al. 2011; Zhang et al. 2012). One
small study that evaluated the frequency of IL-17-producing CD4+ T cells in the
blood of people with MS found no sex difference in this population (Tillack et al.
2013). On the other hand, consistent with a female Th1 bias, CD4+ T cells isolated
from female naïve SJL mice or healthy humans tend to express lower levels of IL-17
compared to male T cells (Zhang et al. 2012; Hewagama et al. 2009). The sex
difference in IL-17 production may relate to higher expression of PPARγ1 in female

T cells. PPARγ1 is a nuclear receptor that represses IL-17 production and is turned
off by androgens (Zhang et al. 2012). Thus, male T cells, on an individual level, may
be more prone to producing IL-17; however, this phenotype may be difficult to see in
EAE because of the overall lower expansion of male myelin-specific T cells.
7.9 Sex Differences in CD4+ Treg Cell Numbers and Activity
FoxP3+ Tregs are crucial for maintaining immune tolerance and preventing autoim-
munity (McGeachy et al. 2005; Zhang et al. 2004) and these cells are functionally
impaired in their ability to suppress T effector cells in people with MS (Carbone et al.
2014; Viglietta et al. 2004). However, it is controversial whether Tregs contribute to
sex differences in RR-MS. For example, studies in a Southern Brazilian MS cohort
found that a polymorphism in the promoter of the FOXP3 gene (rs3761548 FOXP3–
3279 C > A) that results in reduced FoxP3 expression in T cells associated with
increased MS risk, but only in females (Flauzino et al. 2019). This same single
nucleotide polymorphism did not associate with MS in a Polish MS cohort, where
instead another FOXP3 polymorphism (rs3761547 C > T) associated with MS, but
only in males (Wawrusiewicz-Kurylonek et al. 2018).
It is also controversial whether there is a sex difference in the number or function
of Treg in healthy people or in mice. One study in humans reported finding no sex
difference in the frequency of CD127loCD25+ CD4+ Treg in peripheral blood, albeit
these cells were found to be in a higher state of activation in females (Aguirre-
Gamboa et al. 2016). In contrast to this, another study reported detecting a higher
number of Treg in the circulation of male human donors (Afshan et al. 2012). On the
other hand, studies in SJL mice support the idea that specific features of Treg may be
enhanced in males. Though there is no sex difference in the in vitro suppressive
function of SJL Treg, male Treg from this strain are more abundant in peripheral
lymph organs and express higher levels of the inhibitory molecule CTLA-4 com-
pared to female Treg (Hussain et al. 2011). SJL male T cells also exhibit lower
methylation in the FoxP3 promoter compared to female T cells (Voskuhl et al.
2018), However, in contrast to this latter finding, no sex differences in FoxP3
expression have been reported for murine (Golden et al. 2019) or human T cells
(Schmiedel et al. 2018). The FOXP3 gene also does not escape X-inactivation in
humans (Carrel and Willard 2005).
In EAE and MS, there is evidence that Treg expand alongside T effector CD4+ T
cells and may have greater activities in females. In SJL mice, FoxP3 Treg are
increased more so in the spleens of females during EAE (Zhang et al. 2004). Higher
frequencies of CD4+ CD25high and CD4+ CD25+ FoxP3+ cells are also detected in
the CSF of females compared to males with MS (Tejera-Alhambra et al. 2012). In the
TCR1640 model of passive EAE, the remitting phenotype that was seen post-transfer
of the female, but not the male TCR1640 cells correlated with higher expression of
genes involved in Treg function or differentiation (Ctla4, Gpr15, Foxp3) (Dhaeze
et al. 2019).
Taken together, there is no conclusive evidence of a role for Treg in mediating sex
differences in MS incidence or EAE disease activity.
7.10 Sex Differences in T Cell IL-10 Production
Though IL-10 was originally described as a Th2 cytokine (Fiorentino et al. 1991), it
is also expressed by other regulatory T cells including Tr1 cells and has protective
functions in EAE (Bettelli et al. 1998). In this regard, male myelin-specific T helper
cell lines have been reported to produce higher levels of IL-10 compared to female T
cell lines (Bebo et al. 1999). It has been also observed that treatment of SJL mice
with 5-α dihydrotestosterone, the high-affinity agonist of the androgen receptor,
increases the potential of CD4+ T cells to produce IL-10 in response to anti-CD3 and
anti-CD28 (Liva and Voskuhl 2001). Together, these findings hint that male sex and
androgens enhance T cell IL-10 production, which would be expected to counter the
development of autoimmunity. More research is needed to delineate which male T
cell subset is the major producer of IL-10.
7.11 Sex Differences in CD8+ T Cells in MS Risk and MS

Progression
Though CD4+ T cells have been the focus of most MS immunological studies,
pathological studies have revealed that CD8+ T cells outnumber CD4+ T cells in
white matter and cortical demyelinating lesions in MS brain specimens (Fransen
et al. 2020; Skulina et al. 2004; Salou et al. 2015) and that CD8+ T cells exhibit
evidence of more extensive clonal expansion than CD4+ T cells in the CNS (Babbe
et al. 2000; Jacobsen et al. 2002). Males with MS exhibit higher TCR variable beta
chain perturbation in the CD8+ T cell compartment, indicative of increased clonal
expansion compared to females (Schneider-Hohendorf et al. 2018). Pathological
studies have shown that CD8+ T cells persist in the perivascular cuffs at the rim of
the chronic-active lesions and in the normal appearing white matter (Crawford et al.
2004). These CD8+ T cells show features of a tissue resident effector memory
phenotype (Fransen et al. 2020) with high expressions of granzyme B, IL-17,
GM-CSF, and IFN-γ (Ifergan et al. 2011; Annibali et al. 2011; Jilek et al. 2007).
Two large post-mortem studies in MS detected that males had a higher proportion
of chronic-active lesions relative to inactive lesions compared to females (Frischer
et al. 2015; Luchetti et al. 2018), further suggestive of more chronic T cell inflam-
mation in men with long-standing MS disease. Thus, smouldering CD8+ T cell-
mediated inflammation in white matter lesions in the CNS could be a factor
contributing to the more rapid progression seen in men with MS.
Polymorphisms in the perforin gene (Prf1) that associate with reduced perforin
expression and cytotoxic activity contribute to a higher risk of MS, but only in males
(Camina-Tato et al. 2010). It was speculated that this effect relates to defective viral
clearance in the males (Camina-Tato et al. 2010). In this regard, a sex difference in
viral clearance and its impact on CNS autoimmunity has been studied in the
Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination model.
It was shown that male mice of various strains (SJL, C57L/J, SWR/J) are more
susceptible to TMEV-induced demyelination than female mice, and that this related
to a weaker T cell and neutralizing antibody response and chronic viral persistence in
the males (Alley et al. 2003; Butterfield et al. 2003; Fuller et al. 2005; Kappel et al.
1990). Thus, having weaker cytolytic T cell activity in males could increase the
likelihood of MS if indeed MS is triggered by a viral infection.
7.12 Sex Differences in NK Cells as Contributors

to the Increased Tissue Damage in Males with MS
NK cells can be subdivided into two major subtypes according to the expressions of
CD16 and CD56. The CD56low (CD16+) subset has high levels of perforin and
potent cytotoxic and pro-inflammatory activity, whereas the CD56bright (CD16)
subset is considered to have more of a regulatory function (Gross et al. 2016) and
these cells are associated with protection from EAE and MS (Kucuksezer et al.
2021). CD56low NK cells are increased (Plantone et al. 2013), while regulatory
CD56bright NK cells are decreased (Lunemann et al. 2011) in the blood in
MS-affected individuals, suggesting that an inbalance in NK subsets may be a
contributor to the autoimmune attacks in MS.
Only one study has examined sex differences in the proportions of NK subsets
and NKT cells in the blood of male and female healthy and MS donors. Whereas no
sex differences were found in total NK or NKT cells (de Andres et al. 2017),
CD56low NK cells and NKT cells were found to be in a higher state of activation
as assessed by CD69 expression in the males with MS compared to healthy male
counterparts (de Andres et al. 2017). In addition, MS-affected females also had a
higher frequency of CD56bright NK cells compared to healthy females (de Andres
et al. 2017). This finding is consistent with there being more NK regulatory cell
activity in females, and more cytotoxic NK activity in males with MS. This finding
does not explain the sex difference in MS onset, but could go further in explaining
why MS progresses more rapidly in males.
7.13 Sex Differences in B Cells: Humoral Responses Are

More Robust in Females
The remarkable success of anti-CD20 B cell depletion therapies in preventing MS

relapses has highlighted the crucial involvement of B cells in MS (reviewed in Roach
and Cross (2020)). B cells act as APC to DR2a/2b-restricted T cells in MS (Jelcic
et al. 2018) and can infiltrate the brain where they are detected both in the
perivascular cuffs in white matter lesions and in tertiary lymphoid-like structures
located in the submeningeal spaces of the brain (Mitsdoerffer and Peters 2016).
CNS-resident plasma cells are thought to be the source of oligoclonal immunoglob-
ulin (Ig) detected in the CSF of the Ig deposited in white matter lesions (Lucchinetti
et al. 2000).
Despite the importance of B cells in MS, studies examining sex differences in
autoantibody production in either EAE or MS are scarce. Consistent with the idea
that humoral immunity is stronger in females, males with MS are more likely to be
negative for oligoclonal bands in the CSF (Karrenbauer et al. 2021). Furthermore, in
neuromyelitis optica, a CNS autoimmune disorder that is distinct from MS, females
exhibit higher levels of total IgM and anti-MOG and anti-aquaporin-4 IgG anti-
bodies in the CSF (Kunchok et al. 2020). Also consistent with humoral immunity
being more robust in females, healthy females exhibit a higher number of B cells and
IgM producing memory B cells and IgM levels in the circulation compared to
healthy males (Abdullah et al. 2012; Aguirre-Gamboa et al. 2016; Trend et al.
2018). Antibody responses to vaccination are also stronger in female humans
(Klein et al. 2015) and female mice (Eidinger and Garrett 1972) compared to male
counterparts. This stronger humoral immunity in females is likely in part driven by
the more robust T helper immunity in this sex, but could be also driven by known
effects of E2 in decreasing B cell tolerance and promoting antibody production
(reviewed in Cohen-Solal et al. (2008)). More studies are needed to study the effect
of sex on B cell features that are important in MS including antigen presentation,
cytokine, antibody production, and regulatory function.
7.14 Sex Differences in the Trafficking of T Cells Across

the BBB
The BBB is formed by endothelial cells of the CNS micro-vessels, which are
strongly connected to one another by tight junction proteins. This endothelial barrier
is further supported by pericytes and the terminal processes of astrocytes, which
together form the glia limitans (reviewed in Sweeney et al. (2019)). The BBB
functions to prevent toxins, blood cells, and pathogens from entering the CNS,
and alterations in its integrity are an early feature of inflammatory lesions in MS
(Filippi et al. 1998) and EAE (Alvarez et al. 2015). As overviewed earlier, there is
evidence from MRI findings that females with MS show more BBB disruption
compared to male counterparts as determined by the increased presence of

Gd-enhancing lesions (Pozzilli et al. 2003).
There are a number of reasons why females may exhibit increased BBB perme-
ability during CNS autoimmunity. It could relate to the increased myelin-specific
Th1 cell inflammation seen in females. Th1 cells, upon reaching the CNS, can induce
the activity of matrix metalloproteinase (MMP)-9, which is an enzyme that is
important for breaking down the extracellular matrix at the BBB (Abraham et al.
2005). Indeed, the levels of MMP-9 and MMP-1 are increased in the CSF and blood
in females, but not males with RR-MS (Castellazzi et al. 2018; Hamedani et al.
2016). Also, murine studies have shown that female myelin-specific Th1 cells
express higher levels of β1 integrin than male T cells. β1 integrin associates with
α4 integrin to form VLA-4 (Brahmachari and Pahan 2010), a critical molecule
involved in the trafficking of Th1 cells across the BBB in the CNS (Rothhammer
et al. 2011).
Increased BBB permeability could also be mediated by sex differences in the
BBB endothelial cells. In the SJL model of EAE, higher BBB permeability in female
mice is associated with higher expression of the sphingosine-1-phosphate receptor
2 (S1PR2) in female endothelial cells; S1PR2 activates the ROCK signaling path-
way, which can break tight junctions in the BBB (Cruz-Orengo et al. 2014). This
same study also reported that S1PR2 expression was higher in the CNS endothelium
of female compared to male MS brain specimens. In contrast to these findings,
another study that used the same sex TCR1640 T cells to induce EAE in male and
female SJL mice observed no difference in the kinetics of leakage of the BBB as
assessed by staining for the blood product fibrinogen in the CNS parenchyma
(Dhaeze et al. 2019), supporting the concept that the sex difference in BBB leakage
may occur as a result of the sex differences in the phenotype or number of myelin-
specific T cells infiltrating in the CNS.
7.15 Sex Differences in Microglia Phenotype
Microglia are the brain-resident macrophages that function to maintain CNS homeo-
stasis and to clear apoptotic debris (Butovsky and Weiner 2018). In EAE and MS,
these cells sense the presence of invading T cells by purinergic and cytokine
receptors and adopt a more activated phenotype (Butovsky and Weiner 2018).
Upon activation, microglia upregulate MHC-II and can contribute to myelin and
axon damage in EAE or MS through production of TNF or nitric oxide or by
producing chemokines that recruit other immune cells to the lesion site. These
cells also function in the resolution of inflammation by clearing myelin and neuronal
debris (Bogie et al. 2014).
Most post-mortem studies have not distinguished microglia from macrophages in
the CNS; however, those that examined the density of macrophages/microglia in the
MS brain noted no differences between male and female specimens (Kuhlmann et al.
2009; Zrzavy et al. 2017; Jackle et al. 2020). However, certain markers of
macrophage/microglia activation, including chitinase-3-like protein 1 and monocyte

chemoattractant protein were present at higher levels in the CSF in females than in
males living with MS (Martinez et al. 2015). Chitinase-3-like protein 1 is a marker of
microglial activation associated with earlier progression to severe disability in MS,
whereas monocyte chemoattractant protein (also known as CCL2) is a key factor that
attracts inflammatory monocytes into lesions (Martinez et al. 2015). These data
suggest a potential for enhanced macrophage or microglial cell activity in females
in MS.
Since T cell-mediated CNS inflammation tends to be higher in female mice with
EAE (Bebo et al. 1996), it has been difficult to discern whether sex differences in
microglia activation are due to sex differences in T cell recruitment or phenotype or
to an increased reactivity of the female microglia to the invading T cells. In this
regard, it has been reported that MBP-reactive murine Th cells from female SJL mice
are more likely to induce the production of nitric oxide and pro-inflammatory
cytokines by sex-matched microglia in culture (Dasgupta et al. 2005). Interestingly,
the male T cells gained these pro-inflammatory activities on microglia when they
were sourced from castrated male mice, suggesting that the T cells were specifying
the sex difference in the microglia response (Dasgupta et al. 2005).
To gain insights into intrinsic differences in microglia function, studies have
profiled sex differences in microglia gene expression in healthy mice and in murine
models of neurodegenerative disease (reviewed in Hanamsagar and Bilbo (2016)).
Most of these studies report sex differences in microglia phenotype, but the findings
do not appear to be consistent. For example, a study that conducted RNA sequencing
analysis on microglia from healthy young adult male and female mice detected
546 genes to be differentially expressed between male and female microglia
(Guneykaya et al. 2018). Interestingly, more than a third of the genes enriched in
the male microglia were involved in inflammatory processes including cell migra-
tion, cytokine production, and MHC-I and -II expression and were enriched for
binding sites for NF-κB and RUNX1. Female microglia instead exhibited higher
expression of genes associated with pathways of morphogenesis, development, and
cytoskeletal organization. This study further found that these sex-based differences
in microglia gene expression were not sensitive to E2 modulation and were
maintained even after in vitro culture or after transplant into mice of the opposite
sex (Guneykaya et al. 2018), suggesting that they were specified early in develop-
ment, prior to puberty.
Somewhat consistent with these findings, a study that evaluated sex-based dif-
ferences in microglia gene expression at different stages of development (up to post-
natal day 60) reported that male microglia are more reactive than female microglia to
LPS treatment in vitro. These authors attributed this sex difference in microglia
activation to a delay in the maturation status of the male microglia (Hanamsagar et al.
2018). Developmental effects on microglia gene expression were also observed in
another transcriptional profiling study of microglia in mice (Thion et al. 2018).
However, in contrast to the conclusions of these other studies, young adult female
microglia were found to be more pro-inflammatory and pro-immune than male
microglia, expressing higher levels of genes involved in type I IFN signaling
(Thion et al. 2018). Similar to these findings, a study of cultured microglia reported
that microglia harvested from young female mice exhibited higher expression of Tnf,
Il6, Tlr2, and Tlr4 mRNAs compared to male microglia in response to stimulation
with IFN-γ (Guneykaya et al. 2018). In addition, a study that evaluated the mor-
phology, gene expression, and metabolism of microglia in aged WT and APP/PS1
transgenic mice (a model of Alzheimer’s) noted very striking sex differences in the
gene expression and metabolic state of microglia (Guillot-Sestier et al. 2021). While
female microglia had higher glycolytic metabolism, male microglia had a more
amoeboid appearance and increased phagocytic potential (Guillot-Sestier et al.
2021). Furthermore, a 18-kDa translocator protein positron emission tomography
(TSPO-PET) study of aged, but otherwise healthy male and female mice reported
that females exhibited higher microglia activity as detected by TSPO signal and
Iba-1 immunoreactivity in the cortex at 12–14 months of age compared to male mice
(Biechele et al. 2020).
Overall, there is evidence that the metabolic activity of microglia is higher in
females compared to males with age. Further there is evidence that female microglia
exhibit a higher type I IFN signature and are more responsive to the T cell cytokine
IFN-γ production compared to male microglia. On the other hand, male microglia in
the steady state exhibit higher NF-κB activity and are more sensitive to the
LPS-induction of inflammatory gene expression. Thus, there exist sex differences
in microglia gene expression that vary according to the age of mice, and the
inflammatory context or disease being studied. It still remains to be understood
whether these sex differences in microglia phenotype contribute to the sex differ-
ences in EAE or MS.
8 Conclusions
There is a clear female bias in MS development that manifests post-puberty. There is

also evidence that peripheral immune mechanisms leading to BBB breakdown and
white matter lesion formation are more robust in females than males at the earliest
stages of disease. Similar to MS, sex differences in CNS lesion formation are
apparent in EAE in commonly studied mouse strains and appear to be largely driven
by sex differences in gonadal hormone production. Sex differences in the immune
system can explain these sex differences in EAE and MS (summarized in Figs. 3 and
4). Females show an increased expansion and differentiation of Th1 cells in MS and
EAE. Pro-inflammatory Th1 cells expressing adhesion molecules such as VLA-4 are
more likely to traffic across the BBB into the CNS parenchyma. On the other hand,
males exhibit a Th2-skewed immune response and higher IL-10 production, which
are known to protect against autoimmunity. Compared to these immune mecha-
nisms, sex differences in B cells, CD8+ T cells, and other immune cell types are
relatively understudied in MS. It also remains to be determined how the major
genetic risk factor for MS, the HLA-DR15 haplotype, contributes to sex differences
in MS.
Fig. 3 Summary of sex differences in clinical course, CNS mechanisms, and peripheral immune
responses related to the onset of MS or EAE. Red and blue lettering and symbols indicate what
features are higher in female and males, respectively
Fig. 4 Summary of immune mechanisms that differ between the sexes to impact CNS autoimmune
disease. Myelin-reactive Th cells escape negative selection in the thymus and enter the peripheral T
cell pool in both males and females; however, there are sex differences in Aire expression in thymic
epithelial cells that may contribute to sex differences in central tolerance. After immunization,
APCs loaded with myelin peptides and activated by CFA prime myelin-reactive Th cells in the
peripheral lymphoid organs (draining lymph node and spleen) and promote the differentiation of
these cells into Th lineage cells. Female myelin-specific Th cells also expand more than male Th
cells during EAE. Female Th cells are more prone to becoming Th1 cells, whereas male Th cells are
more prone to becoming Th2 or Th17 cells. These sex differences may relate in part to higher
production of IL-12p40 and lower production of IL-10 by female APC and a higher potential of the
female Th cells to secrete IFN-γ. Th1 and Th17 effector cells traffic through the blood and spleen,
among other organs, before reaching the CNS. They cross the BBB and become activated by
dendritic cells presenting myelin antigens and produce cytokines and chemokines, which activate
microglia and facilitate the migration of additional immune cells into the CNS, which mediate
demyelination and axon injury. Female Th1 cells may traffic more efficiently across the BBB
endothelium as a result of higher expression of beta 1 integrin and higher IFN-γ induced MMP-9
expression. The BBB endothelium in females is more disrupted as a result of this inflammation.
Once on site, Th1 cells elicit NO and TNF production by microglia. Females show increased
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Current Topics in Behavioral Neurosciences 62

Sex Differences in Brain

ISSN 1866-3370 ISSN 1866-3389 (electronic)

Nottingham, UK Claire Gibson

8. Zucker I, Prendergast BJ (2020) Sex differences in pharmacokinetics predict adverse drug

Part I Considering Sex and Gender in Research Studies

Part II Sex Differences in Cognitive and Mental Health Across the

Sex Differences in Cognition Across Aging . . . . . . . . . . . . . . . . . . . . . . . 235

Part III Diseases of the Brain and Relevance of Sex

Alana Brown, Laurice Karkaby, Mateja Perovic, Reema Shaﬁ,

A. Brown (*), L. Karkaby, M. Perovic, and R. Shaﬁ

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

Keywords Gender · Qualitative · Quantitative · Research methods · Science · Sex ·

1 Sex and Gender Science: The World Writes on the Body

qualitative research can generate questions to be probed further by quantitative tools

2 Integrating Objective and Subjective Sleep Measures

2.1 The Role of Sex and Gender in Explaining Misalignment

measures, with men demonstrating greater correspondence. Therefore, what we

2.2 Considering Gender Might Explain Objective

In tandem with sex-related factors, gender-related factors – often disregarded – also

experiential accounts with objective measures through qualitative interviewing

3 Integrating Subjective Experiences in Memory Research

This participant mentioned unexpected cognitive impacts of oophorectomy,

4 Neuropathic Pain and Very Mixed Methods

Qualitave Short-Form of Sensory Tesng

5 Sex and Gender in Concussion

5.1 Gender in Repetitive Brain Injuries

While sex-based biological variations can lead to different cascades of damage

5.2 Contextualizing Differences

5.3 The Intersection of Sex and Gender in Concussion

One often-overlooked biological player that contributes to sex differences in recov-

effecting post-injury recovery outcomes, including cognition and quality of life

intricate neuro-landscape. We will never understand this complexity in its totality,

Conﬂict of Interest: We have no known conﬂict of interest to disclose.

Mckee AC, Abdolmohammadi B, Stein TD (2018) The neuropathology of chronic traumatic

prospective elderly cohort study. Aging (Albany NY) 12:21942–21958. https://doi.org/10.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 27

Keywords Biomedical and health research · EU gender equality policy · Gender

biomedicine. Finally I will highlight best practices in the study of gender-related

2 Exposing the Gender Bias in Traditional Biomedical

3. Gendered Innovations framework: coined as term by Londa Schiebinger in 2008

3 Introducing Sex and Gender into Research

4 EU Research Policy 2000–2010

4.1 Gender as a Multidimensional Concept

By 2010 gender was further deﬁned as referring to socio-cultural norms, identities,

5 EU Research Policy 2010–2020

5.1 Comparing Research Policy Initiatives of International

Policies similar to the EU GE policy to support the research community in incorpo-

Various initiatives can be considered as best practices. According to a deﬁnition for

6.1 Gender Medicine as a Scientiﬁc Field of Its Own

Parallel to the EU developments in the ﬁrst decade of the twenty-ﬁrst century,

6.2 Measuring Gender

When science funding bodies launched their initiatives to incorporate attention to

6.3 Curriculum Development

Universitätsmedizin) Berlin; European Institute of Women’s Health Dublin, Ireland;

6.5 (Online) Trainings

6.6 Editorial Policies of Journals

7.1 Diversity and Inclusion

surveillance or unlocking phones. Despite efforts by academic and industrial

7.2 Evaluation Criteria and Monitoring of Policies

Monitoring the implementation of a policy is crucial. The Canadian IGH evaluated

Phoebe D. Edwards, Ilapreet Toor, and Melissa M. Holmes

Abstract Research in the neurobiology of sex differences is inherently inﬂuenced