Article

Preclinical models in the study of sex differences

Abstract and Figures

The biology of sex differences deals with the study of the disparities between females and males and the related biological mechanisms. Gender medicine focuses on the impact of gender and sex on human physiology, pathophysiology and clinical features of diseases that are common to women and men. The term gender refers to a complex interrelation and integration of sex - as a biological and functional determinant - and psychological and cultural behaviours (due to ethnical, social or religious background). The attention to the impact of gender differences on the pathophysiology and, therefore, on the clinical management of themost common diseases, such as cardiovascular diseases (CVD), neurodegenerative disorders, immune and autoimmune diseases as well as several tumours, is in fact often neglected. Hence, studies covering different fields of investigation and including sex differences in the pathogenesis, in diagnostic and prognostic criteria as well as in response to therapy appear mandatory. However, prerequisites for this development are preclinical studies, including in vitro and in vivo approaches. They represent the first step in the development of a drug or in the comprehension of the pathogenetic mechanisms of diseases, in turn a necessary step for the development of new or more appropriate therapeutic strategies. However, sex differences are still poorly considered and the great majority of preclinical studies do not take into account the relevance of such disparities. In this review, we describe the state of the art of these studies and provide some paradigmatic examples of key fields of investigation, such as oncology, neurology and CVD, where preclinical models should be improved.
Content may be subject to copyright.
Clinical Science (2017) 00 1–21
DOI: 10.1042/CS20160847
*Tobeconsideredasfirst
authors.
Received: 7 November 2016
Revised: 12 December 2016
Accepted: 3 January 2017
Accepted Manuscript Online:
xxxx xxxx
Review
Preclinical models in the study of sex differences
Maria Buoncervello1,*, Matteo Marconi2, *, Alessandra Car `
e3, Paola Piscopo4, Walter Malorni2,5
and Paola Matarrese2,5
1Section of Experimental Immunotherapy, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanit`
a, 00161 Rome, Italy; 2Section of Gender
Medicine, Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanit `
a, 00161 Rome, Italy; 3Section of Molecular Oncology, Department of
Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanit`
a, 00161 Rome, Italy; 4Section of Clinical, Diagnosis and Therapy of CNS Degenerative Diseases,
Department of Neurosciences, Istituto Superiore di Sanit `
a, 00161 Rome, Italy; 5Center of Gender-Specific Medicine, Istituto Speriore di Sanita’, 00161 Rome, Italy
Correspondence: Walter Malorni (malorni@iss.it)
The biology of sex differences deals with the study of the disparities between females and
males and the related biological mechanisms. Gender medicine focuses on the impact of
gender and sex on human physiology, pathophysiology and clinical features of diseases
that are common to women and men. The term gender refers to a complex interrelation and
integration of sex – as a biological and functional determinant – and psychological and cul-
tural behaviours (due to ethnical, social or religious background). The attention to the impact
of gender differences on the pathophysiology and, therefore, on the clinical management of
the most common diseases, such as cardiovascular diseases (CVD), neurodegenerative dis-
orders, immune and autoimmune diseases as well as several tumours, is in fact often neg-
lected. Hence, studies covering different elds of investigation and including sex differences
in the pathogenesis, in diagnostic and prognostic criteria as well as in response to therapy
appear mandatory. However, prerequisites for this development are preclinical studies, in-
cluding in vitro and in vivo approaches. They represent the rst step in the development of a
drug or in the comprehension of the pathogenetic mechanisms of diseases, in turn a neces-
sary step for the development of new or more appropriate therapeutic strategies. However,
sex differences are still poorly considered and the great majority of preclinical studies do not
take into account the relevance of such disparities. In this review, we describe the state of
the art of these studies and provide some paradigmatic examples of key elds of investiga-
tion, such as oncology, neurology and CVD, where preclinical models should be improved.
Background
The study of the biology of sex differences as well as the development of gender medicine (GM) is a mile-
stone in the advancement of our knowledge in the different fields of biomedical sciences. In fact, among
thesefieldsofinvestigationarealltheresearcheffortsheadingfortheimprovementofourknowledgeas
concerns the pathogenetic mechanisms of human diseases and the appropriateness of medical interven-
tionsinclinicalpractice.Itmeansthatthedevelopmentorprogressofcurescouldtakeadvantagefrom
the information of sex and gender disparities in order to ascertain and develop differential interventions
between women and men. Since the number of human pathologies displaying a significant sex/gender
disparity is rapidly growing (Tab l e 1), the needs for investigations specifically devoted to point out the
mechanisms underlying sex or gender differences appear mandatory, and the number of works published
in the field underscores this assumption.
Human pathological conditions that have been demonstrated to display a gender disparity are really im-
pressive: transmissible and non-transmissible diseases have been investigated in a series of studies aimed
at the analysis of the incidence, progression or outcome of very important diseases, such as viral infect-
ions, cardiovascular, neurodegenerative, metabolic, respiratory, autoimmune diseases and several forms
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 1
Clinical Science (2017) 00 2–21
DOI: 10.1042/CS20160847
Table 1. Human pathologies displaying a significant sex/gender disparity. Some examples of very important
diseases, either transmissible or non-transmissible, which show significant gender differences in terms of
incidence, progression or symptoms.
Gender differences
Disease Incidence Progression Symptoms Example
Cardiovascular Yes Yes Yes Infarct
Neurodegenerative Yes No Yes Alzheimer’s
Autoimmune Yes Yes No Lupus erythematosus
Infectious Yes Yes No Hepatitis B
Cancer Yes Yes No Melanoma
Respiratory Yes No No Chronic obstructive pulmonary disease (COPD)
of cancer [1]. In addition, numerous works underscored sex/gender differences in response to therapy, either for in-
fections, e.g. antiviral therapy, or for non-transmissible diseases such as cancer. Finally, a disparity has been described
concerning the adverse effects of pharmacological therapies and for iatrogenic diseases. Being out of the scope of the
present work, details of these disparities are simply mentioned here.
A number of works and reviews dealing with these issues can be found in recent literature. For example, the works
of the group of Regitz-Zagrosek extensively tackled the cardiovascular matter, at least partially elucidating the key
mechanisms underlying the delayed and different features of cardiovascular diseases (CVD) in women [24]. In the
same vein, other authors started to shed light on the reasons for gender disparity in autoimmune or neurodegen-
erative diseases [5,6]. However, the biological mechanisms underlying these gender differences are far from being
elucidated and surely need further experimental investigations. In particular, to gain this goal, preclinical studies
need a reappraisal and a ‘re-shaping’, either as concerns in vitro studies or in vivo studies in proper animal models
[7]. In few words, there is room for all research approaches in this new field called ‘GM’ as well as in the ‘biology
of the sex differences’ [8].Ofrelevanceonthismatterarethe‘RecommendationsconcerningthenewU.S.National
Institutes of Health initiative to balance the sex of cells and animals in preclinical research, recently proposed at the
Georgetown Consensus Conference and specifically devoted to the implementation and support of preclinical studies
taking into account sex in NIH research policy [9].
Gender–sex premise
With regards to the terms sex and gender, they are usually referred to the biological or to the socio-cultural issues,
respectively: studies in animals show ‘sex differences’ whereas ‘gender differences’ are found in human studies in
which environment, self-perception, etc., can be taken into account [10], as also stated by the Canadian Institute
of Health Research, http://www.cihr-irsc.gc.ca/e/8673.html. Experimental studies on sex differences are essentially
based on three different approaches: research in in vitro models, i.e. cultured cells or organ cultures; analyses carried
out ex vivo, e.g. in cells of peripheral blood and classical preclinical studies carried out in animal models. In particular,
as far as cultured cells are concerned, they are usually used for drug screening or mechanistic studies (see below).
The cells continuously communicate with each other so that an alteration of one cell can propagate to the others.
Hence, this kind of study requires specific analyses on cell–cell as well as cell–substrate interactions. In particular,
cell adhesion pathways and communication with other cells can include either sibling cells (homotypic interactions)
or other cell types (heterotypic interactions). Two examples: cultured cells normally adhere with each other and to
a substrate, so that the propagation of a proliferating or, conversely, of a death signal is very rapid and effective. As
concerns the ex vivo experimental models, the most paradigmatic example is represented by immune system cells that,
belonging to different subpopulations with different roles, need to continuously communicate. This whole scenario
can be greatly influenced by the sex. Therefore, even in cytology and cytopathology studies, the sex of the cells (XX
or XY) must be taken into account.
In this review, we will recapitulate these issues and discuss the possibility to disclose novel scenarios in the devel-
opment of diagnostic and therapeutic interventions if and when preclinical studies, e.g. pharmacological and toxico-
logicalones,wouldtakeintoaccountbothsexes.
Animal studies
Despite decades of research demonstrating sex differences in animal research, many studies are still performed with
one sex only [9,11]. The bias towards using only animals of one sex is often based on quite practical reasons: male
2c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 3–21
DOI: 10.1042/CS20160847
mice are larger and thus offer easier targets for attaching electrodes, e.g. in neurological studies, and they do not
have oestrous cycles that can complicate pharmacology, toxicological studies in particular. In contrast, females are
less aggressive and easier to handle; they are smaller, requiring less weight-administered drug and they are generally
less expensive. The importance of such differences varies by strain and by what is under analysis. It is possible, in
fact, that males are more responsive in some strains and females in others. For this reason, experimental studies
performed by using animals of one sex only might miss some of these differences. For instance, a research team that
is mainly testing drugs on male animals might miss beneficial aspects restricted to females contributing to a lack
of consistency in experimental results. Also, the costs of not taking sex into account in preclinical studies should
be considered: these include failed clinical trials, misdiagnosis and inappropriate therapies for women, as well as
omission of fundamental biological principles [12]. On these bases, NIH policy recently encouraged applicants to
consider studying both females and males in preclinical biological experiments [13]. This suggestion would avoid
research groups assuming no sex difference or ignoring one sex entirely and underscore the relevance of this issue
in the development of science. Finally, recent editorial issues asking scientific journals to include both sexes in the
experimental analyses or, at least, specify the sex of mice used have recently been published [14]. In few words, the
‘instruction to authors’ of scientific journals taking into account the sex issue and asking for specific attention to use
male and female animals could help in the advancement of experimental medicine.
In vitro studies
In the past century, after the establishment of the first cell lines obtained from human cancers, a series of catalogues
of cell lines of different histotype has been made available. More importantly, the widespread use of disposable ma-
terials allowing easier, fast and, mainly, reproducible in vitro evaluationsystemsmadetheuseofculturedcellsthe
most suitable investigation tool in the field of experimental medicine [15,16]. In Tabl e 2,themostusedandpromin-
ent cell lines in experimental studies are listed in order to point out their uselessness as tools for studies taking into
account the sex issue. For many years, cultured cells, usually tumour cells, represent the first step for the screening of
drugs or toxicants. In addition, a mass of studies aimed at the comprehension of the mechanism of action of drugs
or toxicants, as well as the elucidation of genetic or biochemical cell pathways has been carried out in these cell lines.
More recently, several non-tumour cells have been cultured in vitro, thus providing further information concerning
the physiological and pathological features of various cell types, including vessel cells, e.g. fibroblasts, endothelial or
vascular smooth muscle cells, as well as other cell types, such as neuronal cells. An important limit of these primary
cultures of non-transformed cells in experimental studies is that, unlike tumour cell cultures, they can survive in vitro
for few subseedings (5–7 subseedings). In fact, thereafter, they stop growing and are considered to lose their histo-
specificity and their ‘sex memory’ (e.g. their sex-specific chromosomal features are lost, see below). A further series
of cultured cell models, such as co-cultures of cells of different types, or cells stably or transiently transfected in order
to increase or decrease the expression levels of certain proteins, are also available. These models generally appear
irrespective of the original sexual features of these cells.
In this trivial description of the state of the art of cell cultures, several additional pitfalls must be underscored. One
example is represented by the reproducibility of the results obtained with the same cell line in different laboratories.
Some years ago it was suggested that the same cell line from the same collection could provide dissimilar results in
parallel identical experiments performed in different labs. This was due, at least partially, to the genetic instability
or to post-transcriptional changes occurring after several in vitro subseedings, due to the selection of various clones
of the same cell line in different laboratories. In fact, cell lines with an abnormal number of chromosomes or with
constitutive overexpression or the lack of growth factor or hormone receptors can usually be found. For these reasons,
a growing number of scientific journals require authors to check authentication of the origin and identity of the cells
by DNA profiling. Also, for tumour cells freshly isolated from patient tissue or in the case of a new cell line, the DNA
profileneedstobecross-checkedwiththeDNAprofileofthedonortissue.Thiscouldbeofgreatrelevanceinorder
to develop sex-specific treatments, as recently suggested for chemotherapy [17,18].
On these bases, it appears clear how difficult it is to conduct cellular physiopathology studies in which the cell
functions or dysfunctions, object of the research, may be related to the cell sex by using stabilized cell lines. It means
that a comparative analysis of the same cell type should be conducted on male or female freshly isolated cells to point
out a possible sex-specific pathway. In few words, to get reproducible and sex-tailored results in basic or translational
science, cell models that maintain sexual specificity should be considered. Moreover, in line with the fundamental
review paper by Shah et al. [15], it is clearly inappropriate to assume that results from studies conducted on only one
sex will apply wholesale to the other.
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 3
Clinical Science (2017) 00 4–21
DOI: 10.1042/CS20160847
Table 2. Cell lines of widespread use in experimental studies. The salient features of these cell lines are
listed in order to point out their uselessness as tools for studies taking into account sex differences (see
text for details). Cells indicated as normal or noncancerous are often virally transformed. This table is not
intended to be a comprehensive data set, but rather to highlight the cell lines that are routinely used.
Name Morphology Species, sex, age Origin Isolation date
HeLa Epidermoid Human, female, 31 Adenocarcinoma of the cervix, HPV-18 +1951
HEp-2 Epidermoid Human, male Carcinoma of the larynx, HPV +, 1952
contaminated with HeLa
CHO-K1 Epithelial Chinese hamster, female Ovary 1958
3T3-L1 Fibroblast Mouse embryo, sex unknown Fibroblast 1962
Vero Epithelial Green monkey, sex unknown Kidney 1962
Raji Lymphoblast Human, male, 11 Burkitt’s lymphoma, B-cells EBV +1963
CEM Lymphoblast Human, female ALL, T-cells, EBV +1964
HT-29 Epithelial Human, female, 44 Colorectal adenocarcinoma 1964
MDCK Epithelial Dog, sex unknown Kidney 1966
U-87 MG Epithelial Human, male, 44 Glioblastoma 1968
Jurkat Lymphoblast Human, male, 14 ALL, T-cells 1970
MCF7 Epithelial Human, female, 69 Breast cancer 1970
SH-SY5Y Epithelial Human, female, 4 Neuroblastoma 1970
A549 Epithelial Human, male Lung carcinoma 1972
A-375 Epithelial Human, female, 54 Malignant melanoma 1973
Saos-2 Epithelial Human, female, 11 Osteosarcoma 1973
U937 Monocyte Human, male, 37 Histiocytic lymphoma 1974
K-562 Lymphoblast Human, female, 53 CML 1975
H9c2 Myoblast Rat embryo, sex unknown Heart tissue 1976
PC12 Irregularly Rat, male Pheochromocytoma 1976
shaped cells
CaCo-2 Epithelial Human, male Colon cancer 1977
HL-60 Myeloblastic Human, female, 36 Acute promyelocytic leukaemia 1977
IEC6 Epithelial Rat, male Small intestine 1979
NCI-H292 Epithelial Human, female, 32 Mucoepidermoid pulmonary carcinoma 1985
PNT1A Epithelial Human, male, 35 Prostate 1991
HEP G2 Epithelial Human, male Liver cancer 1994
Cor.4U Cardiomyocyte Human, female, 26 In vitro differentiated stem cells Recently isolated
Some paradigmatic examples
Thesex-tailoredapproachcouldbeofgreatimportanceinthescreeninganddevelopmentofnewdrugsortherapeutic
strategies that could take into account the differences between XX and XY cells. Some specific examples come from
literature data reporting ‘cell-sex’ as a key variable in the response to exogenous agents [1517]. In a series of works,
our group underscored the disparity of cells from males (XY) and females (XX) in their response to an exogenous
stress [4,1922]. Primary cultures of cells of different histotypes from different animal species, such as endothelial
cells, smooth muscle cells, fibroblasts, neuronal cells from mice, rats or humans provided a number of results that
can be summarized as follows: under the same stressing conditions, somewhat mimicking an inflammatory state,
cells from males undergo cell death more easily (apoptotic triggering), whereas cells from females, thanks to a potent
antioxidant power, counteract the stressing molecules, i.e. reactive oxygen species, and survive better. Some works
also refer to the XX and XY cell disparity as a difference in metabolic response. For instance, during starvation con-
ditions, neurons from males more readily decreased mitochondrial respiration undergoing death, whereas neurons
from females mobilize fatty acids, accumulate triacylglycerols, form lipid droplets and survive longer [21,23,24]. This
represents a key issue of in vitro cultured system investigations: the different ‘frailty’ of XX and XY cells, the former
displaying a higher plasticity to environmental stress, that results in a higher rate of survival or proliferation, through
the cytoprotection mechanisms of autophagy and senescence, the latter more easily undergoing the programmed cell
death pathway [19,21,22,24].
4c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 5–21
DOI: 10.1042/CS20160847
Table 3 . The main ex vivo systems useful to investigate the pathogenetic mechanisms of diseases.
Maintaining ‘sex memory’ Experimental
Cell type Animal species (number of passages) tools for
Fibroblasts Human, mouse, rat 10 CVD, autoimmune
Vascular smooth muscle cells (VSMC) Human, mouse, rat 10 CVD, gastroenterology
Keratinocytes Human 8–10 Dermatology
Endothelial cells Human (from umbilical cord) 10 Vascular
Cardiomyocytes Rat, mouse 5CVD
Central nervous system cells Rat, mouse Neurodegeneration
(neurons, glia, astrocytes)
Mouse embryo fibroblasts (MEF) Mouse 15–20 Cell biology, cell pathology
Freshly isolated cancer cells Human, mouse 10–15 Experimental oncology
Peripheral blood lymphocytes Human Immunity, inflammation,
experimental oncology
Platelets Human Haematology, CVD
Red blood cells Human Haematology, CVD
Ex vivo studies
The term ex vivo refers to an experimental model consisting of cells or tissues from an organism kept in an external
environment with minimal alteration of ‘natural’ conditions. Ex vivo systems allow us to experiment on freshly isol-
ated cells under more controlled conditions than in in vivo models. The analysis of peripheral blood cells certainly
represents a great tool for translational studies in various fields of investigation, such as research for diagnostic and
prognostic purposes as well as for investigations of the pathogenetic mechanisms of diseases. Although inflammatory,
immune and autoimmune diseases can be considered as paradigmatic fields of investigation, a number of haematolo-
gic diseases or disorders leading to peripheral blood cell alterations also represents a milestone. It means that platelets
or red blood cell alterations have to be considered in this field [2530]. Even if the main goal of these studies is re-
lated to the improvement of diagnostic matters, the implication of these ex vivo analyses in the advancement of our
knowledge of the pathogenetic mechanisms of diseases in a sex perspective is of great relevance. The main ex vivo
experimental models have been summarized in Tabl e 3.
Some paradigmatic examples
The first point concerns the use of peripheral blood mononuclear cells (PBMC) or lymphocytes (PBL). A good ex-
ample dealing with these tools is represented by the study of autoimmune diseases, often associated with disturbances
of PBL. These cells, directly taken up from the peripheral blood of patients with autoimmune diseases, such as sys-
temic lupus erythematosus (SLE) or rheumatoid arthritis – displaying a significant gender disparity in their incidence
(even 9-11:1 F:M) – can be studied in detail for their integrity and function. However, to date, very few studies have
specifically taken into account the gender issue so that only recently some of the possible determinants of gender
disparity in these diseases have been discovered [3134]. In particular, a role for oestrogen has been envisaged in
SLEinwhichthepresenceofautoantibodiestotheoestrogenreceptor-αhas been observed. These autoantibodies
could interfere with T-lymphocyte homoeostasis thus playing a role in disease activity [27]. Several other examples
suggest that major sex differences exist in the mechanisms underlying immune modulation of various diseases [35].
A recent study reported that the adaptive immune system could be implicated in the development of hypertension in
males but not in females due either to the role of the immune system in the development of high blood pressure in
femalesortoovarianhormones.Infact,inwomen,naturalmenopauseisknowntoresultinsignificantchangesin
the expression of genes regulating the immune system [36]. Unfortunately, confounding factors can complicate these
analyses: (i) lymphocyte activation pathways, (ii) the presence of different lymphocytes subsets and (iii) the interac-
tion among these cell subpopulations. These factors, making data interpretation difficult, impair the advancement of
our knowledge and the proposal of conclusive hypotheses. Hence, albeit the literature data on the implication of cell
sex on immune system function, as well as gender disparity in immunoregulation, are well documented, research in
this area is still at the beginning and needs a strong incentive from funding agencies and scientific journal editors that
couldbeinterestedtothismatter.
As concerns non-nucleated cells taken up by the peripheral blood, i.e. platelets and red blood cells, even these cell
types have been suggested to represent useful tools to dissect sex differences in human pathology. In particular, the
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 5
Clinical Science (2017) 00 6–21
DOI: 10.1042/CS20160847
usefulness of these cell types as real-time biomarkers, as diagnostic or disease progression determinants has also been
proposed in a gender perspective [26,37,38]. A good example is represented by sex variability in platelet aggregation
in response to common agonists [30]. For instance, by using an ex vivo experimental system, it has been observed that
sex can represent ‘a determinant of agonist effects on platelet aggregability even in healthy subjects’. The mechanisms
underlying this disparity could be related to the relevance of the oestrogen–oestrogen receptor system in platelet
homoeostasis (reviewed in [37]).
Here below we will examine the main experimental models used in the study of some important human diseases
focusing on sex and gender. Such information comes from research done on the PubMed website using the search
strings listed in Supplementary Figures S1 and S2.
Oncology
Experimental models
For ethical reasons, the number of animals used in biomedical experiments has strongly been reduced in the last
20 years. However, this number had to be adequate for the statistical power required to generate robust data. Many
preclinical animal models in oncology drug development fail to accurately predict the clinical efficacy of novel anti-
cancer agents, mainly due to their inability to reflect the complexity and heterogeneity of human tumours. The most
commonly used models for the study of cancer are the human tumour xenografts, humanized mice models, in which
immunodeficient mice are engrafted with human cells or tissues, which are considered extremely useful for functional
research in vivo studies.
Cancercelllineshavewidelybeenusedforresearchpurposesandprovedtobeusefultoolsinthegeneticapproach.
Results show that their characterization is, in fact, an excellent model for understanding the biological mechanisms
involved in cancer [15]. The use of cancer cell lines allowed increased information about the deregulated genes and
signalling pathways underlying this disease. Furthermore, cell models represent the first step in the development and
testing of anticancer drugs presently used, and in the development of innovative therapies, but also an alternative
to transplantable animal tumours in chemotherapeutics testing. In fact, the use of the appropriate in vitro model in
cancer research is crucial for the investigation of genetic, epigenetic and biochemical pathways, for the study of prolif-
eration deregulation, apoptosis and cancer progression, to define potential molecular markers, and for the screening
and characterization of cancer therapeutics. Very recently, the relevance of sexual dimorphism has been discussed in
detail as concerns basic science research in the field of oncology [17].
Although gender disparity in the incidence, aggressiveness and disease prognosis have been observed for a variety
of cancers, clinical trials and research in animal models are still gender unbalanced [39]. Gender-specific oncology
needs to reconstruct a correct equilibrium in order to understand the molecular basis underlying the gender dif-
ferences in the outcome and response to therapies. The interactive database GenderMedDB [40], which enables the
retrieval of pre-selected publications containingsex- and gender-specific analyses, shows that in the field of oncology,
sex-related publications have increased in the last 10 years (Figure 1A). In particular, melanoma, bladder, stomach
and thyroid cancers are the most represented (Figure 1B).
Here, we explore sex differences in preclinical studies of two important forms of cancer: melanoma, as a solid
tumour example, and leukaemia/lymphomas as blood cancers, focusing on the experimental models used.
Melanoma
Metastatic melanoma is one of the fastest growing cancers, causing more than 8650 deaths in 2009 alone, and this
numberisprojectedtoincreaseovertime[41]. Gender-specific differences in melanoma epidemiology are well es-
tablished. The probability of developing melanoma during one’s lifetime is 1.72% in men and 1.22% in women. In the
Netherlands, a large population-based cohort study including 10538 melanoma patients from 1993 to 2004 analysed
the gender difference in melanoma survival after adjusting for tumour-related variables (Breslow thickness, histo-
logy, tumour site, and metastatic and nodal status). It has been found that the relative excess risk of mortality was
2.70 in males compared with females. The female survival advantage remained after adjusting for multiple confound-
ing variables including tumour thickness. Gamba and colleagues recently analysed data from 26107 individuals, age
15–39 years, from the US National Cancer Institute’s Surveillance, Epidemiology and End Results registry. They re-
portedthatyoungmenhada55%lowerrateofmelanomasurvivalcomparedwithage-matchedyoungwomen,and
concluded that male sex, within all specific age groups and across all tumour thickness categories, histologic subtypes
and anatomic sites, is associated with a disproportionate burden of melanoma deaths [42]. Localized melanomas in
women showed a lower risk of metastasis, resulting in a better survival when compared with men, even after first
disease progression. In localized melanoma, men generally had worse characteristics at diagnosis, such as older age,
6c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 7–21
DOI: 10.1042/CS20160847
Figure 1. Animal models for melanoma studies
(A) Oncology-related publications per year (data from PubMed.gov). (B) Publication distribution within diseases (extrapolated data from
GenderMedDB database). (C) Number of publications showed in MEDLINE in the last 10 years where scientists report the use of mice or
rats and the gender for melanoma studies. (D) As a result of the large number of published melanoma models, we can discuss only an
arbitrary selection of the currently available models in this review considering the major signalling networks deregulated in melanoma by
mutation or other genomic alterations, which include: (i) xeno-transplantation models; (ii) syngeneic transplantation models and (iii) models
involving genetically modied animals (mutant BRAF or mutant NRAS), and/or via inactivation of key tumour suppressors, including CDKN2A,
PTEN or BRAF V600E that cooperate with PTEN loss to induce metastatic melanoma. The relevance of each particular model depends on
how closely it represents the genetic and epigenetic aberrations, histology, physiological effects and metastatic pattern observed in human
melanoma.
increased likelihood of having an ulcerated or thicker primary tumour, melanomas more commonly located on the
head, neck and trunk, and less often on the extremities. However, even after diagnosis, men continue to have dis-
advantages compared with women. In various studies, women showed a longer delay before relapse and higher cure
rate compared with men [43]. On the basis of these results, we identified, by optimizing the search of the MEDLINE
database incorporating Medical Subject Headings (MeSH R
) (Supplementary Figure S1), the number of publica-
tions related to animal studies performed with male or female mice. As shown in Figure 1(C), female mice are the
most used animals for preclinical studies in melanoma. Several mouse melanoma models have been developed and
are used: (i) to determine the function of particular proteins in melanoma progression; (ii) to approximate certain
biological aspects of human melanomas and (iii) to critically evaluate novel drugs/therapies. In particular, C57Bl/6,
immunodeficient mice (SCID, NOD and NOD/SCID) and engineered mice (such as BRAF and p53) strains are the
most used (Figure 1D).
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 7
Clinical Science (2017) 00 8–21
DOI: 10.1042/CS20160847
Figure 2. Most used cell lines for melanoma studies
(A) Pie chart depicting the relative distribution of the scientic papers in which the 10 most used melanoma cell lines have been used as an
in vitro model (left panel). These 10 examined cell lines represent 91% of the cell lines used in the literature for in vitro studies on melanoma.
Pie chart depicting the sex distribution of the 10 most used cell lines in the literature (right panel). (B) Pie charts depicting the same analysis
of panel A but restricted to the ve most used cell lines. These ve examined cell lines represent 71% of the cell lines used in the literature
for in vitro studies on melanoma.
As far as the in vitro studieswereconcerned,weanalysedthemaincancercelllinesusedintheliteraturethrough
a specific search-string on PubMed (Supplementary Figure S1). In particular, we have analysed 50 cell lines of com-
mercially available melanoma. Within this list we examined the 10 most used cell lines representing 91% of the cell
lines used in the literature for in vitro studies on melanoma, as shown in Figure 2(A) (left panel). All of these cell lines
have been isolated in the 1970s from patients with a mean age of 51 years old (y.o.). A-375 is the most used cell line,
being reported in 38% of the total number of selected papers. It was isolated in 1973 from a 54 y.o. female patient,
and originates from a primary lesion of a malignant melanoma showing a BRAF mutation [44]. In this top 10 list, cell
lines from males and females are equally represented (Figure 2A, right panel). Going to restrict our analysis to the top
five most used cell lines (Figure 2B,leftpanel),weobservedthattheseareemployedin71%ofthepublishedpapers.
In addition, although the percentage of male and female cell lines remains unchanged (Figure 2B, right panel), we
can notice that the whole amount of studies using cell lines from females can be attributed to one cell line, the A-375
cell line.
A very interesting point has recently been underlined as concerns genetic differences between men and women by
an analysis of mutation spectra in 266 metastatic melanomas. These analyses demonstrate for the first time a gender
difference in the mutation burden of cutaneous melanoma [18]. A recent study also found that female patients with
melanoma had a significantly higher frequency of tumour-associated, antigen-specific CD4+T-cells than t heir male
counterparts [45]. In consideration of the relevance of recent advances in the field of molecularly targeted therapy
and immunotherapy, these results could open new scenarios also in the field of GM.
8c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 9–21
DOI: 10.1042/CS20160847
Leukaemia
Since sex differences have been observed also for leukaemia, the main features of this disease are first very briefly
described here. There are four main types of leukaemia: (i) acute myelogenous leukaemia (AML), a fast-growing
formofcancerofthebloodandbonemarrow,thatcanoccurinchildrenandadults,(ii)acutelymphocyticleukaemia
(ALL) mostly occurring in children [46], (iii) chronic myelogenous leukaemia (CML), a form of cancer that affects
the bone marrow and blood, that mostly affects adults. It begins in the blood-forming cells of the bone marrow and
then, over time, spreads to the blood; (iv) chronic lymphocytic leukaemia (CLL) very rarely seen in children and is
most likely to affect people over the age of 55. CLL is a typically slow-growing cancer, which begins in lymphocytes
in the bone marrow and extends into the blood.
As reported in the Surveillance, Epidemiology and End Results Program, an authoritativesource of information on
cancer incidence and survival in the US, the number of new cases of leukaemia was 13.5 per 100000 men and women
per year. The number of deaths was 6.9 per 100000 men and women per year (these rates are age-adjusted and based
on 2009–2013 cases and deaths). Importantly, the number of new cases is 17.3 per 100000 males compared with 10.5
per 100000 females. Differences in sex-specific incidence and prognosis have long been recognized for CML. In this
pathology, male predominance was demonstrated in clinical trials, and the Sokal score for younger patients (<45 years
old) identified female sex as a favourable prognostic factor [47]. In the context of B-cell lymphoproliferative disorders,
a marked preponderance of men (4:1 male to female ratio) is reported in diseases such as hairy cell leukaemia [48].
Further differences between men and women have referred to sex variation in drug efficacy and toxicity, particularly
sex differences in pharmacokinetics [49].AsreportedbyCatovskyandcolleagues,womenaffectedbyCLLshowa
better overall response to treatment than men but greater gastrointestinal toxicity. No good hypotheses have been
advanced to explain the observed trend for a better outcome in women. There are three possible factors that may
contribute to the better treatment response and longer survival in women: (i) the association with good prognostic
factors, (ii) pharmacokinetic differences between the sexes and (iii) the effect of oestrogens [50].
Preclinical studies with animal models provided important insights for the understanding of the molecular determ-
inants of leukaemogenesis also exploring new drugs or combinations [51]. As shown in Figure 3,thereareseveral
animal models, mainly female mice, for use in leukaemia research (Figures 3Aand3B). However, the lack of hetero-
geneity and inter-case variability of animal leukaemia models limits the transferability of the results into the clinical
setting.
Concerning the in vitro cell models of leukaemia, the 10 most commonly used cell lines (among 60 commercially
available), represent the great majority (92%) of the cell lines used in preclinical studies on leukaemia (Figure 4A, left
panel). All these cell lines have been isolated from 1964 to 1980, from patients with a mean age of 29 y.o. HL-60 is
the most used one, being reported in 36% of the total selected papers. HL-60 was isolated in 1977 from a 36-year-old
woman with acute promyelocytic leukaemia at the National Cancer Institute [52]. As clearly depicted in Figure 4(A)
(right panel), cell lines from males and females are not equally represented. In fact, in 70% of studies, cell lines from
female patients have been used, and in several studies the ‘sex’ of the analysed cell lines was not mentioned at all. When
we restricted our analyses to the first five cell lines used in these studies (Figure 4B, left panel), we observed that they
represented 82% of all published papers. Furthermore, the male/female imbalance further increased the disadvantage
of males (75% female and 25% male, Figure 4B, right panel). Given the high intrinsic variability of leukaemias, an
increase in the number of stabilized cell lines and the isolation of new freshly isolated cells from males and females
seem to be unpostponable.
Lymphoma
Lymphomas comprise a heterogeneous group of cancers with diverse aetiologies, treatment pathways and outcomes,
varying both for the type of malignant cells and tumour location [53]. They most frequently originate from B-cells,
and the two main groups of B-cell lymphomas, B-cell non-Hodgkin and Hodgkin, account, for 80% and 15% of
alllymphomas,respectively[54]. As for many other cancers, the likelihood of an individual being diagnosed with
lymphoma markedly increases with age, the median age at diagnosis being 67.2 years for all patients combined, as
described by Smith et al. [55]. Hodgkin and Burkitt lymphomas dominate the paediatric age range (<15 years). By
contrast, in those aged 60 years or more, diffuse large B-cell, marginal-zone and follicular lymphomas accounted for
over 80% of diagnoses.
As concerns gender, males tend to be diagnosed with B-cell lymphomas at younger ages than females, the
male/female sex rate ratio being highest in those diagnosed before the age of 15 years. Indeed, males are almost three
times as likely to develop some B- and T-cell cancer subtypes [55]. However, when MEDLINE analyses was carried
out considering preclinical studies performed on animals in this field (Supplementary Figure S1C), we found that
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 9
Clinical Science (2017) 00 10–21
DOI: 10.1042/CS20160847
Figure 3. Leukaemia and lymphoma studies
(A) Number of publications shown in MEDLINE in the last 10 years where scientists report the use of mice or rats for leukaemia studies. (B)
Here, we discuss only a selection of the currently available models considering the major signalling networks deregulated in leukaemia by
mutation or other genomic alterations, which include: knockout models of AML1, PLZF and PML; xenograft models and third-generation
immunodecient hosts combining additional mutations on the NOD/SCID background. Newborn OD/SCID/Il2rg/(‘NOG’) hosts support the
highest level of engraftment to date for primary, unmanipulated human AML cells. (C) Number of publications shown in MEDLINE in the
last 10 years where scientists report the use of mice or rats for lymphoma studies. The most used mice models for lymphoma studies are
reported in (D).
differences reported above were ignored so that only male or female mice were taken into consideration.Figures 3(C)
and 3(D) show the number of publications reported in MEDLINE in the last 10 years including mice or rats for in vivo
lymphomastudiesandthemostusedmicemodelsforlymphomastudies.Asfarasthein vitro studies were concerned,
following the same criterion of literature analysis through a specific search-string on PubMed, we selected the 10 most
used cell lines in the literature among 70 cell lines commercially available (Figure 5A, left panel). These 10 cell lines
represented 81% of the total cell lines used for preclinical studies on lymphoma. All of them were isolated between
1963 and 1983 from patients with a mean age of 26 y.o. The most commonly used is the Raji cell line analysed in 32%
of the total number of the selected papers. Isolated in 1963 from an 11 y.o. male patient, Raji cells are lymphoblastoid
derived from a Burkitt lymphoma and they are the first continuous human cell line of haematopoietic origin [56].
Alsointhecaseoflymphoma,celllinesfrommalesandfemalesarenotequallyrepresented.Infact,92%ofthestud-
ieshaveconsideredcelllinesfrommalepatientsonly(Figure 5B, left panel). Restricting our analysis to the five most
commonly used cell lines in lymphoma studies (top five represented 70% of published papers), this imbalance has
become even more marked with this percentage reaching almost 100%. Of notice, DNA profiles for the aforemen-
tioned lines have not usually been counterchecked with the DNA profile of the donor tissue and compared with the
DNA profile of other continuous cell lines provided by the data bank.
10 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 11–21
DOI: 10.1042/CS20160847
Figure 4. Most used cell lines for leukaemia studies
(A) Pie chart depicting the relative distribution of the scientic papers in which the 10 most used leukaemia cell lines have been used as an
in vitro model (left panel). These 10 examined cell lines represent 92% of the cell lines used in the literature for in vitro studies on leukaemia.
Pie chart depicting the sex distribution of the 10 most used cell lines in the literature (right panel). (B) Pie charts depicting the same analysis
of panel A but restricted to the ve most used cell lines. These ve examined cell lines represent 82% of the cell lines used in the literature
for in vitro studies on leukaemia.
All in all these bibliometric analyses in the field of cancer research underscore the fact that ‘cell sex’ is an under-
considered issue. Only very recently, the work by Clocchiatti and colleagues raised this question in a leading journal
possibly encouraging a sex-tailored research in the field of in vitro studies on cancer [17].
Neurological diseases
The interactive database GenderMedDB [38] showed an increased trend of neurological studies linked to gender
disparity (Figure 6A). In particular, Alzheimers, epilepsy, multiple sclerosis, Parkinson’s and stroke are the most rep-
resented neurological diseases (Figure 6B). As for oncology, we explored sex differences in preclinical studies of two
important, and very different, neurological diseases: stroke and Alzheimer’s.
Stroke
Stroke, the second leading cause of death worldwide, is a cerebrovascular disease due to a focal neurologic deficit
caused by interrupted or reduced blood cerebral circulation. According to the World Health Organization, 15 mil-
lion people suffer stroke worldwide each year. Of these, 5 million die and another 5 million are permanently disabled.
Europe averages 650000 stroke deaths each year and the overall rate of strokes remains high due to aging of the popu-
lation. Approximately 610000 of these are first events and 185000 are recurrent stroke events. Control of diabetes mel-
litus and high cholesterol and smoking cessation programmes, particularly in combination with hypertension treat-
ment, contributed to the decline in stroke mortality observed in the last years [57]. According to data from the 2013
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 11
Clinical Science (2017) 00 12–21
DOI: 10.1042/CS20160847
Figure 5. Most used cell lines for lymphoma studies
(A) Pie chart depicting the relative distribution of scientic papers in which the 10 most used lymphoma cell lines have been used as an
in vitro model (left panel). These 10 examined cell lines represent 81% of the cell lines used in the literature for in vitro studies on lymphoma.
Pie chart depicting the sex distribution of the 10 most used cell lines in the literature (right panel). (B) Pie charts depicting the same analysis
of panel (A) but restricted to the ve most used cell lines. These ve examined cell lines represent 70% of the cell lines used in the literature
for in vitro studies on lymphoma.
Behavioural Risk Factor Surveillance System, each year 55000 more women than men have a stroke. Epidemiologic
studies have revealed a clear age-by-sex interaction leading to several mechanistic hypotheses. Stroke risk, symptom
presentation and long-term recovery differ in males and females. In younger population, more strokes occur in men
than women, but with advancing age (>60 year), in which stroke morbidity is higher, women have a greater risk
with worse outcomes (reviewed in [58]). Premenopausal women appear less vulnerable to stroke than similarly aged
men. However, in menopausal women, probably due to the lack of protective activity of oestrogen, prevalence and
incidence of stroke are increased. Premenopausal women are most probably protected against stroke because of sex
steroid hormone-dependent mechanisms. Oestrogen, testosterone and progesterone affect different functions of the
cerebral circulation. Oestrogen promotes blood flow by decreasing vascular reactivity whereas testosterone has op-
posite effects. Both are involved in the development of atherosclerosis. Other factors, such as anatomic and genetic
factors may also contribute to the observed differences [59].
Sex differences in response to experimental ischaemic stroke are also well established in animal models. Young fe-
males sustain smaller infarcts and better cerebral blood flow than age-matched males [60]. In a very recent work, it was
reported that circadian rhythm disruption exacerbates these sex differences in stroke impairments because exposure
to shifted light-dark cycles dramatically increased stroke-induced mortality in male but not female rats [61]. Ovarian
hormones, especially oestrogen, may mediate the female advantage with regard to the lethal pathological effects of cir-
cadian rhythm disruption on stroke outcomes. This hypothesis is indirectly supported by studies demonstrating that
infarct volume is increased in ovariectomized females relative to intact animals or oestrogen-replaced animals, and
12 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 13–21
DOI: 10.1042/CS20160847
Figure 6. Neurological diseases studies
(A) Neurology-related publications per year (data from PubMed.gov). (B) Publication distribution within diseases (extrapolated data from
GenderMedDB database). (C) Number of publications shown in MEDLINE where scientists report the use of mice or rats for stroke studies.
The most used mice models in the last 10 years for AD preclinical studies are showed in (D) which include: C57Bl/6 wild-type mice; Tg2576
mice (the rst presentation of cognitive decits is seen at 5 months of age in spatial working memory); the APP23 mouse model (the
cognitive decits begin to rst appear in both recognition memory and spatial working memory at 3 months of age, the decits appear to be
progressive with age); TgCRND8 model exhibits early cognitive impairment; the cognitive decits in the APP/PS1 mouse model (cognitive
decits are rst seen at 3 months of age in the RAWM spatial working memory task and are also reported by 6 months of age in the MWM);
the APP/PS1 knockin (APP +PS1) mouse model uses endogenous promoters to drive the expression of humanized amyloid βsequence,
and AD-like pathology and cognitive decits develop in the absence of APP or PS1 overexpression.
that exogenous oestrogen treatmentalso promotes neuroprotection in response to stroke in males [62]. Other factors,
in addition to oestrogen alone, could play a critical role in stroke neuroprotection observed in young females after
circadian rhythm disruption. By the way, insulin growth factor-1 (IGF-1) plays a key role in modulating neuropro-
tective responses to stroke in young females. In fact, low levels of IGF-1 are associated with increased morbidity and
mortality in ischaemic heart disease and stroke. In addition, exogenous IGF-1 reduces ischaemic injury, stimulates
stroke-induced neurogenesis and promotes neuronal survival, neuronal myelination and angiogenesis [61].
Although women received comparable stroke care to men, as measured by specified ‘Get With The Guidelines
Metrics’, other studies reported sex-specific disparities in stroke care, including thrombolytic therapy and early hos-
pital admission [63]. A further possible cause that can contribute to the gender differences observed in stroke is the
access to treatments. In fact, women are older at stroke onset and more likely to live alone, therefore evidencing longer
prehospital delays after symptom onset compared with men [63].
In recent years, the use of animal models for stroke research has improved our understanding of the physiopath-
ology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger mod-
els, such as rabbits and even non-human primates, is increasing in order to better understand the disease and its
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 13
Clinical Science (2017) 00 14–21
DOI: 10.1042/CS20160847
treatment. However, the applicability of the results obtained with animals to the treatment of human diseases is lim-
ited. The use of older animals can provide information about stroke-induced damage and the recovery process, not
well represented in younger animal models [64]. By using the MeSHs terms search (Supplementary Figure S1D), rats
are more used than mice and male animals are underrepresented in most preclinical studies (Figure 6C). Importantly,
the group of McCullough, that extensively studied gender disparity in stroke, strongly recommended performing
stroke studies in animals of both sexes. These authors also underscore the relevance of this approach to point out
gender-specific therapies [27,65]. In fact, much remains to be learned about differences in stroke between women
and men and further research is needed to improve stroke risk profiles and gender-specific treatments.
Alzheimer’s disease
Alzheimer’s disease (AD) is manifested by a progressive loss of memory and cognition. It is the main cause of dementia
and, in fact, 60% of all cases of dementia are AD patients. The prevalence of AD is higher in women than in men.
The prevalence of Alzheimer’s disease in Europe was estimated at 5.05%. The prevalence in men was 3.31% and in
women 7.13% and this gender disparity increased with age. This sex difference may be due to the higher longevity
women generally experience. However, actually, the incidence also differs between men and women: it has been found
to be 7.02 per 1000 person-years (95% CI, 6.06–8.05) in men and 13.25 per 1000 person-years (95% CI, 12.05–14.51)
in women; again these rates increased with age [66]. In fact, increasing evidence suggests that longevity alone is not
a sufficient explanation and other factors may be involved. Women have a broader spectrum of dementia-related
behavioural symptoms with a predominance of depression, whereas aggressiveness is more frequent in men than
in women. Biological explanations for gender-specific differences in the phenotype of AD include different brain
morphology and function, with higher susceptibility for pathological lesions in women and greater cognitive reserve
in men [67]. A further hypothesis for the higher incidence of dementia in women is that they suffer higher rates of
obesity, diabetes and other conditions, which increase the likelihood of developing AD [68]. However, the normal
age-related depletion of sex steroid hormones also represents an important AD risk factor. Steroid hormone decrease
is associated with vulnerability to disease in all the hormone-responsive tissues, including the brain. Few studies have
addressed the effects of aging on brain hormone levels [69,70]. The literature suggests that both loss of oestrogen and
progesterone at menopause in women and the gradual decrease in testosterone in aging men could be AD risk factors.
As such, therapeutic strategies that counteract age-related depletion of sex steroid hormones may offer significant
protection from the development and perhaps treatment of AD [71]. Further points of interest also derive from a
very recent study pointed to children’s air pollution exposures as associated with systemic and brain inflammation
and the early hallmarks of AD [72]. These authors indicate the Apolipoprotein E (APOE) 4 allele as the most prevalent
genetic risk for AD, with higher risk for females, suggesting that sex, BMI, APOE and metabolic variables in healthy
children with high exposure to ozone and fine particulate matter (PM2.5) influence cognition, with glucose probably
being a key player.
Preclinical animal models, especially in mice, have been extremely useful to test mechanistic hypotheses about
AD pathophysiology and to predict outcomes from pharmacological interventions. However, no animal model re-
capitulates the entirety of AD in humans, thus making it important to understand both the utility and limitations
of particular animal models. For instance, the progression of cognitive impairment seen in human diseases was not
detectable in AD mice models. Each transgenic (Tg) mouse model of AD provides different insights into aspects
of AD pathogenesis and the cognitive deficits associated with the disease. In particular, animal models used in pre-
clinical studies for AD, including Tg models in mice, and non-Tg models also in rats, dogs and monkeys can be
distinguished in: (i) Tg models of AD, consisting of single or multi-Tg animals overexpressing the amyloid precursor
protein (APP), presenilin (PS) and/or Tau mutations; (ii) non-Tg models obtained by toxin injection into the brain,
including direct injection of Aβoligomer or tau, and models of aging. The most commonly used mice models of AD
are characterized by APP mutations [73]. Moreover, it could be useful to note that C57Bl/6 represents the most diffuse
wild-type background of the mouse Tg models [74].Asobserved,malemiceareoftenusedmoreoftenthanfemale
mice (Figure 6D). 5XFAD mice, a well-characterized double Tg mouse model of AD, exhibit an early onset of ro-
bust AD pathology and memory deficits, thus representing a good animal model to investigate AD pathology. These
mice have five distinct human mutations in two genes, the APP and presenilin1 (PS1) engineered into two transgenes
driven by a neuron-specific promoter (Thy1), and thus develop severe amyloid deposition by 4 months of age. In
particular, BACE1.5XFAD haploinsufficiency [β-Site APP-cleaving enzyme 1 (BACE1) ( +/-); i.e. 50% reduction]
represents a therapeutic relevant model to evaluate the efficacy of partial β-secretase inhibition. BACE1 initiates the
generation of amyloid-β(Aβ), thus representing a prime therapeutic target for AD. However, it is unclear whether the
14 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 15–21
DOI: 10.1042/CS20160847
extent of Aβreductions in APP Tg mice with BACE1 (+/-) gene ablation may vary with sex or disease progression
[75].
SynapsesappeartobetheinitialtargetofADpathogenesis;aslossofsynapsesatafinestructurallevelaswell
as reduction in synaptic markers have been documented at early and late stages of AD [76]andshowntocorrelate
with the extent of cognitive deficits [77]. Progressive decline of synaptic proteins occurs also in the 5XFAD mouse
model and appears to be correlated with the progression of memory deficits [78]. For instance, by using this model
it has been documented that along with the improvement of cognitive abilities, neuronal matrix metalloproteinase-9
(MMP-9) overexpression was accompanied by increased levels of the pre-synaptic marker synaptophysin in the brain
of 5XFAD female animals. MMP-9 has been shown to participate in receptor-mediated sAPPαrelease [79]andto
be able to degrade Aβfibrils in vitro and Aβplaques in situ. Consistently, it was demonstrated that overexpression
of MMP-9 restored the levels of mature brain-derived neurotrophic factor (BDNF) in the brains of 5XFAD females.
Such an increase could contribute to the preservation of cognitive functions observed in 5XFAD/TgMMP-9 animals.
As far as the in vitro model systems are concerned, although there are many in vitro studies on the topic of neuro-
pathology, particularly on AD, a careful analysis of the cell lines used in the field of neurodegenerative diseases clearly
indicated that the authors do not specify if cell lines or primary cultures were derived from males or females. A num-
ber of mechanistic studies on AD are achieved by using the human neuroblastoma cell line SH-SY5Y, genetically
female with two X and no Y chromosome (Table 2). This cell model has two important features that make it eligible
for this purpose: (i) following retinoic acid treatment, it acquires a neuronal phenotype evidenced by axonal growth
and expression of neuronal markers and (ii) it is easily transfectable, therefore suitable for exploring the effect of dif-
ferentgenes,i.e.theAPPgene,involvedinthepathogenesisofAD.However,thesearehighlyproliferatingtumour
cells and, as usual, they are cultured cells that have experienced a number of subseedings without any check of their
karyotype.Thesameoccursforanothercellline,thePC12cells,derivedfromamaleratpheochromocytoma,widely
used for studies on neuroprotection. For example, PC12 cells were used as a preclinical model to investigate some
forms of Parkinson’s disease with finds that support interference after vesicle docking and prior to vesicle release
[80]. These studies suggest that, in women, higher physiological striatal dopamine levels may delay the development
of symptomatic PD and that this is possibly due to the activity of oestrogens. This could explain the epidemiological
observations of lower incidence and higher age at onset in women. However, also in this case, the model system,
although appropriate, deals with tumour cells from rat adrenal medulla that, having an embryonic origin from the
neural crest, only partially mimic neuronal pathophysiology. Primary cultures of neurons should be considered in
order to correctly investigate neurodegenerative diseases, especially in sex-oriented research.
Cardiomyopathy and heart failure
Cardiology is of critical importance and represents a milestone in the field of GM. It has in fact been investigated
for many years [1,81,82]. Major differences between men and women exist in epidemiology, manifestation, patho-
physiology, treatment and outcome of CVD [83,84]. Women have less cardiovascular disorders than men in the pre-
menopausal period with risks increasing in the postmenopausal period reaching and even exceeding that of men [85].
In addition, women have more frequent diastolic heart failure (HF), associated with the major risk factors of diabetes
and hypertension whereas men have more frequent systolic HF because of coronary artery disease [79]. Under stress,
male hearts develop pathological hypertrophy with dilation and poor systolic function more easily than female hearts
[86]. As far as acute myocardial infarction (AMI) is concerned, females, at least in some studies, have been reported to
experience a longer delay to reach hospital and less frequently receive invasive therapy, e.g. stents, and cardiac bypass
surgery, than males [87]. For both sexes, age represents a demographic predictor of short- and long-term mortality
in AMI [88]. An analysis from the American National Registry of Myocardial Infarction underlined an interaction
between sex and age with regard to 30-day mortality. In particular, this parameter was significantly higher in fe-
males. Interestingly, there was a progressive decrease in the male/female difference with increasing age until the age
of 75 years [89,90]. A very recent study based on retrospective collection of data compared females and males over
the age of 75 years with regard to the diagnosis of AMI and survival follow-up data for more than 7 years [91]. In
contrast with that observed with regard to 30-day mortality, after 7 years mortality was as high as 80% for both sexes,
but males had a significantly higher age-adjusted 7-year mortality than females [91]. These are just some of the sex
differences observed in cardiovascular disease onset and features.
As concerns preclinical studies with animals, sexual dimorphism is a well-established phenomenon [92]. However,
its degree varies dramatically among species. In fact, for example, the aim of animal modelling of HF is to simplify
an extremely complex syndrome into more manageable research questions. A key decision is the choice of the an-
imal system, which is often a trade-off between convenience/cost and physiological applicability. Usually, animal
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 15
Clinical Science (2017) 00 16–21
DOI: 10.1042/CS20160847
studies use single sex homogeneous groups maintained on standard diets in carefully controlled environments and
this makes these models not suitable to detect sex differences. The relatively complete annotation and simple manip-
ulation of the mouse genome have allowed significant mechanistic insights into human disease. Although mice are
relatively inexpensive and convenient, there are significant differences between mouse and human hearts that lower
the relevance of preclinical analyses. Mouse hearts are obviously very small and beat very quickly [400–600 beats per
minute (bpm)] compared with humans (60–90 bpm), leading to important differences in calcium handling and ion
currents between mice and human hearts. There are also important differences in the predominant myosin isoforms
expressed in adult human and mouse hearts [93]. Human data indicate that sex differences are particularly noticeable
at lower heart rates, so unsurprisingly, data on sex differences in mice are difficult to interpret overall in the field of
electrophysiology. The dog, with a heart size and cardiac action potential (AP) characteristics similar to humans, has
been extensively used as a model for HF and sudden cardiac death. However, in contrast with humans, sex differences
have not been observed in the Corrected QT Interval (QTc) or other ECG parameters in this animal model [94]. This
issue is of importance as we have known for many years that there are marked differences in QT intervals of men and
women. In addition, only recently we also appreciated the profound implications of sex-based electrophysiological
differences in QT interval on the shown sex differences with respect to arrhythmia risk, drug sensitivity and treat-
ment modalities. Little is known about the fundamental mechanisms responsible for sex differences in the electrical
substrateofthehumanheart,inlargepartduetothelackoftissueavailability.Althoughanimalmodelsrepresent
an important research tool, species differences in the sexual dimorphism of the QT interval, the ionic currents un-
derlying the cardiac repolarization and effects of sex steroids advice against the possibility to extrapolate sex-related
results from animals to humans. In fact, studies performed on different animal models often yield conflicting data.
Each model has its strength, such as ease of genetic manipulation in mice or proper size in dogs. The New Zealand
white rabbit is a frequently used animal strain. It exhibits similar sex differences in LQT2-related arrhythmias as in
humans, for both adult and prepubertal rabbits, and approximately the same combination of ionic currents underlie
rabbit and human AP [95].
As concerns HF, the most commonly used mice models use the response to a surgical intervention, such as banding
an aorta or clipping a coronary artery, to model the multisystem effects of HF [96]. These surgical models of HF have
the advantage of very closely replicating specific disease situations of myocardial infarction (coronary artery ligation)
and HF owing to hypertension or aortic valve stenosis (aortic banding). However, all surgical models are relatively
expensive and technically demanding with high rates of intraoperative mortality, which reduces reproducibility. In-
terventions such as long-term pacing of the heart at high rates are usually used in larger animals, for example dogs
and rabbits, but this is not technically feasible in mice. Administration of a single toxin or drug is a theoretically at-
tractive method of modelling HF. For example, the antineoplastic drug doxorubicin [97] is known to be cardiotoxic in
humans and has been used in mice to induce HF syndromes in which, as in humans, a sex disparity has been detected
[85]. An additional useful approach offered by mice models could be represented by the changes in expression of
somegenesinvolvedinHFbytargetedmutagenesis/transgenesis. Unfortunately, some lines of evidence demonstrate
that diverse gene expression patterns are associated with HF due to differing causes [98]. Thus, the deletion of a single
gene has a high chance of leading to artifactual phenotypes not representative for human disease [99].
Further, large animal models that approximate human physiology, function and anatomy, recapitulating the clinical
HF phenotype and translating basic science to clinical applications have successfully been used. In particular, due to
anatomical dimensions, for cardiac mechanical devices and surgical procedures, swine are recognized as the most
suitable animal model also for clinician training in optimizing new technical procedures [100]. However, the high
economic costs and the difficulties of stabulating make this animal model unsuitable, also from the point of view of
statistical significance.
Analysing literature of the last 10 years in the MEDLINE database incorporating MeSH R
of HF, by using the list of
strings shown in Supplementary Figure S2(A), we found that most of the studies dealing with HF used rats or mice. In
particular, the use of male mice and male rats is higher than female small size animals (Supplementary Figure S2B).
Currently, the limiting factor is the size of the mouse heart even if new technologies in molecular imaging of mouse
hearts are developing in parallel with human technology. Validation of the effect of drug treatment in mice models
against human CVD remains therefore of primary importance.
As far as the in vitro model systems are concerned, as mentioned in the neuropathology section, even in the case
of CVD, it was not possible to make an accurate analysis of the cell lines most used in experimental research. A
number of studies in the CVD field used the H9c2 rat cardiomyoblast cell line, which has the advantage of being an
animal-free alternative. A careful evaluation by Watkins et al. [101] demonstrated that H9c2 cells could accurately
mimic the hypertrophic responses of primary cardiac myocytes. This finding validates the importance of H9c2 cells
16 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 17–21
DOI: 10.1042/CS20160847
as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell
lines for prospective molecular studies in heart development and disease [101]. In spite of this, there is no informa-
tion on grounds of sex of this important cell line (Table 2), which originates from myocardium of Rattus norveg icus
[102]. However, in vitro differentiated human cardiomyocytes still represent an essential tool not only for general
cardiovascular research, but also to address key unmet needs in drug development and preclinical research. New
testing systems, such as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, enable the assessment of the
safety pharmacology core battery at the preclinical stage [103,104]. In fact, a major constraint for the development of
adequate therapies has been the lack of suitable cell-based assays with physiological relevance. Human-induced pluri-
potent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential
tools to advance cardiac drug safety screening, in particular to examine the effects of compounds on cardiac elec-
trical activity. Although this model system has been validated for many applications (among these calcium transient
analysis, voltage sensitive dyes, cell metabolism analysis, hypertrophy disease modelling, cell contraction force and
3D organotypic cell culture), currently only hiPSC-CM originated from a female are available (Table 2). Thus, also in
this area of research, the in vitro models used so far appear inadequate for sex-oriented studies.
Conclusions
The experimental matter
As recently suggested by some papers analysing literature data, very few works take into account both males and fe-
males in experimental settings or discuss the problem of sex differences. Preclinical studies, either carried out in vitro
or ex vivo or in animal models, barely consider both sexes. More often only one sex is taken into account. In addition,
frequently, the analysed experimental models do not reflect the incidence in humans so that a disease, which is pre-
dominant in females, has been analysed in preclinical studies carried out in male mice or vice versa. A paradigmatic
example is that of pharmacological studies in the field of neurodegenerative diseases, more frequent in females but
studied mainly in males.
The editorial needs
The fundamental role of editors in disseminating research output is well evident. The rules and policies of journals
can directly affect research. For example, ethical review procedures are now a universal requirement for human and
animal research at least in part because of journal editorial requirements. In the same vein, a specific work [105]
proposed a series of recommendations aimed at the inclusion of information regarding sex in the context of the work.
For example, papers should specify the sex of animals or cells, tissues and other material derived from these, or the
sex/gender of human participants. Authors should report how sex/gender was taken into account in the design of the
study, whether they ensured adequate representation of males and females, or justify the reasons for any exclusion of
males or females. Altogether the required information could provide useful clues in the development of the biology
of sex differences and GM and the improvement of our knowledge of disease onset and progression. Nowadays,
information on the sexual origin of cells or animals is too often disregarded so that the great majority of works still
lack this important information either in in vitro or in animal studies. As a consequence, comparisons between the
two sexes are very rare and almost barely investigated and discussed. We hope that this brief discussion on the state
of the art of the models used in different fields of experimental research contributes to a reappraisal of the ‘sex issue’
in preclinical studies.
Funding
This work was supported by the Ministry of Health [grant number RF-2011-02346986 (to P.M.)]; the ARCOBALENO Onlus and Peretti
Foundation [grant number 2011-20 (to W.M.)]; and the Italian Association for Research against Cancer (AIRC) [grant numbers 11610
(to L.G.) and 18526 (to W.M.)].
Abbreviations
AD, Alzheimer’s disease; ALL, acute lymphocytic leukaemia; AML, acute myelogenous leukaemia; AP, action potential; APOE,
Apolipoprotein E; APP, amyloid precursor protein; BDNF, brain-derived neurotrophic factor; CLL, chronic lymphocytic leukaemia;
CML, chronic myelogenous leukaemia; CVD, cardiovascular disease; EBV, Epstein-Barr virus; GM, gender medicine; HF, heart
failure; HPV, Human Papilloma virus; iPSC , induced pluripotent stem cell; IGF-1, insulin growth factor-1; MeSHR
, Medical Subject
Headings; PBL, peripheral blood lymphocytes; PBMC, peripheral blood mononuclear cells; PS, presenilin; Tg, transgenic.
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 17
Clinical Science (2017) 00 18–21
DOI: 10.1042/CS20160847
References
1 Regitz-Zagrosek, V. (2012) Sex and gender differences in health. Science & Society Series on Sex and Science. EMBO Rep. 13,596603CrossRef
2 Regitz-Zagrosek, V. and Seeland, U. (2012) Sex and gender differences in clinical medicine. Handb. Exp. Pharmacol. 2012,322
3 Regitz-Zagrosek, V., Oertelt-Prigione, S., Prescott, E., Franconi, F., Gerdts, E., Foryst-Ludwig, A. et al. (2016) Gender in cardiovascular diseases: impact
on clinical manifestations, management, and outcomes. Eur. Heart J. 37,2434CrossRef
4 Pierdominici, M., Ortona, E., Franconi, F., Caprio, M., Straface, E. and Malorni, W. (2011) Gender specific aspects of cell death in the cardiovascular
system. Curr. Pharm. Des. 17, 1046–1055 CrossRef
5 Joel, D., Berman, Z., Tavor, I., Wexler, N., Gaber, O., Stein, Y. et al. (2015) Sex beyond the genitalia: the human brain mosaic. Proc. Natl. Acad. Sci.
U.S.A. 112, 15468–15473 CrossRef
6 Ortona, E., Margutti, P., Matarrese, P., Franconi, F. and Malorni, W. (2008) Redox state, cell death and autoimmune diseases: a gender perspective.
Autoimmun. Rev. 7, 579–584 CrossRef
7 Miller, L.R., Marks, C., Becker, J.B., Hurn, P.D., Chen, W.J., Woodruff, T. et al. (2016) Considering sex as a biological variable in preclinical research.
FASEB J. 31, 29–34
8 Ortona, E., Pierdominici, M., Maselli, A., Veroni, C., Aloisi, F. and Shoenfeld, Y. (2016) Sex-based differences in autoimmune diseases. Ann. Ist Super
Sanit `
a52, 205–212
9 Sandberg, K. and Umans, J.G. (2015) Recommendations concerning the new U.S. National Institutes of Health initiative to balance the sex of cells and
animals in preclinical research. FASEB J. 29, 1646–1652 CrossRef
10 Miller, V.M. (2014) Why are sex and gender important to basic physiology and translational and individualized medicine? Am. J. Physiol. Heart Circ.
Physiol. 306, H781–H788 CrossRef
11 Ebert, S.N., Liu, X.K. and Woosley, R.L. (1998) Female gender as a risk factor for drug-induced cardiac arrhythmias: evaluation of clinical and
experimental evidence. J. Womens Health 7, 547–557 CrossRef
12 Fields, R.D. (2014) NIH policy: mandate goes too far. Nature 510, 340 CrossRef
13 Clayton, J.A. and Collins, F.S. (2014) Policy: NIH to balance sex in cell and animal studies. Nature 509,282283CrossRef
14 Heidari, S., Babor, T.F., De Castro, P., Tort, S. and Curno, M. (2016) Sex and Gender Equity in Research: rationale for the SAGER guidelines and
recommended use. Res. Integrity Peer Rev. 1,2
15 Shah, K., McCormack, C.E. and Bradbury, N.A. (2014) Do you know the sex of your cells? Am. J. Physiol. Cell Physiol. 306, C3–C18 CrossRef
16 Kararigas, G.S.U., Barcena de Arellano, M.L., Dworatzek, E. and Regitz-Zagrosek, V. (2016) Why the study of the effects of biological sex is important.
Ann. Ist Super Sanit `
a52, 149–150
17 Clocchiatti, A., Cora, E., Zhang, Y. and Dotto, G.P. (2016) Sexual dimorphism in cancer. Nat. Rev. Cancer 16,330339CrossRef
18 Gupta, S., Artomov, M., Goggins, W., Daly, M. and Tsao, H. (2015) Gender disparity and mutation burden in metastatic melanoma. J. Natl. Cancer Inst.
107, djv221 CrossRef
19 Straface, E., Gambardella, L., Brandani, M. and Malorni, W. (2012) Sex differences at cellular level: “cells have a sex”. Handb. Exp. Pharmacol. 2012,
49–65
20 Lista, P., Straface, E., Brunelleschi, S., Franconi, F. and Malorni, W. (2011) On the role of autophagy in human diseases: a gender perspective. J. Cell
Mol. Med. 15, 1443–1457 CrossRef
21 Maselli, A., Matarrese, P., Straface, E., Canu, S., Franconi, F. and Malorni, W. (2009) Cell sex: a new look at cell fate studies. FASEB J. 23,
978–984 CrossRef
22 Matarrese, P., Colasanti, T., Ascione, B., Margutti, P., Franconi, F., Alessandri, C. et al. (2011) Gender disparity in susceptibility to oxidative stress and
apoptosis induced by autoantibodies specific to RLIP76 in vascular cells. Antioxid. Redox. Signal. 15, 2825–2836 CrossRef
23 Manole, M.D., Tehranian-DePasquale, R., Du, L., Bayir, H., Kochanek, P.M. and Clark, R.S. (2011) Unmasking sex-based disparity in neuronal
metabolism. Curr. Pharm. Des. 17, 3854–3860 CrossRef
24 Du, L., Hickey, R.W., Bayir, H., Watkins, S.C., Tyurin, V.A., Guo, F. et al. (2009) Starving neurons show sex difference in autophagy. J. Biol. Chem. 284,
2383–2396 CrossRef
25 Mathur, P., Ostadal, B., Romeo, F. and Mehta, J.L. (2015) Gender-related differences in atherosclerosis. Cardiovasc. Drugs Ther. 29, 319–327 CrossRef
26 Patti, G., De Caterina, R., Abbate, R., Andreotti, F., Biasucci, L.M., Calabro, P. et al. (2014) Platelet function and long-term antiplatelet therapy in
women: is there a gender-specificity? A ‘state-of-the-art’ paper. Eur. Heart J. 35, 2213–223b CrossRef
27 Roy-O’Reilly, M. and McCullough, L.D. (2014) Sex differences in stroke: the contribution of coagulation. Exp. Neurol. 259, 16–27 CrossRef
28 Jaremo, P., Eriksson-Franzen, M. and Milovanovic, M. (2015) Platelets, gender and acute cerebral infarction. J. Transl. Med. 13, 267 CrossRef
29 Pietraforte, D., Vona, R., Marchesi, A., de Jacobis, I.T., Villani, A., Del Principe, D. et al. (2014) Redox control of platelet functions in physiology and
pathophysiology. Antioxid. Redox Signal. 21, 177–193 CrossRef
30 Straface, E., Gambardella, L., Mattatelli, A., Canali, E., Boccalini, F., Agati, L. et al. (2011) The red blood cell as a gender-associated biomarker in
metabolic syndrome: a pilot study. Int. J. Cell Biol. 2011, 204157 CrossRef
31 Maselli, A., Conti, F., Alessandri, C., Colasanti, T., Barbati, C., Vomero, M. et al. (2016) Low expression of estrogen receptor beta in T lymphocytes and
high serum levels of anti-estrogen receptor alpha antibodies impact disease activity in female patients with systemic lupus erythematosus. Biol. Sex
Differ. 7,3CrossRef
32 Pierdominici, M., Vomero, M., Barbati, C., Colasanti, T., Maselli, A., Vacirca, D. et al. (2012) Role of autophagy in immunity and autoimmunity, with a
special focus on systemic lupus erythematosus. FASEB J. 26, 1400–1412 CrossRef
18 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 19–21
DOI: 10.1042/CS20160847
33 Colasanti, T., Maselli, A., Conti, F., Sanchez, M., Alessandri, C., Barbati, C. et al. (2012) Autoantibodies to estrogen receptor alpha interfere with T
lymphocyte homeostasis and are associated with disease activity in systemic lupus erythematosus. Arthritis Rheum. 64,778787CrossRef
34 Barragan-Martinez, C., Amaya-Amaya, J., Pineda-Tamayo, R., Mantilla, R.D., Castellanos-de la Hoz, J., Bernal-Macias, S. et al. (2012) Gender
differences in Latin-American patients with rheumatoid arthritis. Gend. Med. 9, 490–510, e5 CrossRef
35 Klein, S.L. and Flanagan, K.L. (2016) Sex differences in immune responses. Nat. Rev. Immunol. 16, 626–638 CrossRef
36 Sandberg, K., Ji, H., Einstein, G., Au, A. and Hay, M. (2016) Is immune system-related hypertension associated with ovarian hormone deficiency? Exp.
Physiol. 101, 368–374 CrossRef
37 Del Principe, D., Ruggieri, A., Pietraforte, D., Villani, A., Vitale, C., Straface, E. et al. (2015) The relevance of estrogen/estrogen receptor system on the
gender difference in cardiovascular risk. Int. J. Cardiol. 187, 291–298 CrossRef
38 Otahbachi, M., Simoni, J., Simoni, G., Moeller, J.F., Cevik, C., Meyerrose, G.E. et al. (2010) Gender differences in platelet aggregation in healthy
individuals. J. Thromb. Thrombolysis 30, 184–191 CrossRef
39 Gabriele, L., Buoncervello, M., Ascione, B., Bellenghi, M., Matarrese, P. and Car`
e, A. (2016) The gender perspective in cancer research and therapy:
novel insights and on-going hypotheses. Ann. Ist Super Sanit `
a52, 213–222
40 Oertelt-Prigione, S., Gohlke, B.O., Dunkel, M., Preissner, R. and Regitz-Zagrosek, V. (2014) GenderMedDB: an interactive database of sex and
gender-specific medical literature. Biol. Sex Differ. 5,7CrossRef
41 Howell, Jr, P.M., Li, X., Riker, A.I. and Xi, Y. (2010) MicroRNA in melanoma. Ochsner. J. 10, 83–92
42 Gamba, C.S., Clarke, C.A., Keegan, T.H., Tao, L. and Swetter, S.M. (2013) Melanoma survival disadvantage in young, non-Hispanic white males
compared with females. JAMA Dermatol. 149, 912–920 CrossRef
43 Roh, M.R., Eliades, P., Gupta, S. and Tsao, H. (2015) Cutaneous melanoma in women. Int. J. Womens Dermatol. 1,2125CrossRef
44 Giard, D.J., Aaronson, S.A., Todaro, G.J., Arnstein, P., Kersey, J.H., Dosik, H. et al. (1973) In vitro cultivation of human tumors: establishment of cell
lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51, 1417–1423
45 Wesa, A.K., Mandic, M., Taylor, J.L., Moschos, S., Kirkwood, J.M., Kwok, W.W. et al. (2014) Circulating type-1 anti-tumor CD4( +) T cells are
preferentially pro-apoptotic in cancer patients. Front. Oncol. 4, 266 CrossRef
46 Bearison, D.J. and Mulhern, R.K. (1994) Psychological Perspectives on Children with Cancer. Pediatric Psychooncology 1–272
47 Berger, U., Maywald, O., Pfirrmann, M., Lahaye, T., Hochhaus, A., Reiter, A. et al. (2005) Gender aspects in chronic myeloid leukemia: long-term results
from randomized studies. Leukemia 19, 984–989 CrossRef
48 Morton, L.M., Wang, S.S., Devesa, S.S., Hartge, P., Weisenburger, D.D. and Linet, M.S. (2006) Lymphoma incidence patterns by WHO subtype in the
United States, 1992–2001. Blood 107, 265–776 CrossRef
49 Gandhi, M., Aweeka, F., Greenblatt, R.M. and Blaschke, T.F. (2004) Sex differences in pharmacokinetics and pharmacodynamics. Annu. Rev.
Pharmacol. Toxicol. 44, 499–523 CrossRef
50 Catovsky, D., Wade, R. and Else, M. (2014) The clinical significance of patients’ sex in chronic lymphocytic leukemia. Haematologica 99,
1088–1094 CrossRef
51 Cook, G.J. and Pardee, T.S. (2013) Animal models of leukemia: any closer to the real thing? Cancer Metastasis Rev. 32, 63–76 CrossRef
52 Collins, S.J., Gallo, R.C. and Gallagher, R.E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.
Nature 270, 347–349 CrossRef
53 Campo, E., Swerdlow, S.H., Harris, N.L., Pileri, S., Stein, H. and Jaffe, E.S. (2011) The 2008 WHO classification of lymphoid neoplasms and beyond:
evolving concepts and practical applications. Blood 117, 5019–5032 CrossRef
54 Donnou, S., Galand, C., Touitou, V., Sautes-Fridman, C., Fabry, Z. and Fisson, S. (2012) Murine models of B-cell lymphomas: promising tools for
designing cancer therapies. Adv. Hematol. 2012, 701704 CrossRef
55 Smith, A., Crouch, S., Lax, S., Li, J., Painter, D., Howell, D. et al. (2015) Lymphoma incidence, survival and prevalence 2004–2014: sub-type analyses
from the UK’s Haematological Malignancy Research Network. Br. J. Cancer 112, 1575–1584 CrossRef
56 Drexler, H.G. and Minowada, J. (1998) History and classification of human leukemia-lymphoma cell lines. Leuk. Lymphoma 31, 305–316 CrossRef
57 Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M. et al. (2016) Heart disease and stroke statistics-2016 update: a report
from the American Heart Association. Circulation 133, e38–e360 CrossRef
58 Sohrabji, F., Welsh, C.J. and Reddy, D.S. (2015) Sex Differences in Neurological Diseases. In Chapter 12 in Sex Differences in the Central Nervous
System (Rebecca Shansky, M. ed.), pp. 297–323, Academic Press, London
59 Haast, R.A., Gustafson, D.R. and Kiliaan, A.J. (2012) Sex differences in stroke. J. Cereb. Blood Flow Metab. 32, 2100–2107 CrossRef
60 Selvamani, A., Williams, M.H., Miranda, R.C. and Sohrabji, F. (2014) Circulating miRNA profiles provide a biomarker for severity of stroke outcomes
associated with age and sex in a rat model. Clin. Sci. (Lond) 127, 77–89 CrossRef
61 Earnest, D.J., Neuendorff, N., Coffman, J., Selvamani, A. and Sohrabji, F. (2016) Sex differences in the impact of shift work schedules on pathological
outcomes in an animal model of ischemic stroke. Endocrinology 157, 2836–2843 CrossRef
62 Selvamani, A. and Sohrabji, F. (2010) Reproductive age modulates the impact of focal ischemia on the forebrain as well as the effects of estrogen
treatment in female rats. Neurobiol. Aging 31, 1618–1628 CrossRef
63 Asdaghi, N., Romano, J.G., Wang, K., Ciliberti-Vargas, M.A., Koch, S., Gardener, H. et al. (2016) Sex disparities in ischemic stroke care: FL-PR CReSD
study (Florida-Puerto Rico Collaboration to Reduce Stroke Disparities). Stroke 47, 2618–2626 CrossRef
64 Casals, J.B., Pieri, N.C., Feitosa, M.L., Ercolin, A.C., Roballo, K.C., Barreto, R.S. et al. (2011) The use of animal models for stroke research: a review.
Comp. Med. 61, 305–313
65 Ahnstedt, H., McCullough, L.D. and Cipolla, M.J. (2016) The importance of considering sex differences in translational stroke research. Transl. Stroke
Res. 7, 261–273
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 19
Clinical Science (2017) 00 20–21
DOI: 10.1042/CS20160847
66 Niu, H., Alvarez-Alvarez, I., Guillen-Grima, F. and Aguinaga-Ontoso, I. (2016) Prevalence and incidence of Alzheimer’s disease in Europe: a
meta-analysis. Neurologia, in press
67 Schmidt, R., Kienbacher, E., Benke, T., Dal-Bianco, P., Delazer, M., Ladurner, G. et al. (2008) [Sex differences in Alzheimer’s disease]. Neuropsychiatry
22,115
68 Vina, J. and Lloret, A. (2010) Why women have more Alzheimer’s disease than men: gender and mitochondrial toxicity of amyloid-beta peptide. J.
Alzheimers Dis. 20 Suppl 2, S527–S533
69 Rosario, E.R., Chang, L., Head, E.H., Stanczyk, F.Z. and Pike, C.J. (2011) Brain levels of sex steroid hormones in men and women during normal aging
and in Alzheimer’s disease. Neurobiol. Aging 32, 604–613 CrossRef
70 Bixo, M., Backstrom, T., Winblad, B. and Andersson, A. (1995) Estradiol and testosterone in specific regions of the human female brain in different
endocrine states. J. Steroid Biochem. Mol. Biol. 55, 297–303 CrossRef
71 Barron, A.M. and Pike, C.J. (2012) Sex hormones, aging, and Alzheimer’s disease. Front. Biosci. (Elite Ed) 4, 976–997
72 Calderon-Garciduenas, L., Jewells, V., Galaz-Montoya, C., van Zundert, B., Perez-Calatayud, A., Ascencio-Ferrel, E. et al. (2016) Interactive and additive
influences of gender, BMI and apolipoprotein 4 on cognition in children chronically exposed to high concentrations of PM2.5 and ozone. APOE 4
females are at highest risk in Mexico City. Environ. Res. 150, 411–422 CrossRef
73 Webster, S.J., Bachstetter, A.D., Nelson, P.T., Schmitt, F.A. and Van Eldik, L.J. (2014) Using mice to model Alzheimer’s dementia: an overview of the
clinical disease and the preclinical behavioral changes in 10 mouse models. Front. Genet. 5,88CrossRef
74 Puzzo, D., Gulisano, W., Palmeri, A. and Arancio, O. (2015) Rodent models for Alzheimer’s disease drug discovery. Expert Opin. Drug Discov. 10,
703–711 CrossRef
75 Devi, L. and Ohno, M. (2015) Effects of BACE1 haploinsufficiency on APP processing and Abeta concentrations in male and female 5XFAD Alzheimer
mice at different disease stages. Neuroscience 307, 128–137 CrossRef
76 Masliah, E., Rockenstein, E., Veinbergs, I., Sagara, Y., Mallory, M., Hashimoto, M. et al. (2001) beta-amyloid peptides enhance alpha-synuclein
accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer’s disease and Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A. 98,
12245–12250 CrossRef
77 Terry, R.D., Masliah, E., Salmon, D.P., Butters, N., DeTeresa, R., Hill, R. et al. (1991) Physical basis of cognitive alterations in Alzheimer’s disease:
synapse loss is the major correlate of cognitive impairment. Ann. Neurol. 30, 572–580 CrossRef
78 Oakley, H., Cole, S.L., Logan, S., Maus, E., Shao, P., Craft, J. et al. (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss
in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J. Neurosci. 26,
10129–10140 CrossRef
79 Fragkouli, A., Tzinia, A.K., Charalampopoulos, I., Gravanis, A. and Tsilibary, E.C. (2011) Matrix metalloproteinase-9 participates in NGF-induced
alpha-secretase cleavage of amyloid-beta protein precursor in PC12 cells. J. Alzheimers Dis. 24, 705–719
80 Gillies, G.E. and McArthur, S. (2010) Independent influences of sex steroids of systemic and central origin in a rat model of Parkinson’s disease: a
contribution to sex-specific neuroprotection by estrogens. Horm. Behav. 57, 23–34 CrossRef
81 Maas, A.H. and Appelman, Y.E. (2010) Gender differences in coronary heart disease. Neth. Heart J. 18, 598–602 CrossRef
82 Mosca, L., Barrett-Connor, E. and Wenger, N.K. (2011) Sex/gender differences in cardiovascular disease prevention: what a difference a decade makes.
Circulation 124, 2145–2154 CrossRef
83 Patrizio, M. and Marano, G. (2016) Gender differences in cardiac hypertrophic remodeling. Ann. Ist Super Sanit `
a52, 223–229
84 Regitz-Zagrosek, V. and Kararigas, G. (2017) Mechanistic pathways of sex differences in cardiovascular disease. Physiol. Rev. 97,137CrossRef
85 Moulin, M., Piquereau, J., Mateo, P., Fortin, D., Rucker-Martin, C., Gressette, M. et al. (2015) Sexual dimorphism of doxorubicin-mediated
cardiotoxicity: potential role of energy metabolism remodeling. Circ. Heart Fail. 8, 98–108 CrossRef
86 Regitz-Zagrosek, V., Oertelt-Prigione, S., Seeland, U. and Hetzer, R. (2010) Sex and gender differences in myocardial hypertrophy and heart failure.
Circ. J. 74, 1265–1273 CrossRef
87 Fang, J. and Alderman, M.H. (2006) Gender differences of revascularization in patients with acute myocardial infarction. Am. J. Cardiol. 97,
1722–1726 CrossRef
88 Karlson, B.W., Lindqvist, J., Sjolin, M., Caidahl, K. and Herlitz, J. (2001) Which factors determine the long-term outcome among patients with a very
small or unconfirmed AMI. Int. J. Cardiol. 78, 265–275 CrossRef
89 Berger, J.S. and Brown, D.L. (2006) Gender-age interaction in early mortality following primary angioplasty for acute myocardial infarction. Am.J.
Cardiol. 98, 1140–1143 CrossRef
90 Puddu, P.E., Terradura Vagnarelli, O., Mancini, M., Zanchetti, A. and Menotti, A. (2014) Typical and atypical coronary heart disease deaths and their
different relationships with risk factors. The Gubbio residential cohort study. Int. J. Cardiol. 173, 300–304 CrossRef
91 Thang, N.D., Karlson, B.W., Karlsson, T. and Herlitz, J. (2016) Characteristics of and outcomes for elderly patients with acute myocardial infarction:
differences between females and males. Clin. Interv. Aging 11, 1309–1316 CrossRef
92 Miller, V.M., Kaplan, J.R., Schork, N.J., Ouyang, P., Berga, S.L., Wenger, N.K. et al. (2011) Strategies and methods to study sex differences in
cardiovascular structure and function: a guide for basic scientists. Biol. Sex Differ. 2,14CrossRef
93 Swynghedauw, B. (1986) Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol. Rev. 66, 710–771
94 Fulop, L., Banyasz, T., Szabo, G., Toth, I.B., Biro, T., Lorincz, I. et al. (2006) Effects of sex hormones on ECG parameters and expression of cardiac ion
channels in dogs. Acta Physiol. (Oxf) 188, 163–171 CrossRef
95 Salama, G. and Bett, G.C. (2014) Sex differences in the mechanisms underlying long QT syndrome. Am. J. Physiol. Heart Circ. Physiol. 307,
H640–H648 CrossRef
96 Mayosi, B.M., Kardos, A., Davies, C.H., Gumedze, F., Hovnanian, A., Burge, S. et al. (2006) Heterozygous disruption of SERCA2a is not associated with
20 c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
Clinical Science (2017) 00 21
DOI: 10.1042/CS20160847
impairment of cardiac performance in humans: implications for SERCA2a as a therapeutic target in heart failure. Heart 92, 105–
109 CrossRef
97 Delgado, 3rd, R.M., Nawar, M.A., Zewail, A.M., Kar, B., Vaughn, W.K., Wu, K.K. et al. (2004) Cyclooxygenase-2 inhibitor treatment improves left
ventricular function and mortality in a murine model of doxorubicin-induced heart failure. Circulation 109, 1428–1433 CrossRef
98 Huang, X., Pan, W., Grindle, S., Han, X., Chen, Y., Park, S.J. et al. (2005) A comparative study of discriminating human heart failure etiology using gene
expression profiles. BMC Bioinformatics 6, 205 CrossRef
99 Breckenridge, R. (2010) Heart failure and mouse models. Dis. Model Mech. 3, 138–143 CrossRef
100 Suzuki, Y., Yeung, A.C. and Ikeno, F. (2011) The representative porcine model for human cardiovascular disease. J. Biomed. Biotechnol. 2011, 195483
101 Watkins, S.J., Borthwick, G.M. and Arthur, H.M. (2011) The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic
responses in vitro.. In Vitro Cell Dev. Biol. Anim. 47, 125–131 CrossRef
102 Kimes, B.W. and Brandt, B.L. (1976) Properties of a clonal muscle cell line from rat heart. Exp. Cell Res. 98, 367–381 CrossRef
103 Blinova, K., Stohlman, J., Vicente, J., Chan, D., Johannesen, L., Hortigon-Vinagre, M.P. et al. (2016) Comprehensive translational assessment of human
induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias. Toxicol. Sci.
104 Hortigon-Vinagre, M.P., Zamora, V., Burton, F.L., Green, J., Gintant, G.A. and Smith, G.L. (2016) The use of ratiometric fluorescence measurements of
the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived
cardiomyocytes. Toxicol. Sci. 154, 320–331
105 De Castro, P. (2016) HSaBT. Sex and gender equity in research (SAGER): reporting guidelines as a framework of innovation for an equitable approach to
gender medicine. Ann. Ist Super Sanit `
a52, 154–157
c
2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society. 21
... In general, CRC incidence and mortality seems to be lower for women than for man [4,5], with also a higher survival rate [6]. Nevertheless, even if the literature shows that gender is the single significant predictor of the relative advantage of survival [6,7], several pre-clinical and clinical studies do not focus on it [8], and female participants rate assesses around 38.8% [9]. Moreover, hormone balance may be an influencing factor with therapeutic potential [10]. ...
Article
Full-text available
Colorectal cancer (CRC) incidence and mortality seems to be lower in women than in men. The present study aims to evaluate the impact of gender on CRC diagnosis, treatment, and survival. This is a retrospective cohort study based on a single-center dataset of CRC patients from the University Hospital of Trieste (Italy). Data of 1796 consecutive CRC patients referred to our center from November 11th, 2004, to December 31st, 2017, were analyzed. Right-sided carcinomas are more frequent in women than in men; furthermore, women had a lower surgical complication rate. Men showed a higher 5- and 10-year mortality. This survival benefit for women was observed independently of the tumor localization. The 5-year hazard ratio (HR) for women vs men was 0.776 ( p 0.003), and after 10-year 0.816 ( p 0.017). Regarding the disease-free survival (DFS), 5 and 10-year HR was 0.759 ( p 0.034) and 0.788 ( p 0.07), respectively. On multivariable analysis, respecting tumor localization, the odds of female gender were higher than man with right colon disease. Male gender was more independently associated with age at the surgery time. Women survival advantage was higher than men, except for patients older than 80. Surgical outcome and survival after CRC surgical treatment seem to be gender related. For this reason, gender could play an important role in CRC diagnosis and therapy, allowing an earlier diagnosis in women.
... Third, despite the comprehensive nature of the GBD dataset, data on certain childhood cancers from some regions or countries might be sparse or even absent. For example, the GBD 2019 did not report data for chronic lymphocytic leukemia in children younger than 5 years, as this sub-type of leukemia mostly affects adults (53); the study also lacked sufficient data for non-Hodgkin's lymphoma. For this reason, we were unable to accurately quantify the total number of children with leukemia or compare all incident cases between the cancers of interest in this study. ...
Article
Full-text available
Objectives To quantify the burden and variation trends of cancers in children under 5 years at the global, regional, and national levels from 1990 to 2019.Methods Epidemiological data for children under 5 years who were diagnosed with any one childhood cancer were obtained from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) from 1990 to 2019. The outcomes were the absolute numbers and rates of incidence, prevalence, mortality, and disability-adjusted life-years (DALYs) for different types of cancer.ResultsIn 2019, 8,774,979.1 incident cases (95% uncertainty interval [UI]: 6,243,599.2 to11,737,568.5) and 8,956,583.8 (6,446,323.9 to 12,364,520.8) prevalent cases of cancer in children under 5 years were identified worldwide; these cancers resulted in 44,451.6 (36,198.7 to 53,905.9) deaths and 3,918,014.8 (3,196,454.9 to 4,751,304.2) DALYs. From 1990 to 2019, although the numbers of incident and prevalent cases only decreased by −4.6% (−7.0 to −2.2) and −8.3% (−12.6 to −3.4), respectively, the numbers of deaths and DALYs clearly declined by −47.8% (−60.7 to −26.4) and −47.7% (−60.7 to −26.2), respectively. In 2019, the middle sociodemographic index (SDI) regions had the highest incidence and prevalence, whereas the low SDI regions had the most mortality and DALYs. Although all of the SDI regions displayed a steady drop in deaths and DALYs between 1990 and 2019, the low-middle and low SDI regions showed increasing trends of incidence and prevalence. Leukemia remained the most common cancer globally in 2019. From 1990 to 2019, the burdens of leukemia, liver cancer, and Hodgkin's lymphoma declined, whereas the incidence and prevalence of other cancers grew, particularly testicular cancer.Conclusions The global childhood cancer burden in young children has been steadily decreasing over the past three decades. However, the burdens and other characteristics have varied across different regions and types of cancers. This highlights the need to reorient current treatment strategies and establish effective prevention methods to reduce the global burden of childhood cancer.
... Women have a slower gastric emptying time and a bigger distribution volume for lipophilic drugs. Chemotherapeutical compounds show, thus, a 1.7-fold increase in adverse effects in women, although the longer half-life provides a benefit in terms of survival rate [3][4][5][6][7][8]. Analogously, women are more sensitive to toxicity, especially at the gastrointestinal and mucosal level, when treated with 5-fluorouracile. ...
Article
Full-text available
Purpose Given the biological differences between females and males, sex-specific evaluations should be carried out to obtain better cancer prevention, diagnosis, and treatment strategies. To this purpose, our aim was to evaluate sex differences for toxicity in a cohort of colorectal cancer (CRC) patients undergoing chemotherapy. Methods We performed a retrospective study in 329 CRC patients. Differences between males and females were tested performing the Mann-Whitney U test or the Fisher exact test. Multivariate logistic regression models were computed to evaluate the association between sex and risk of chemotherapy agent-related toxicity. Results According association sex toxicity, significant differences were observed in the median number of episodes of nausea (p = 0.044), vomit (p = 0.007), heartburn (p = 0.022), thrombocytopenia (p = 0.005), mucositis (p = 0.024). Moreover, statistically significant differences between males and females were observed in the distribution of the highest toxicity grades of nausea (p = 0.024), heartburn (p = 0.016), and thrombocytopenia (p = 0.034). Females have an increased risk of vomit (p = 0.002), alopecia (p = 0.035), heartburn (p = 0.005), mucositis (p = 0.003), and lower risk for thrombocytopenia (p = 0.005). Conclusion According to the association of sex chemotherapy agent-related toxicities, females resulted on average at a significant increased risk of more common adverse events (constipation, dysgeusia, alopecia, heartburn, vomit, asthenia, nausea, pain events, and mucositis). Sex-tailored CRC chemotherapy treatment is necessary to obtain efficacy avoiding toxicity, based on patients’ biological and genetic characteristics, a vision that would change CRC setting, a stable disease but still orphan of a real tailored approach.
... In medicine, gender differences are receiving more and more attention both with regard to the choice of therapy and side effects [1,2]. In oncology, the gender-dependent induction of cancer is also an important topic and clear sex differences are observed. ...
Article
Full-text available
Gender is increasingly recognized as an important factor in medicine, although it has long been neglected in medical research in many areas. We have studied the influence of gender in advanced rectal cancer with a special focus on radiosensitivity. For this purpose, we studied a cohort of 495 men (84.1% ≥ T3, 63.6% N1, 17.6%, M1) and 215 women (84.2% ≥ T3, 56.7% N1, 22.8%, M1) who all suffered from advanced rectal cancer and were treated with radiochemotherapy. The energy deposited, DNA double-strand break (dsb) repair, occurrence of chromosomal aberrations, duration of therapy, tumor regression and tumor-infiltrating lymphocytes, laboratory parameters, quality of life and survival were assessed. The residual DNA dsb damage 24 h after irradiation in lymphocytes was identical in both sexes. Furthermore, chromosomal aberrations accurately reflecting radiosensitivity, were similar in both sexes. There were no gender-dependent differences in tumor regression, tumor-infiltrating lymphocytes and outcome indicating no differences in the radiosensitivity of cancer cells. The irradiated tumor volume in women was slightly lower than in men, related to body weight, no difference was observed. However, when the total energy deposited was calculated and related to the body weight, women were exposed to higher amounts of ionizing radiation. During radiochemotherapy, decreases in blood lymphocyte counts and albumin and several quality-of-life parameters such as nausea and vomiting, loss of appetite, and diarrhea were significantly worse in women. There is no difference in radiation sensitivity between men and women in both normal tissue and tumors. During radiochemotherapy, the quality of life deteriorates more in women than in men. However, women also recover quickly and there are no long-term differences in quality of life.
... Another justification is that they do not have estrous cycles that strongly alter various physiological variables. There are also specific considerations such as the possibility of a differential response to treatment between men and women, and it is specifically for this reason that gender disparities should not be overlooked (Buoncervello et al., 2017). From preclinical studies (in animal models) to clinical studies, the study of the mechanisms involved in the appearance of many pathologies must consider the existence of different behavior and regulatory mechanisms between sexes, which is why there are explicit recommendations to include both sexes in preclinical studies (Sandberg et al., 2015) and to consider it in clinical treatment and evaluation (Mauvais-Jarvis et al., 2020). ...
Article
Full-text available
Within human physiology, systemic interactions couple physiological variables to maintain homeostasis. These interactions change according to health status and are modified by factors such as age and sex. For several physiological processes, sex-based distinctions in normal physiology are present and defined in isolation. However, new methodologies are indispensable to analyze system-wide properties and interactions with the objective of exploring differences between sexes. Here we propose a new method to construct complex inferential networks from a normalization using the clinical criteria for health of physiological variables, and the correlations between anthropometric and blood tests biomarkers of 198 healthy young participants (117 women, 81 men, from 18 to 27 years old). Physiological networks of men have less correlations, displayed higher modularity, higher small-world index, but were more vulnerable to directed attacks, whereas networks of women were more resilient. The networks of both men and women displayed sex-specific connections that are consistent with the literature. Additionally, we carried out a time-series study on heart rate variability (HRV) using Physionet’s Fantasia database. Autocorrelation of HRV, variance, and Poincare’s plots, as a measure of variability, are statistically significant higher in young men and statistically significant different from young women. These differences are attenuated in older men and women, that have similar HRV distributions. The network approach revealed differences in the association of variables related to glucose homeostasis, nitrogen balance, kidney function, and fat depots. The clusters of physiological variables and their roles within the network remained similar regardless of sex. Both methodologies show a higher number of associations between variables in the physiological system of women, implying redundant mechanisms of control and simultaneously showing that these systems display less variability in time than those of men, constituting a more resilient system.
... Human pathological conditions that show differential incidence rates, clinical manifestations, and progression according to sex can be extremely interesting as a focus of research [31]. In particular, sex is a significant variable in the prevalence and incidence of neurological disorders [32]. ...
Article
Full-text available
Sex is a significant variable in the prevalence and incidence of neurological disorders. Sex differences exist in neurodegenerative disorders (NDs), where sex dimorphisms play important roles in the development and progression of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the last few years, some sex specific biomarkers for the identification of NDs have been described and recent studies have suggested that microRNA (miRNA) could be included among these, as influenced by the hormonal and genetic background. Failing to consider the possible differences between males and females in miRNA evaluation could introduce a sex bias in studies by not considering some of these sex-related biomarkers. In this review, we recapitulate what is known about the sex-specific differences in peripheral miRNA levels in neurodegenerative diseases. Several studies have reported sex-linked disparities, and from the literature analysis miR-206 particularly has been shown to have a sex-specific involvement. Hopefully, in the near future, patient stratification will provide important additional clues in diagnosis, prognosis, and tailoring of the best therapeutic approaches for each patient. Sex-specific biomarkers, such as miRNAs, could represent a useful tool for characterizing subgroups of patients.
Article
Disordered eating can underpin a number of debilitating and prevalent chronic diseases, such as obesity. Broader advances in psychopharmacology and biology have motivated some neuroscientists to address diet-induced obesity through reductionist, pre-clinical eating investigations on the rodent brain. Specifically, chemogenetic and optogenetic methods developed in the 21st century allow neuroscientists to perform in vivo , region-specific/projection-specific/promoter-specific circuit manipulations and immediately assess the impact of these manipulations on rodent feeding. These studies are able to rigorously conclude whether a specific neuronal population regulates feeding behaviour in the hope of eventually developing a mechanistic neuroanatomical map of appetite regulation. However, an artificially stimulated/inhibited rodent neuronal population that changes feeding behaviour does not necessarily represent a pharmacological target for treating eating disorders in humans. Chemogenetic/optogenetic findings must therefore be triangulated with the array of theories that contribute to our understanding of appetite. The objective of this review is to provide a wide-ranging discussion of the limitations of chemogenetic/optogenetic circuit manipulation experiments in rodents that are used to investigate appetite. Stepping into and outside of medical science epistemologies, this paper draws on philosophy of science, nutrition, addiction biology and neurophilosophy to prompt more integrative, transdisciplinary interpretations of chemogenetic/optogenetic appetite data. Through discussing the various technical and epistemological limitations of these data, we provide both an overview of chemogenetics and optogenetics accessible to non-neuroscientist obesity researchers, as well as a resource for neuroscientists to expand the number of lenses through which they interpret their circuit manipulation findings.
Article
Full-text available
Severe outcomes from SARS-CoV-2 infection are highly associated with preexisting comorbid conditions like hypertension, diabetes, and obesity. We utilized the diet-induced obesity (DIO) model of metabolic dysfunction in K18-hACE2 transgenic mice to model obesity as a COVID-19 comorbidity. Female DIO, but not male DIO mice challenged with SARS-CoV-2 were observed to have shortened time to morbidity compared to controls. Increased susceptibility to SARS-CoV-2 in female DIO was associated with increased viral RNA burden and interferon production compared to males. Transcriptomic analysis of the lungs from all mouse cohorts revealed sex- and DIO-associated differential gene expression profiles. Male DIO mice after challenge had decreased expression of antibody-related genes compared to controls, suggesting antibody producing cell localization in the lung. Collectively this study establishes a preclinical co-morbidity model of COVID-19 in mice where we observed sex- and diet-specific responses that begin explaining the effects of obesity and metabolic disease on COVID-19 pathology.
Article
Pancreatic carcinoma incidence showed a significant increase in men over the last few years and the prognosis remains poor. Patients are treated with different pharmacological plans with no evidence about gender-specific adverse effects. We aimed to investigate differences in the incidence of chemotherapy side effects in the treatment of pancreatic cancer, in order to provide insights toward a personalized assistance based in individual needs. The sample population is composed of 207 patients. Regression model highlighted the predictive role of female gender for alopecia, constipation, hand-foot syndrome and epigastric pain. Also considering single therapeutic schemes, gender differences have been reported. Moreover, evaluating the effect of age, a general reduced risk of toxicity has been reported in younger patients. In order to personalize chemotherapy and increase patients survival rate and life quality during the therapy, gender medicine and pharmacology studies are recommended.
Article
Resumen Objetivo Analizar los contenidos sobre las diferencias por sexo en las leucemias en libros de texto de hematología y medicina interna utilizados en el Grado de Medicina en España, en el curso 2019-2020, por comparación con las diferencias reconocidas en la literatura científica. Método Análisis del contenido manifiesto de los capítulos sobre leucemias en los libros de medicina interna, hematología clínica y pregrado de hematología. Categorías de análisis: epidemiología, etiopatogenia, diagnóstico, tratamiento y pronóstico de las leucemias. Resultados La información epidemiológica de los libros revisados tiene mayor consideración de las diferencias por sexo en la incidencia y el pronóstico, pero no en la mortalidad y la supervivencia. La etiopatogenia está descrita en todos los libros como un proceso fisiopatológico igual para ambos sexos, pero ninguno describe diferencias por sexo en su presentación clínica. Tres libros describen un tratamiento único, asumido igual para ambos sexos; dos libros mencionan el tratamiento de la leucemia mieloide aguda en las embarazadas y uno el tratamiento de la leucemia mieloide crónica. Ningún libro menciona diferencias por sexo en la farmacocinética, la eficacia y la toxicidad del tratamiento, aunque existe amplia evidencia. Conclusiones Los contenidos sobre las diferencias según sexo y de género en los capítulos sobre leucemias analizados son insuficientes por comparación con las evidencias en la literatura científica. La inclusión de conocimiento sobre la interacción sexo-género en las diferentes secciones de los capítulos de leucemias de los libros de texto de hematología y medicina interna incrementará su calidad científica en ediciones futuras, contribuyendo a unas mejores prácticas profesionales, más eficientes y equitativas.
Article
Full-text available
Background A disease of unknown aetiology, Alzheimer's disease (AD) is the most common type of dementia. As the elderly population grows worldwide, the number of patients with AD also increases rapidly. The aim of this meta-analysis is to evaluate the prevalence and incidence of AD in Europe. Methodology We conducted a literature search on Medline, Scopus, and CINAHL Complete using the keywords “Alzheimer”, “Alzheimer's disease”, and “AD” combined with “prevalence”, “incidence”, and “epidemiology”. A Bayesian random effects model with 95% credible intervals was used. The I² statistic was applied to assess heterogeneity. Results The prevalence of Alzheimer's disease in Europe was estimated at 5.05% (95% CI, 4.73-5.39). The prevalence in men was 3.31% (95% CI, 2.85-3.80) and in women, 7.13% (95% CI, 6.56-7.72), and increased with age. The incidence of Alzheimer's disease in Europe was 11.08 per 1000 person-years (95% CI, 10.30-11.89). Broken down by sex, it was 7.02 per 1000 person-years (95% CI, 6.06-8.05) in men and 13.25 per 1000 person-years (95% CI, 12.05-14.51) in women; again these rates increased with age. Conclusions The results of our meta-analysis allow a better grasp of the impact of this disease in Europe.
Article
Full-text available
Background:: Sex and gender differences are often overlooked in research design, study implementation and scientific reporting, as well as in general science communication. This oversight limits the generalizability of research findings and their applicability to clinical practice, in particular for women but also for men. This article describes the rationale for an international set of guidelines to encourage a more systematic approach to the reporting of sex and gender in research across disciplines. Methods:: A panel of 13 experts representing nine countries developed the guidelines through a series of teleconferences, conference presentations and a 2-day workshop. An internet survey of 716 journal editors, scientists and other members of the international publishing community was conducted as well as a literature search on sex and gender policies in scientific publishing. Results:: The Sex and Gender Equity in Research (SAGER) guidelines are a comprehensive procedure for reporting of sex and gender information in study design, data analyses, results and interpretation of findings. Conclusions:: The SAGER guidelines are designed primarily to guide authors in preparing their manuscripts, but they are also useful for editors, as gatekeepers of science, to integrate assessment of sex and gender into all manuscripts as an integral part of the editorial process.
Article
Full-text available
Background: Gender disparity in melanoma outcome is consistently observed, suggesting that gender is as an important prognostic factor. However, the source of this gender disparity in melanoma remains unclear. Objective: This article reviews advances in our understanding of gender differences in melanoma and how such differences may contribute to outcomes. Methods: A broad literature search was conducted using the PubMed database, with search terms such as ‘gender differences in melanoma’ and ‘sex differences in melanoma.’ Additional articles were identified from cited references. Results: Herein, we address the gender-linked physiologic differences in skin and melanoma. We discuss the influence of estrogen on a woman’s risk for melanoma and melanoma outcomes with regard to pregnancy, oral contraceptives, hormone replacement therapy, and UV tanning. Conclusions: The published findings on gender disparities in melanoma have yielded many advances in our understanding of this disease. Biological, environmental, and behavioral factors may explain the observed gender difference in melanoma incidence and outcome. Further research will enable us to learn more about melanoma pathogenesis, with the goal of offering better treatments and preventative advice to our patients.
Article
Full-text available
BACKGROUND: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the Comprehensive in Vitro Proarrhythmia Assay (CiPA). METHODS AND RESULTS: We studied the effects of 26 drugs and 3 drug combinations on two commercially available iPSC-CM types using high-throughput voltage-sensitive dye (VSD) and microelectrode-array (MEA) assays being studied for the Comprehensive in Vitro Proarrhythmia Assessment (CiPA) initiative and compared the results to clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced APDc and FPDc prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the 4 remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to hERG potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. CONCLUSION: Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted.
Article
Full-text available
Objectives This study analyzed age-adjusted sex differences among acute myocardial infarction (AMI) patients aged 75 years and above with regard to 7-year mortality (primary end point) and the frequency of angiograms and admission to the coronary care unit (CCU) as well as 1-year mortality (secondary end points). Methods A retrospective cohort study comprised 1,414 AMI patients (748 females and 666 males) aged at least 75 years, who were admitted to Sahlgrenska University Hospital in Gothenburg, Sweden, during two periods (2001/2002 and 2007). All comparisons between female and male patients were age adjusted. Results Females were older and their previous history included fewer AMIs, coronary artery bypass grafting procedures, and renal diseases, but more frequent incidence of hypertension. On the contrary, males had higher age-adjusted 7-year mortality in relation to females (hazard ratio [HR] 1.16 with corresponding 95% confidence interval [95% CI 1.03, 1.31], P=0.02). Admission to the CCU was more frequent among males than females (odds ratio [OR] 1.38 [95% CI 1.11, 1.72], P=0.004). There was a nonsignificant trend toward more coronary angiographies performed among males (OR 1.34 [95% CI 1.00, 1.79], P=0.05), as well as a nonsignificant trend toward higher 1-year mortality (HR 1.18 [95% CI 0.99, 1.39], P=0.06). Conclusion In an AMI population aged 75 years and above, males had higher age-adjusted 7-year mortality and higher rate of admission to the CCU than females. One-year mortality did not differ significantly between the sexes, nor did the frequency of performed coronary angiograms.
Article
Full-text available
In June 2015, the National Institutes of Health (NIH) released a Guide notice (NOT-OD-15-102) that highlighted the expectation of the NIH that the possible role of sex as a biologic variable be factored into research design, analyses, and reporting of vertebrate animal and human studies. Anticipating these guidelines, the NIH Office of Research on Women's Health, in October 2014, convened key stakeholders to discuss methods and techniques for integrating sex as a biologic variable in preclinical research. The workshop focused on practical methods, experimental design, and approaches to statistical analyses in the use of both male and female animals, cells, and tissues in preclinical research. Workshop participants also considered gender as a modifier of biology. This article builds on the workshop and is meant as a guide to preclinical investigators as they consider methods and techniques for inclusion of both sexes in preclinical research and is not intended to prescribe exhaustive/specific approaches for compliance with the new NIH policy.-Miller, L. R., Marks, C., Becker, J. B., Hurn, P. D., Chen, W.-J., Woodruff, T., McCarthy, M. M., Sohrabji, F., Schiebinger, L., Wetherington, C. L., Makris, S., Arnold, A. P., Einstein, G., Miller, V. M., Sandberg, K., Maier, S., Cornelison, T. L., Clayton, J. A. Considering sex as a biological variable in preclinical research.
Article
Full-text available
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from two commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors two wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229±15ms vs. 427±49ms [mean±SD, P<0.01]; average spontaneous cycle length: 0.99±0.02s vs. 1.47±0.35s [mean±SD, P<0.01], Cor.4U vs. iCell CMC, respectively). The 10%–90% rise time of the AP (Trise) was ~6ms and was normally distributed when expressed as 1/Trise² in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.
Article
Major differences between men and women exist in epidemiology, manifestation, pathophysiology, treatment, and outcome of cardiovascular diseases (CVD), such as coronary artery disease, pressure overload, hypertension, cardiomyopathy, and heart failure. Corresponding sex differences have been studied in a number of animal models, and mechanistic investigations have been undertaken to analyze the observed sex differences. We summarize the biological mechanisms of sex differences in CVD focusing on three main areas, i.e., genetic mechanisms, epigenetic mechanisms, as well as sex hormones and their receptors. We discuss relevant subtypes of sex hormone receptors, as well as genomic and nongenomic, activational and organizational effects of sex hormones. We describe the interaction of sex hormones with intracellular signaling relevant for cardiovascular cells and the cardiovascular system. Sex, sex hormones, and their receptors may affect a number of cellular processes by their synergistic action on multiple targets. We discuss in detail sex differences in organelle function and in biological processes. We conclude that there is a need for a more detailed understanding of sex differences and their underlying mechanisms, which holds the potential to design new drugs that target sex-specific cardiovascular mechanisms and affect phenotypes. The comparison of both sexes may lead to the identification of protective or maladaptive mechanisms in one sex that could serve as a novel therapeutic target in one sex or in both.