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Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing
Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing Flavia Franconi, Giuseppe Rosano, Ilaria Campesi PII: DOI: Reference:
S0167-5273(14)02161-5 doi: 10.1016/j.ijcard.2014.11.019 IJCA 19175
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
8 September 2014 27 October 2014 3 November 2014
Please cite this article as: Franconi Flavia, Rosano Giuseppe, Campesi Ilaria, Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing, International Journal of Cardiology (2014), doi: 10.1016/j.ijcard.2014.11.019
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ACCEPTED MANUSCRIPT Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing
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Flavia Franconi1 , Giuseppe Rosano2, Ilaria Campesi3
Department of Biomedical Sciences, University of Sassari, National Laboratory of Gender
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Medicine of the National Institute of Biostructures and Biosystems, Osilo-Sassari, Italy, , Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Italy;
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Author had the idea to indagate the effect of sex and gender on pre-analytical phase of research and
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Cardiovascular and Cell Sciences Research Institute, St George's University of London. The
author wrote and discussed the review
Department of Biomedical Sciences, University of Sassari, National Laboratory of Gender
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3
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wrote and discussed the review.
Medicine of the National Institute of Biostructures and Biosystems, Osilo-Sassari, Italy. The author
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wrote the review and took care of bibliography
Corresponding author: Flavia Franconi, Dipartimento di Scienze Biomediche Via Muroni 23,
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Sassari, Italy ; e-mail [email protected], phone +39079228717
Conflict of Interest The authors report no relationships that could be construed as a conflict of interest.
Acknowledgement: This research was partially funded by a grant of Sardinia Region “Effetti avversi e farmaci utilizzati nel trattamento del diabete di tipo 2: un approccio di genere” and by a grant of Istituto Superiore di Sanità “Piattaforma italiana per lo studio sulla polimorbidità”. We express deep gratitude to the Italian Pharmacological Society that provided the award “Gender Innovation” to Ilaria Campesi
ACCEPTED MANUSCRIPT Abstract Many international organisations encourage studies in a sex-gender perspective. However, research with a gender perspective presents a high degree of complexity, and the inclusion of sex-gender
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variable in experiments presents many methodological questions, the majority of which are still neglected. Overcoming these issues is fundamental to avoid erroneous results. Here, pre-analytical
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aspects of the research, such as study design, choice of utilised specimens, sample collection and processing, animal models of diseases, and the observer’s role, are discussed. Artefacts in this stage
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of research could affect the predictive value of all analyses. Furthermore, the standardisation of
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research subjects according to their lifestyles and, if female, to their life phase and menses or oestrous cycle, is urgent to harmonise research worldwide. A sex-gender-specific attention to preanalytical aspects could produce a decrease in the time for translation from the bench to bedside.
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Furthermore, gender-specific pre-clinical pharmacological testing will enable adequate assessment of pharmacokinetic and pharmacodynamic actions of drugs and will enable, where appropriate, an
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adequate gender-specific clinical development plan. Therefore, gender-specific pre-clinical research
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will increase the gender equity of care and will produce more evidence-based medicine.
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Key words: sex-gender, research complexity, pre-analytical conditions
ACCEPTED MANUSCRIPT 1. Introduction Although Hippocrates of Cos (460-370 BC) evidenced that “A woman does not take the gout unless her menses has stopped,” describing a sex-gender difference in the susceptibility of the
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development of disease [1] , it is indisputable that the differences between men and women were ignored until the two last decades of the last century [2]. However, over the past 20-30 years,
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research has shown, from single cells to more complex biological systems, that biological differences (sex) between men and women are numerous and involve all branches of biomedical
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and are organ specific [7] and cell specific [8].
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sciences [2-5]. In the last few years, it has emerged that sex-gender differences starts in utero [6]
Thus, it is not surprising that remarkable sex-gender differences have been described in the prevalence, progression, treatment and outcome of numerous diseases including diabetes mellitus
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[9], cancer, depression and brain disorders, and infectious, cardiovascular, renal, hepatic, pulmonary, inflammatory and autoimmune diseases [3].
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Indeed, the biological differences between sexes should be considered across the entire
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range of research, starting from the pre-analytic conditions embodying research in genetics, epigenetics [10], developmental biology, biochemistry, physiology, pharmacology, toxicology, and
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epidemiology as well as social sciences adopting all available technologies including omics [10]. All ages should be considered, including pre-natal life, because sex differences start in utero [6]. It is also important to recall that the placenta may play a key role not only in buffering environmental effects transmitted by the mother but also in expressing and modulating effects due to preconception exposure of both the mother and the father to stressful conditions [6]. In addition, in designing experiments, epigenetic modifications should also be taken into consideration. Inevitably, the stress response may play a role . The pioneer paper of Critchlow and colleagues [11] showed that the stress response is sexually dimorphic. Consistently, social status, domestic violence and caregiver role are related to the stress response and can lead to depression, cardiovascular diseases, and diabetes mellitus [12-14]. In this regard, we have to highlight that the
ACCEPTED MANUSCRIPT definitions of sex (biological differences in male and female body) and gender (environmental and social influences) may assume different meanings in biomedical fields and in social sciences. As a result, gender is sometimes mistakenly employed as an updated version of the term sex or to
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indicate only female sex [15]. This viewpoint is widespread, and some studies that mention the word gender in their title incorrectly use the term gender as a synonym for women [16, 17]. Of
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course, this attitude has important consequences, and men’s gender-specific needs may not be sufficiently considered.
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Furthermore, there is a relevant debate regarding the stability and validity of the binaries of
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nature and culture that underpin the concepts of sex and gender [18, 19], and there are some difficulties in segregating sex and gender influences on health, given the constant and dynamic interactions between genes and environment [20]. Some authors [21, 22] reject the discourse of
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biological versus social determinism and advocate a deeper analysis of how interactions between the biological being and the social environment impact on ‘‘individual’’ capacities, suggesting
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instead the use of the term sex-gender. Thus, through this review, the term sex-gender will be used
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to give equal status to the two concepts [23] and to indicate that sex-gender is a domain of complex and highly integrated phenomena and, as consequence of sex-gender research, requires
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intersectionality and integration with other disciplines, including social sciences. It is important to recognise that gender applies to all vertebrates and humans and that sexual dimorphism varies in the species and in the strains of animals. However, in numerous biomedical fields, almost all cellular studies [24]do not differentiate between genetic male or female cells. Furthermore, the majority (68–76%) of preclinical studies use only males or do not report the sex of the animals [25, 26], and the same occurs in some clinical studies [27]. Even fewer studies have been designed to address influences of psycho-social status (gender) of the donors on physiological outcomes [28].
ACCEPTED MANUSCRIPT Randomised clinical trials are still enrolling fewer women than men, especially in phase 1 and 2 trials. Nevertheless, the U.S. Food and Drug Administration recently recommended, for the first time, the use of different dosages of zolpidem in women and in men [29].
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Overall, existing knowledge suggests that:
a) it is inappropriate to assume that results obtained only in one sex can be applied to the
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other [30]:
b) sex-gender demands a lot of attention in the construction of measurements and variables,
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especially because of different body sizes and body composition of males and females;
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c) some baseline questions such as diet, housing, breeding, and cross fostering are still without a clear response;
d) characteristics of donors should be known, because donor lifestyles could affect the
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functions of the organs, cells, biomarkers and indicators [31, 32]; e) and, finally, sex-gender is not adequately considered in preclinical and clinical
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interventions involving diagnostics, medical devices, medications and clinical setting, and yet it is
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unknown if sex-gender influences placebo and nocebo effects and drug adherence [33, 34]. To overcome the scarce enrolment of females versus males, it is necessary to perform more
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studies on females comparing directly the two sexes and to incorporate a sex-gender-focused approach in the entire process of the research, which is something more than the simple enrolment of both sexes (see below). This can facilitate the aims of tailored medicine and further translational science, elevating basic knowledge upon which to build translational approaches that shorten the time from bench to patient bed [35, 36]. Furthermore, the awareness of the differences and similarities between male and females can improve the prevention, the efficacy, the safety and the appropriateness of the treatments [3, 33, 37-39]. A certain number of papers on methodological issues in sex-gender research are available [26, 28, 40-43]. However, they are mainly focused on defining differences between males and females and the origin of these differences such as the role of hormones [44] and on discussing animal
ACCEPTED MANUSCRIPT models of diseases and issues that sex-gender research meets after sampling [26, 28, 40-43], neglecting pre-analytic issues, which also include the influences of environment, with few exceptions [23].
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Indeed, pre-analytic procedures can be the source of a multitude of errors. It has been calculated that in the clinical laboratory, about 62% of errors happen during pre-analytical phases
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[45]. Pre-analytic variations (study design, compliance of the investigated subjects, compliance of the technical staff in adherence to protocols, choice of utilised specimens, sample collection and
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processing) are of critical relevance and importance, being encompassed in all sample preparations.
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Consequentially, an artefact in this stage could affect the predictive value of all analyses. They can be classified [46] in:
a) actions performed in animals or in humans before sample collection (animal handling,
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fasting, stress, restraint, analgesia and anaesthesia, dosing, diet, etc.) b) actions performed at collection (time of collection, blood sampling, collection technique,
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amount of blood, etc.)
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c) actions performed on the specimen such as dimension of collection tubes, sample separations, storage time, temperature, etc.
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Theoretically, all pre-analytic variations may be sex-gender dependent. Thus, pre-analytic variations are of special interest in sex-gender research. Considering that we are focusing on gender research, we include in the pre-analytical aspect the importance of the professionals and researchers’ sexgender, and the reason for focusing on this variable is illustrated below. Inclusion of XX cells and female animals in experiments and analysis of data by sex may contribute to resolve, at least in part, the issue of irreproducibility seen in preclinical biomedical research. This requires a special attention to methodological questions. Therefore, we suggest that it is no longer reasonable to ignore the methodological issues in sex-gender research, because only awareness of these issues can lead to sex-gender innovations. Although many studies tend to simply compare males and females on a number of health indicators, it emerges that there is an urgency to
ACCEPTED MANUSCRIPT use more sophisticated experimental designs, to redefined old methods and to develop new ones to produce new measures to study the influence of sex-gender on health. Here we discuss the pre-
2.
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analytic variables that have a special interest in sex-gender research through the lens of gender.
Complexity of sex gender research
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There are numerous criticisms regarding the ways in which sex-gender issues are addressed in health research because the various relationships among biological sex, gender and health are
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complex and numerous and may affect the manner in which sex-gender influences outcomes in
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health research. Indeed, sex-gender research has to consider the biosocial approach of medicine, which is composed of the interactions of biology and social environments [21], and the intersectional approaches [47]. Intersectionality is defined by Davis [48] as ‘the interaction
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between gender, race, and other categories of difference in individual lives, social practices, institutional arrangements, and cultural ideologies and the outcomes of these interactions in terms
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of power’. In other words, intersectionality: a) considers simultaneously interactions among various
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aspects of social life [49-51]; b) examines how social life and structural forces interact to shape and
our lives [53].
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influence human experiences [50, 52]; c) and explains how social organisation shapes all aspects of
Importantly, sex-gender researchers must also consider the similarities between women and men [54, 55]. In this regard, a sex-and gender-based analysis has been developed in Canada [56]. This analysis prioritises examinations of similarities and differences between women and men. Furthermore, sex-gender research should also include the boundaries of gender (masculine/feminine) and sex (male/female) [57], because the perpetuation of the dichotomies between sex and gender discourages going beyond two definable sexes and genders in view of the fact that intersex and transgendered people destabilise the dichotomy [58]. In this respect, FaustoSterling’s dynamic system theory [21] and Bekker’s Multi-Facet Gender and Health Model [59] clearly evidence that sex and gender must be integrated.
ACCEPTED MANUSCRIPT However, if the relevance of intersectionality is theoretically clear, it is less clear how it could be translated into research practice, and in this regard, we agree completely with Bowleg, who wrote that researchers “often have to self-teach and learn from trial and error” [49]. Indeed, if
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it is not possible to consider all factors, it is possible to say which factors are included or excluded, explaining the reason of the choices made. Actually, no single study is able avoid these problems
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completely, but it is possible to plan different studies which address different aspects [60]. Finally, researchers and professionals should have the expertise to recognise, perceive and
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incorporate existing sex-gender differences into their decision to make experimental designs and
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actions [50]. As a consequence, a scientist with sex-gender expertise cannot be a reductionist. Reductionism has permitted a great progression of science but has failed to answer numerous questions. Most life events at the organismal and cellular levels, in fact, cannot be only understood
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by understanding molecular functions in the cells and/or in the cell-free condition. Organisms, cells and cellular organelles, before becoming objects of research, live a life characterised by specific
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events (diet, stressors, lifestyle etc.) that may affect the way they perform in individual experiments
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and may even change in the genome, by both genetic and epigenetic mechanisms [61, 62]. Thus,
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social and biomedical sciences have to work together in controlling for pre-analytical variations.
3. Effets of sex-gender of researchers and professionals on research outcomes The recognition and the awareness of the difficulties that researchers meet to capture sex and gender aspects are fundamental. Nevertheless, recommendations and guidelines to make sex-gender research and analysis ([23]and quoted literature) often do not consider the sex-gender of the researcher, forgetting that he/she is a person and every individual is sexed and gendered. The potential influences of the sex-gender of the scientist on the results of the research have recently been shown in both preclinical [63, 64] and clinical studies [65]. In particular, experimental pain in humans is better tolerated when the researcher is of the opposite sex of the examined subject, while higher pain intensity has been measured for subjects tested by female experimenters [66].
ACCEPTED MANUSCRIPT Furthermore, men report lower pain to female experimenters than to male experimenters in the absence of significant changes in automatic response (heart rate and skin conductance), suggesting that the effect of the experimenter’s sex-gender is due to psychosocial factors [67]. The previous
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results indicate the importance of the relationship between research and subjects of research. Further confirmation of this principle comes from the work of Hadjistavropoulos and co-workers
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[68], which examines patients with low back pain, that showed that an observer is significantly influenced by patient gender and by the patients’ physical attractiveness. Rats and mice responses to
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pain depend on the sex of researchers [69]. Furthermore, male and female researchers focus on
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different aspects of behaviour and interpret the very same results in a different ways [70-72]. Indeed, sex-gender modulates visual attention during listening, and this could affect the response of the subject under study [73].
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In conclusion, a research team should be constituted by women and men, and the leadership should include men and women in order to reduce or to avoid inappropriate conclusions linked to
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the sex-gender of the researchers.
4. Experimental design
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Experimental planning should define outcomes and independent measures a priori in order to explain the biological basis of sex differences. Further experimental design should develop paradigms that include a sufficient number of samples that involve social and biosocial aspects including appropriate control groups for all hypotheses. Small sample size may prevent the emergence of important biological and biosocial interactions [74]. Finally, sensitivity analyses should be performed. Experimental planning should consider, with the highest priority, the animal welfare and the relationship between researcher and subject. As illustrated below, experimental errors could occur in a sex-gender specific manner and have profound consequences on outcomes.
ACCEPTED MANUSCRIPT The experimental plan should include social and environmental situations, because these factors may influence disease vulnerability, therapeutic response and experimental outcomes [42], depending on the sexual dependence of the phenomena being studied [75, 76].
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As already mentioned, the stress response is essentially sexually dimorphic [11]. Thus, stressful events can have different features in males and females. Notably, this sexually dimorphic
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stress response seems to occur in a stressor-, species- and strain-specific manner [11, 77], and may also depend on bedding materials (see below). Thus, animal studies should take into account the
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potential role of environmental stress and sex-gender differences. In other words, studies with
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animals should consider that they have a social interaction.
The definition of primary endpoints and their variability are required for group size calculations, which also require the definition of power for detecting a difference [78]. Primary and
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secondary endpoints should be defined before starting the study; further, data should be analysed by a blinded observer and, if needed, statisticians should be consulted. If possible, studies should use
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balanced sex-gender and sex-gender X age factors. In addition, protocols should analyse data by
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sex-gender and interactions, rather than simply adjusting for gender. Power should be adequate, or lack of power (if inevitable) should be clearly mentioned. Inclusion and exclusion criteria need to
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be defined for each sex, and studies should include only individuals who match defined diagnostic criteria. Theoretically, more than one animal model should be used to represent the diversity in human patients.
5. Actions performed before sample collection 5.1 Age and sex differences The effect of age on data has been well recognised. Sexual dimorphism starts in utero and seems to occur at a pre-gonadal stage [10]. In line with the previous observations, in multiparous rodents such as mice, rats and gerbils, the position of male and female foetuses within the uterus can generate long lasting sex-gender differences in novelty seeking at puberty [79]. Furthermore, a
ACCEPTED MANUSCRIPT male mouse foetus that develops between female foetuses is more sexually active as an adult, less aggressive and has smaller seminal vesicles [80]. The intrauterine position of female mice can predict reproductive traits including genital morphology, sexual attractiveness, behaviour, and
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timing of puberty [81]. This effect seems to depend on sexual hormones, because a male mouse foetus located between males experiences greater concentrations of testosterone than a foetus of the
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same sex positioned between females. Likewise, a female foetus positioned between females experiences greater concentrations of oestradiol than a foetus of the same sex positioned between
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males [81]. These results suggest that standardisation even in experimental animals is very difficult
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to achieve, because the influence of intrauterine life is difficult to control adequately. This effect can be amplified by sex-gender aspects. Notably, prenatal and neonatal events can perpetuate into adulthood and in the next generation, and this effect seems to occur in a sex-gender-dependent
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manner [82, 83], increasing the complexity of research.
The interaction between sex and age is seen at all ages also depends on the stress effect.
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Aged male and female Fischer 344 rats, without stress and when given 21 days of restraint for 6
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h/day, present significant differences. In particular, female rats loose less weigh than male rats when stressed. Furthermore, unstressed males are more anxious than unstressed female. Interestingly,
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stress increases anxiety-related behaviour in male rats, but decreases it in female rats [84]. Previously, it has been shown that young unstressed female rats are generally less anxious than young control male rats. However, stress exerts anxiogenic and anxiolytic effects on young female rats and young male rats, respectively [85, 86]. Interestingly, the sexual dimorphism observed after stress induction appears to be reversed during aging. These results suggest that sexual dimorphism needs to be studied considering the influence of age. Remarkably, the previous observations suggest that sexual dimorphism is highly dependent on the sex and age combination, indicating that these factors should be studied together to obtain the optimisation of standardisation.
ACCEPTED MANUSCRIPT 5.2 Environmental and social factors and handling of animals Animals are widely used in toxicological and pharmaceutical research using methods developed in the last 50–60 years [87]. Animal use includes the breeding of laboratory animals as
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well as the housing of animals in the animal facilities, both of which are complex processes that can influence the results of the experiments in a significant manner. Indeed, animal tests as a surrogate
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for human research are subjected of numerous criticisms because their use involves a number of assumptions and extrapolations. In view of the fact that more male animals are used in comparison
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to female ones [25, 26, 44], more criticisms have to be made for extrapolation of results to women.
5.3 Cross fostering
The method of cross fostering, in which a litter of rodents born to one dam is suckled by
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another dam, is largely used in animal facilities and by animal producers, although cross fostering has little relevance for humans. However, it is not yet clear whether the process of fostering is
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harmless or if cross fostering itself is influenced by sex-gender. Rare findings suggest that cross
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fostering is not innocuous, because the procedure for group formation, which involves housing of unfamiliar and unisexual subjects, can be stressful [88]. For example, in mice, cross fostering is
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linked with reduced growth and altered behaviours [88-90], while in rats, cross fostering alters nociception and emotional behaviour [91, 92]. Furthermore, cross fostering per se influences cardiovascular and metabolic function in adulthood, programming a ‘thrifty’ phenotype, especially in male mice [93]. Notably, alterations induced by cross fostering are sex-gender-dependent [94]. In conclusion, the previous results suggest that cross fostering procedures may have different effects in male and female animals, indicating that cross fostering should be indicated, at least, in the method section of papers that enrol male and female animals. In our opinion, in preclinical testing of drugs, if animals are grouped through cross fostering, appropriate controls should be given, because prenatal and neonatal events including environmental changes can have long-term effects on adult phenotypes, predisposing individuals to diseases [95-99].
ACCEPTED MANUSCRIPT 5.4 Maternal care and weaning Experiments in rats, mice and monkeys show that maternal care in the first 2 weeks of life
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has critical effects on the neuroendocrine stress response [100, 101]. Offspring, deprived of maternal care, increase glucocorticoid secretion, and this is thought to be a risk factor for mental
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and cardiometabolic disorders [95, 96, 99]. Notably, in adult male mice but not in female ones, postnatal handling (maternal deprivation plus sham injection) induces hormonal and metabolic
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conditions similar to mild metabolic syndrome/type-2 diabetes, suggesting that female sex exerts
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effective protection against the hypothalamus-pituitary-adrenal homeostasis disruption induced by postnatal handling [99].
The amount of maternal care that a pup receives can be affected by litter size. Multiple
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siblings can result in a slower growth curve, lower dominance status, delayed onset of weaning and lower probabilities of survival for particular progeny [102-104]. For example, large siblings induce
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stereotypic behaviour in 2 inbred strains (C57BL/6N and C57BL/6J) and an outbred stock (CD1
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[ICR]) in female but not in male mice, indicating the saliency of the sex and gender [102]. Weaning is one of the most important events in the very early stage of life. Shorting weaning
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can produce long-lasting consequences in adulthood, such as anxiety- and aggression-augmenting effects [97, 98]. Early-weaned male rats have a higher sympathetic response to stressors [105], and early-weaned mice have elevated endocrine responses to mild stress [106]. Further, in early-weaned Balb/c mice, at 3 weeks of age, a reduction in the number of open-arm entries in an elevated plusmaze for both male and female mice is observed, but this effect is long-lasting only in males [107]. Kikusui et al [107] also show that myelin formation increases in normally weaned male and female mice and in early-weaned male mice, but not in early-weaned female mice, in which myelin formation increases strongly at week 3 and then declines at week 5. In conclusion, caution is necessary to evaluate experiments performed with animals subject to early and late weaning and maternal deprivation, because these procedures exert long lasting
ACCEPTED MANUSCRIPT effects on adult phenotypes, and this effect seems to occur in a sex-gender dependent manner. The sex-gender dependency of a phenomenon could produce artefacts that can invalidate results. At least, the modality of maternal care, litter sizes and the time of weaning should be clearly indicated
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in the method section of papers. In our opinion, in preclinical testing of experimental drugs,
5.5 Diets and gut microbiota differences
observed
between
males
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Physiological
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appropriate controls should be given.
and
females
can
influence
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pharmacokinetics as well as pharmacodynamics of endogenous and exogenous compounds such as foods, beverages [22] and drugs [108]. Regarding foods, the sex-gender differences observed at the gastrointestinal level assume a higher relevance in the bio-availability of food and drug [108-110]
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which is also influenced by food form (males eat pelleted food better than females) [111-113], suggesting that sex can play a role. Indeed, for gender studies, the materials where foods and
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beverages are held are also important, because oestrogenic compounds can be present in water
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bottles and cages and may be leaching, especially when cages and bottles are old and visibly worn [114]. However, little attention has been given to this aspect, even in studies focused on the
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investigation of endocrine disruptors [115]. Moreover, commercial diets can contain different compounds such as casein or soya protein. The soya diet contains a large amount of oestrogenic isoflavones (daidzein and genistein) [115118], which may have different pharmacodynamic effects in males and females. For example, in rabbit aortas, isoflavones inhibit formation of neointimal cells and are more active in males than in the females ([119] and quoted literature). Furthermore, genistein may afford greater cardioprotection in female guinea pigs than in males, and a soya-based diet worsens hypertrophic cardiomyopathy, especially in male mice ([119] and quoted literature). Finally, soya phytoestrogen supplementation augments cardiac growth in male mice but not in female ones ([119] and quoted literature). Regarding the importance of sex-gender on the pharmacokinetic of polyphenols, there
ACCEPTED MANUSCRIPT are no univocal data ([119] and quoted literature). After the administration of [14C] genistein, the maximal plasma concentrations of radioactivity are significantly lower in females than in males, whereas the concentration of radioactivity is greater in the liver of female rats than in male rats
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([119] and quoted literature). However, not all authors found sex-gender differences in plasma levels of genistein and daidzein and their metabolites ([119] and quoted literature). Notably,
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genistein’s oral bioavailability appears to be dependent on oestrous phase, being lower when oestrogen is higher ([119] and quoted literature). Polyphenol’s excretion as conjugated forms occurs
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by renal (7-30 %) and biliary routes (10%) ([119] and quoted literature). Considering that renal
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excretion is lower in women than in men [108], this could be of some importance in the elimination of polyphenols.
Finally, isoflavones may modify the metabolism of endogenous and exogenous compounds
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through the inhibition or induction of phase 1 and phase 2 enzymes. In particular, pregnane X receptors, which are implicated in the activation of CYP3A, CYP2B6, CYP2C9, sulfotransferase,
and inhibited by coumestrol ([119] and quoted literature). These results
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daidzein and equol
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UGT1A1, glutathione S-transferases, and multidrug resistance protein-2 , are activated by genistein,
indicate that isoflavone-rich diets, such as soya-derived diets, could alter the pharmacokinetics of
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other xenobiotics including medications, and this point is of extraordinary relevance in pharmacokinetic studies.
Additionally, the isoflavones of a dam’s diet reach the foetus through the placenta and the neonate through the dam’s milk [117, 120], and, consequentially, they could affect development of offspring in a sex-gender-dependent manner. This maternal effect could have salient consequences on predisposing one sex over the other to later adult-onset of diseases [121]. In the absence of awareness of the implication of the previous results, it is possible to unwittingly test drugs in animals that have different risks to develop diseases as a consequence of maternal diet [122, 123]. Finally, sex-gender also influences the outcomes of mild and severe food restriction, because female mice are more impulsive than male mice under mild food restriction [124]. In addition, food
ACCEPTED MANUSCRIPT restriction disrupts the oestrous cycle in female rats [125] without adversely affecting male fertility [126, 127]. Male rats maintain body weight better than females and do not have altered activity or cognitive ability after starvation [128]. Finally, mild starvation diminishes some of the sex-gender
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differences evidenced in hepatic transcript profiles and miRNAs [129].
Another important aspect regards whether interactions in overall gene expression responses
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as a result of diet occur in a sex- and tissue=specific manner. Actual information on this point is lacking. However, this information is necessary to interpret results and to generalise and extrapolate
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effects between tissues. This point is also relevant for surrogate tissues such as peripheral blood
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mononuclear cells. because they are largely used in mechanistic studies in humans [130]. Changes in dietary composition are associated with changes in the composition of gut microbial populations [131]. Recently, Markle et al. [132] suggested that the gut microbiota could
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be a major contributor in the development of sex-gender differences. In fact, the researchers showed that the pronounced sensitivity of female mice versus resistance of male mice to type 1 diabetes
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mellitus (non-obese diabetic (NOD) mouse model), could be directly attributed to the commensal
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microbiota, which differs in males and females at puberty. Indeed, the removal of microbiota increases and decreases plasma testosterone in female and male mice, respectively. Saliently,
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Markle et al. [132] also showed that the interaction between the host and microbes on disease susceptibility can be transferred to the next generation. This study revealed a new source of sexgender differences which could have unexpected consequences. Gut microbiota can be affected by foods and drugs such as antimicrobials [133, 134], and this could induce variations in body weight, metabolism and systemic inflammation in a sex-gender specific way [135-137] either in humans or in animals [132, 138, 139]. For example, olanzapine, an atypical antipsychotic agent, induces specific alterations to the gut microbiota which are associated with an increase in body weight in female but not in male rats suggesting that variations in gut microbiota can affect drug response in a sex-gender specific way [140].
ACCEPTED MANUSCRIPT In conclusion, for sex-gender studies, it appears that the use of diets with low amounts of oestrogenic compounds and oestrogen-free bedding, cages, and water bottles is critical. Additionally, one should not forget that high-fat foods or mild and low caloric intake could affects
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numerous parameters in a sex-gender specific manner. Furthermore, in view of the importance of the very early life events, it would be of relevance to know the diet of dams. This information
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should be included in the method section of papers. Of course, maximal caution should be practiced in the evaluation of endocrine disruptors and sexual hormones, and this is of extraordinary
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relevance in bioassays focused on drug action. Finally, it is necessary to also consider gut
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microbiota, because the microbiome could have heavy consequences on experimental outcomes, and this can occur in a sex-specific manner.
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5.6 Travelling, stabilisation, and acclimatisation periods Some laboratories have facilities to maintain their own breeding colony, whereas others
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purchase the animals from outside vendors, and animal transportation from the supplier to the
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laboratory induces stress responses [141, 142]. Indeed, during transportation, animals leave socially established environments and are exposed to overcrowding in a confined space, unfamiliar and loud
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noises, vibrations and jolting, variation in temperature and humidity, inhalation of gases (from urine, faeces and carburant), food and water deprivation, and change in bacterial environment among other stressors. The manner of shipping is not identical among animal producers [143]. In fact, it is not known if male and female animals require the same travelling conditions. Indeed, a recent paper indicated that shipping-induced stress responses could be scarcely affected by the sex of animals [143]. Upon their arrival into the laboratory, animals require a period of stabilisation and acclimation [144]. It is not clear whether sex-gender influences the duration of stabilisation and acclimation periods, though it is known that animals vary in these requirements according to species and strain. Considering that the acclimation period is of fundamental importance for the outcomes of experiments, it is mandatory to investigate whether these stress responses are sex-gender specific
ACCEPTED MANUSCRIPT parameters. This finding could exert a major influence on tests, because just moving a cage from
5.7 Housing, isolation, spacing and environmental enrichment
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one room or floor to another is stressful [145].
Animal welfare also depends on the housing environment. Recently, it has been shown that
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housing social context differently affects males and females [146]. Thus, in selecting a suitable social environment, sex-gender should have a role [147-150]. Social housing can induce a
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hierarchical structure, which could either be different or have different consequences in males and
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females [151-153]. In general, social housing may enhance coping with stress in female rats, whereas, in male rats, social housing may not have a positive influence on stress-sensitivity [154]. The social context also plays a role in inflammation [155]. For example, group housing exacerbates
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inflammatory responses and sickness behaviours in females, but attenuates these responses in males [156]. Isolation also modifies drug response: in particular, isolation housing reduces barbiturate-
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induced sleeping more in females than in males, and the difference is less evident in castrated males
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[157].
Over the past decade, housing of laboratory animals has largely evolved, and recently individually
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ventilated cages have been developed to maintain optimal hygienic conditions. However, recent data suggest that housing mice in individually ventilated cages impacts animal behaviours. Indeed, this could occur in a sex-gender specific manner. Female mice kept in individually ventilated cages become more anxious when compared to FILTER-housed females, while cognitive tests are not influenced by sex-gender [158]. A peculiar aspect of isolation is the use of metabolic cages: implicitly, there is the presumption that animal functions are relatively normal in animals housed in metabolic cages. However, this is not always true, because isolation in metabolic cages seems to alter, in a sexgender-dependent way, neurohumoral control [159] and renal [160] and cardiovascular function [161, 162], and may also induce significant weight loss in pregnant rats [163].
ACCEPTED MANUSCRIPT Metabolic cages are largely used in pharmacokinetic studies, but it less known that the pharmacokinetics of xenobiotics may be altered by the use of metabolic cages [164]. For example, rats acclimatised to metabolic cages for 21 days have a different response to non-acclimatised rats:
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the LD50 of ammonium diuranate is 360 and 6 mg/kg, respectively, in these mice [165]. However, it is not known whether alterations induced by metabolic cages are influenced by sex and gender.
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Nonetheless, there are some indications which suggest that living in a small space may have a different effect in male and female animals [166]. For example, female mice have a major body
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weight effect, present alterations in inflammatory response [167] and show an increase in grooming
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and sitting behaviours in comparison with male mice [168].
Environmental enrichment is a technique used to increase animal satisfaction. Nevertheless, most experimental paradigms are built on information obtained from studies performed on males.
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There are some suggestions that indicate the influence of sex-gender in the effects of environmental enrichment [169]. In particular, an enriched environment ameliorates spatial memory only in female
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mice [170]. In rats, environmental enrichment decreases baseline level of adrenocorticotropic
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hormone (ACTH) and corticosterone in a sex-gender-specific manner: the decrease in ACTH is greater in female rats than in male rats [171]. Others have found that environmental enrichment
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increases plasma testosterone and ACTH in males but not in females [172]. Furthermore, environmental enrichment raises the hippocampal brain-derived neurotrophic factor, especially in females, and attenuates oxytocin and aldosterone only in females [172]. Although they are not all in agreement, the results suggest that environmental enrichment exerts a sex-gender-specific effect. In a mouse model of Huntington's disease, environmental enrichment associated with handling significantly improved cognitive performance in the Morris water maze in female mice, but not in male mice [173]. Finally, bedding material may have a role in inducing sex differences or blunting them. For example, corncob bedding, which has estrogenic activity, blunted response to defeat in female mice but not in male mice [174].
ACCEPTED MANUSCRIPT In conclusion, experimental design must consider confounding variables such as group size, strain, age, and quality of space as well as social aspects such as environmental enrichment and isolation. In addition, reported results suggest that the response to the previous factors of
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environmental enrichment could be a sex-gender-associated phenomenon, indicating that male and female animals could have different needs and suggesting that the mutual needs of males and
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females should meet in order to arrive at a standardisation that it is optimum for the single sexgender considering the age and the strain. As also suggested by Girbovan and Plamondon [175], an
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accurate description of animal environment should be reported in the method sections of
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publications.
5.8 Timing of sample collection and drug administration
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It is well documented that some biological activities are characterised by prominent rhythms of various periods [176-179]. Biological rhythms can involve only one sex, such as oestrus or
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menstrual cycles [180-183], or both sexes [178, 184-186]. The expression of S100b, a soluble
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protein released by glial cells, changes during the day in a sex-gender specific way, as males and females have an inverse rhythm with changes larger than 30% from 5 to 21 h [187]. Moreover,
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Francavilla and colleagues [188] showed that nuclear oestrogen receptors in the liver are highest early in the morning and lowest in the middle afternoon, which parallels diurnal changes in oestradiol levels in the serum. Finally, we recall that sex-gender differences exist in the circadian rhythmicity of the CYP family of liver genes and corresponding nuclear receptors [189], suggesting that xenobiotic metabolism could be different at diverse times of the day. Oestrus cycle can also influence organ functions. In fact, it has been shown that oestrous cycle impacts myocardial electrical and contractile functions and modifies Ca2+ handling [190]. Additionally, tolerance to severe hypoxia is better in the pro-oestrus female rodent [191]. During oestrus and menses cycles, brain volume, plasma and blood volume change [192-194]. The variations in plasma and blood volume imply changing distributions volume of drugs and levels of
ACCEPTED MANUSCRIPT metabolites. Indeed, the pharmacokinetics of oral ranitidine varies with gender and stage of the menstrual cycle [195]. Biological rhythms can affect numerous pharmacological targets such as receptor number or
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conformation and signalling. For example, the antineoplastic action of IFN-β and the antiviral action of IFN-α in nocturnally active mice is higher in early light phase and is closely related to that
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of IFN receptors and ISGF expression in tumour cells or lymphocytes [196, 197]. The therapeutic-to-toxicity ratio of a drug changes predictably according to biological
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rhythm. For example, the body weight loss induced by irinotecan hydrochloride in nocturnally
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active mice is more severe in the late active phase and the early rest phase [198]. Preclinical and clinical studies show that the foetal toxicity of drugs varies not only with developmental stage but also with circadian time of administration [199]. The above results indicate
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the importance of timing in drug administration, while evidences on sex-gender differences in administration time are not yet sufficient to have clear conclusions. Nevertheless, these issues
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should be accurately investigated.
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In conclusion, special caution should be used for variations induced by menstrual and oestrous cycles, because existing data show that they affect body composition and renal,
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cardiovascular, haematological and immune systems; thus, these changes could impact pharmacokinetics and/or pharmacodynamics [200, 201]. The question of sex-gender-dependent influence on biological rhythms that involve both sexes is still obscure. However, the acknowledgement of this possibility is necessary to conduct high-quality animal and human research and to adopt more appropriate therapies for both sexes. This point is also fundamental because numerous drug delivery systems are under development to be employed for the treatment of chronotherapeutic diseases such as hypertension and asthma.
5.9 Pre-anaesthesia and anaesthesia
ACCEPTED MANUSCRIPT Anaesthesia has the aim to reduce pain relief, to induce the loss of consciousness and to induce muscle relaxation [202]. Appropriate care prior to anaesthesia is necessary [203], and drugs used in pre-anaesthesia, which present some sex-gender differences [204], are aimed to reduce fear,
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pain, oral and respiratory secretions and the quantity of other anaesthetic agents required and to block the vaso-vagal reflex associated with intubation and surgery and analgesics [205]. The central
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nervous system, the main target of anaesthesia, is sexually dimorphic [206]. In view of the huge differences in the male and female central nervous system, it is surprising to find that females are
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often excluded from preclinical and clinical investigation, even in the field of anaesthesia, and this
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bias has led to a large knowledge gap with relevant consequences. Anaesthesia is a large diffuse experimental procedure and is performed for dissecting organs, to isolate cells and to help to restrain animals.
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Anaesthesia in rodents inevitably induces autonomic nervous system depression, alters cardiovascular functions, induces respiratory depression, hormonal and metabolic alterations that
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induce hypothermia [207] and modifies hepatic and renal functions [208-210].The effects of
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anaesthetics on biochemical parameters are often molecule- and animal-strain specific. Therefore, caution is required when interpreting data from anaesthetised animals. Once again, the influence of
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sex-gender on variations induced by anaesthetics is not well known. However, sodium pentobarbital and other barbiturates have a longer ‘‘sleep time’’ in female rats than in male rats [211-213]. Plasma pentobarbital concentrations are lower in male rats than in female rats and decrease more rapidly in male rats [214], depending on different distribution and metabolism in males than in females. The difference in phenobarbital anaesthesia is not surprising, because in 1932, it was described that the hypnotic effect of hexobarbital lasts longer in female rats than in male rats [215], and this effect is dependent on sexual dimorphism in hexobarbital metabolism [216]. Notably, the differing effects of pentobarbital are not only linked to biological differences between male and a female rats (sex), but also depend on environment [157] as described above. Furthermore, the effect of phenobarbitone, at least in mice, is affected by foods, bedding materials,
ACCEPTED MANUSCRIPT and even the temperature used in the experiments [217]. Notably, Lovell [217] reported that environmental influence on pentobarbitone effect could be species-, strain- or possibly sex-genderspecific. However, no sex-gender differences with phenobarbital are found in mice [216, 218].
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The effect of ketamine, a non-competitive antagonist of the N-methyl-D-aspartate glutamate receptor, presents some sex-gender-specific differences, as liver metabolism of ketamine
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is more efficient in males than in females [219, 220]. Furthermore, male rats are less vulnerable to the neurotoxic and to some behavioural effects of ketamine in comparison with female rats [221,
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222]. Sex-gender differences in response to propofol have been reviewed elsewhere [223, 224].
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Notably, at least in women, the EC50 of propofol required to induce loss of consciousness is higher in the follicular phase than in the luteal phase. Emergence time is also longer in the follicular phase than in the luteal phase, and this seems to depend on variation in progesterone levels [225]. Some
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sex-gender differences are also described in the use of steroid anaesthetics: the male rat requires about four times more steroid anaesthetic than the female rat, and this sex-gender difference is age-
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dependent and disappears upon administration of oestrogen to the male [226]. On the other hand,
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the sex-gender differences observed with alfaxalone treatment seem to be dependent on formulation [227]. Notably, a single exposure in prenatal life to either the inhalant isoflurane or the injectable
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phenobarbital leads to significant decrements in cognitive abilities and a reduction in volume and neuron number in the hippocampus in adulthood, and this reduction is significantly greater in males than in females [228].
Finally, opioid analgesics behave differently in males and females. Different efficacy and safety profiles are observed with opioid analgesics in males and females. However, animal and human studies show that the direction and magnitude of sex-gender differences in response to a single compound can depend on numerous variables such as age, dose, route and time of administrations, type of pain, hormonal status, and species [229]. In conclusion, sex-gender differences in pre-anaesthetic and anaesthetic effects should be considered in all protocols requiring anaesthesia. Moreover, it is of value to know the recovery time
ACCEPTED MANUSCRIPT of anaesthesia for ethical considerations as well as for the possibility of drug interactions that might alter the results. The details are essential to understand what influence this may have on either the
post mortem, as also stated in the ARRIVE guidelines [230].
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6. Action performed at collection of the samples
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data obtained from in vivo experiments or from in vitro experiments performed on tissue obtained
One of the most critical issues in biomedical research is sample preparation [231, 232]. Blood
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and cellular fragments, plasma and serum.
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samples are used widely, and blood is generally collected via venipuncture in order to obtain cells
6.1 Blood collections
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Blood collection, handling, processing, and storage can impact final results, and this could occur in a sex-gender-specific manner [233]. Therefore, knowledge of these effects is a prerequisite
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to control their impact on research findings in order to reduce variability and decreasing the
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possibility of errors. There are numerous variables (the selection of blood collection tubes, which are much more complex devices than is commonly appreciated, anticoagulants, lag time before
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centrifugation, anticoagulants, temperature, etc.) that impact sampling procedures of plasma/serum and blood cells [234, 235].
6.2 Anticoagulants Various salts of heparin, EDTA, and sodium citrate are widely used in the clinical laboratory. Heparin salts are intended for determination of electrolyte levels and other routine chemistry values. The choice of anticoagulants is important, because the compound chosen can impact biochemical results. Numerous peptides differ in amount, not only between serum and plasma specimens, but also among plasma specimens prepared with different anticoagulants [236].
ACCEPTED MANUSCRIPT 6.3 Handling and processing from blood collection to specimen analysis Beyond anticoagulants, the time and temperature of storage of the specimen and the
temperature) can introduce pre-analytic variables [237-241].
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preparation of serum/plasma and cell separation (for istance centrifugation time, speed,
It is relevant to establish a standard protocol to monitor the stability of plasma/serum
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samples upon storage, and storage temperature should at least be considered. Storage temperature can induce pre-analytical variability depending on the specific analyte [237]. A recent paper
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indicates that proteins are sensitive to storage temperature in a protein-specific manner [242],
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though at the moment it is not known if this process is sex-gender-dependent. In this context, it is very important to establish standard protocols to monitor the stability of plasma/serum samples upon storage. It is not well known if these pre-analytical procedures
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performed on the specimen are associated with donor sex and age.
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6.4 Volume and frequency of blood collections
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Rodents are widely used in biomedical research. However, due to the animals’ relatively small body size, blood collection can be difficult. Males weigh significantly more than females and
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have more available blood volume than females. Consequentially, blood collection is more difficult in females than in males, and this can produce more artefacts in females. For example, blood cell damage occurs more often in blood obtained from females [243]. Obviously, repeated blood collections are technically difficult in small animals, especially in females that have smaller amounts of blood; indeed the volume and frequency of blood collections should be carefully considered in the female, because excessive blood withdrawal can negatively affect animal wellbeing [244, 245] . In particular, the volume of blood removed and the frequency of sampling will be based on the purpose of the scientific procedure and the total blood volume of the animal. Total blood volume is generally estimated as 55-70 ml/kg body weight. Thus, total blood volume is lower in females than in male, because female animals weigh less than male animals [246].
ACCEPTED MANUSCRIPT In conclusion, minor changes in procedures can cause large changes in biologically relevant variables, highlighting the importance of establishing uniform and validated procedures to
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improve reproducibility of animal phenotypic data. This should occur in both sexes.
6.5 Sampling of blood
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The manoeuvres (handling, restraint, and anaesthesia) needed for collection of blood from animals are stressful, and the operator should keep stress at the lowest possible level in order to
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preserve animal welfare and to produce good results. For example, stress associated with the
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handling of mice during bleeding increases glucose serum concentrations proportionally to the handling time [247]. As already mentioned, anaesthetics (enflurane and halothane) and animal discomfort can modify serum biochemical parameters [247, 248].
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Notably, biochemical parameters also depend on the site of blood sampling [249-252], and this phenomenon seems to occur in a sex-gender-specific manner [243]. Additionally, in rodents,
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significant differences in leukocyte, red cell and platelet counts have been described in blood
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samples collected from different sites [243, 253-255]. Indeed, a study performed in male and female mice and rats using the techniques of cardiac puncture and venous puncture of the tail, foot and
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saphenous veins indicates that total peripheral leukocyte counts are lowest in blood collected from the heart in both male and female rodents [253-259], and this effect seems to occur in a sex-genderdependent manner [259]. The above results suggest that the phlebotomy method and phlebotomy site are important factors affecting haematological and biochemical outcomes, and the practice of utilising different sites of sampling should be avoided, because the introduction of different techniques in a single study introduces a source of error. The cannulation technique can be used to remove blood. Cannulation decreases the stress of multiple samplings, reducing the stress linked to repeated restraint and needle sticks . However, this technique can cause pain, and therefore it may require analgesic medications and appropriate monitoring for the duration of the cannulation period. Furthermore, infections may arise despite the
ACCEPTED MANUSCRIPT use of sterile equipment [260]. Cannulation also permits access to deep vessels, ambulatory infusions and administration of drugs [245]. The influence of sex-gender on cannulation is not well studied, but considering the small diameter of female animal blood vessels, sex-gender differences
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are plausible.
In conclusion, the available data suggest that numerous pre-analytical variables could affect
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haematological and biochemical results, and this seems to depend on the analyte being studied and possibly on the sex of the animal. Thus, in experimental design, it is necessary to establish the
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normal range of analytes for both males and females based upon the procedure adopted for
6.6 Urine collection and processing
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preparation of the samples.
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Urine collection and processing remains a major in vitro diagnostic screening test and has a special role in pharmacokinetics. The pre-analytical phase of planning should include sample
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collection, specimen transport and preparation of samples for testing, and it is important to consider
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potential sources of error. It is relevant to recall that female and male animals may have different urine volumes. Rodent females drink more and have a larger urinary volume which is less
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concentrated [261-263]. Recently, it has been shown that spiny female mouse excrete more urine than male mice, but the urine excretion of sodium and proteins does not differ in the two sexes [264]. It has also been also reported that glomerular filtration rate (GFR) is higher in males than in females due to higher renal plasma flow and lower renal vascular resistance [265-267]. The differences between males and females are lessened when correcting for body weight, which is lower in females [264-266].
6.7 Clinical chemistry, haematological data, and reference values In clinical or pre-clinical research, the normal range of haematological components and biochemical parameters of blood are used for diagnosis and as target for pharmacological response.
ACCEPTED MANUSCRIPT Thus, acknowledgment of reference values is of great relevance. Many sources of variation can affect the results of clinical biochemistry assays in both animals and humans [233, 247, 268-270]. These sources of variation are significantly influenced by environmental factors and characteristics
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of the individual, including sex. Although, in the past 20 years, awareness of the influence of sexgender on blood, plasma, serum and urinary constituent levels is growing, these factors are still
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neglected in clinical chemistry and haematology [248, 271, 272]. Rats, mice, dog, and primates present sexual dimorphism in serum, urine, cerebrospinal and bronchoalveolar lavage fluid
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proteomes in both control situations and during some diseases (Table 1, 2) [273]. Biomarkers of
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lipid metabolism vary in Meishan pigs in a sex-gender- and an age-dependent manner [271]. In 4 months old pigs, total and free cholesterol, LDL and triglycerides are higher in females than in males [271]. On the contrary, in multiple strains of mice, total cholesterol and triglycerides are
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higher in males than in females [274]. A Japanese study [272], which analyses historical control data of clinical pathology provided by sixty-seven companies of the Japan Pharmaceutical
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Manufacturers, notes that differences for haematocrit, haemoglobin concentration, white blood cell
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count, glucose, cholesterol, triglycerides, total protein, albumin/globulin ratio, urea nitrogen, and inorganic phosphorus in rats and in mice are sex-dependent. Indeed, sex-gender differences have
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been found by many authors [275-278]. Notably, some sex-gender differences and parameters are age- [279], species- and strain-specific [280, 281]. For example lionesses have higher haemoglobin values than those found in the lion [282]. However, reference values of haemoglobin in Old World primates, which have pre-menses, breeding and menopausal phases [283, 284]and menstruation frequencies which are very similar to humans [284], do not present significant differences between females and age-matched males [283]. Notably, in studying sex-gender differences, it is also necessary to study the distribution of data, because different distributions could be present in male and female cohorts. Generally, normally hydrated, conscious female rats and women have lower haematocrit levels than their male counterparts, indicating that the volume of red cells and plasma may differ
ACCEPTED MANUSCRIPT between sexes [192, 285]. When comparing the values obtained in males and females, it is relevant to recall that men have a larger total blood volume (about 6–8% greater) than women [286]. Thus, metabolite concentration in blood is diluted more in men than in women, resulting in even greater
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differences between sexes than if this factor is not considered. Considering that reference range values in animals and humans have great value as biomarkers in testing xenobiotic efficacy and
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safety beyond their value in prevention in the natural course of diseases and in outcomes, it is fundamental to reduce errors by determining the normal range, which should be obtained for each
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sex at a single age, taking also into consideration the oestrous and menses cycles and reproductive
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phase of females. Additionally, a number of drugs and their metabolite interfere with analytes. This interference may cause false-positive and false-negative values in clinical patient material, and this could occur in a sex-gender-dependent manner, because drug distribution and metabolism can be
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sex-dependent [108].
In conclusion, changes in blood values are used as end points and/or biomarkers in
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pharmacological studies. Thus, a laboratory must set up and maintain a reference range database to
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minimise environmental interferences. Databases could help to firmly establish which parameters
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are different between sexes and to obtain an international harmonisation.
6.8 Collection and isolation of the cells It is difficult to determine the sex of cells used in scientific papers (Table 3), and within those publications that do indicate the sex of the cells used, some do not perform sex-gender analysis. However, sex-gender should be considered in all cell studies, because cells obtained from males and females can behave differently [8, 31, 32, 287-293]. Many of the differences observed could depend on genetic, epigenetic and hormonal processes [294, 295]. As example of the complexity of these phenomena, we recall that CYP3A4 activity is greater in female cryopreserved human hepatocytes than in male counterparts, whereas, in freshly cultured hepatocytes, induction of CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 does not appear to be influenced by sex-
ACCEPTED MANUSCRIPT gender or by the age of the donor [296], suggesting that freezing exerts a sex-dependent effect. Primary osteoblasts isolated from pre- and post-menopausal women show age-dependent biochemical changes that are not observed in the same bone cells from younger or older men [297].
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Macrophages from female arthritis-susceptible B10.RIII or B10.G mice synthesise more prostaglandins and thromboxane and have higher circulating anti-type II collagen antibodies than
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male counterparts [287]. Sex-gender-dependent differences in vascular smooth muscular cells (VSMC) from the rat aorta are summarised in Table 4. In particular, VSMC from rat aortas have
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different cell migration phenotypes and expression of oestrogen receptors. The differences in steroid
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receptor expression disappear after 14 passages post-isolation [291], indicating the importance of cell passage for studying sex-gender differences and suggesting that these differences could be better-studied in primary culture rather than in cell lines. Furthermore, XX and XY VSMC from the
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aortas of rats present a sexual dimorphism in cell fat; male cells are more prone to apoptosis, anoikis, and autophagy than female cells [289, 290]. Furthermore, XY neurons are more susceptible
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to glutamate exposure than are XX cells, whereas XX cells are more sensitive to pro-apoptotic
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stimuli [298].
Jog and Caricchio [299] demonstrate that bone marrow-derived macrophages obtained from
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male mice are more prone to necrosis through a PARP-1 dependent mechanism than female mice. Conversely, female cell the death is PARP-1-independent. Notably, oestrogens increase survival only in female bone marrow-derived macrophages, independently from oestrogen receptor expression. Human macrophages derived from monocytes isolated from men and women retain the effects of smoking in a sex-gender specific manner [31], and female human macrophages derived from monocytes retain the effects of oral contraceptive (OC) use [32], indicating that the lifestyles of donors can affect cell function. Thus, cells not only have a sex also have a gender, either in basal conditions or after stimulation. Indeed, it is not yet well known whether cessation of OC and hormonal replacement therapy affects isolated cells. However, it has been reported that past OC users have lower (about 5%-10%) levels of endogenous estradiol, estrone, androstenedione,
ACCEPTED MANUSCRIPT testosterone, and sex hormone-binding globulin compared with never-users independently from age, body mass index, smoking, physical activity, and reproductive factors [300]. Past users of hormonal replacement therapy
have lower levels of testosterone and 17alpha-hydroxyprogesterone,
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suggesting that the use of exogenous hormones produces long-lasting effects that persist for years after the cessation of treatments. These changes could also affect the function of blood cells isolated
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to perform ex vivo experiments.
Additionally, samples need to be lysed to measure some components localised inside cells.
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Thus, it is necessary to determine the type of lysis buffer to be used, because this choice could have
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consequences on the biochemical parameters to be determined. The influence of sex-gender on the previous parameters has not been studied. Obviously, it is time to overcome this lack of knowledge to improve research and to increase its translational value.
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In conclusion, fresh isolated cells retain memory of the sex and the gender of the donor, and thus it is crucial to use cells obtained from males and females to characterise mechanisms and to
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avoid erroneous conclusions. Furthermore, it is of relevance to administer a questionnaire (Table 5)
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to human donors to collect more relevant information about lifestyle and xenobiotic use, while, for cells obtained from animals, information on age, sex, diet and housing cross fostering should be
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collected in order to verify the influence of environmental factors in generating sexual differences (Table 6). Journal editors should encourage the publication of questionnaires in “Materials and Methods” sections as standard practice, and scientific societies should participate in the validation process of questionnaires.
6.9 Collection and isolation of cell organelles Sex-gender differences are described in subcellular organelles and their enzyme activities. In particular, mitochondria obtained from rat, mouse and rabbit brain, cardiac tissue, liver, and skeletal muscle present sexual dimorphism [301-308]. Sex-gender differences, particularly regarding calcium handling, oxygen consumption and reactive oxygen species production, have
ACCEPTED MANUSCRIPT been reported. However, mitochondria obtained from B6 (C57Bl/6J) mice of both sexes do not present significant variations in heart, skeletal muscle and liver mitochondrial oxygen consumption, ATP content, H2O2 generation, oxidative stress levels or apoptosis [309]. Previous data suggest that
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mitochondria can have a role in sex-gender differences, but more data are needed to reach a firm conclusion.
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Sex-gender differences have been also observed in lysosomes, which are involved in multiple physiological and pathological cell processes. In particular, sex-and strain-based
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differences in the size of lysosomes in the proximal tubular epithelium have been identified in mice
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[310, 311]. For example, the DBA/2Cr strain possesses giant lysosomes that predominate in the proximal convoluted tubules and in the proximal straight tubules of males and females, respectively [312]. The appearance of giant lysosomes in males and females is controlled by testosterone and
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oestrogen, respectively [312]. Whereas liver cholesterol ester hydrolase activity is decreased by progesterone concentrations >100 µM, the activity of this enzyme is not changed by estradiol in
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vivo or in vitro [313]. Furthermore, beta-galactosidase, beta-glucosidase and acid phosphatase
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acidity are higher in female rat cardiac lysosomes in comparison with male counterparts [314]. A recent study performed in rats revealed that male livers are enriched in LAMP-1, a constitutive
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protein of lysosomes, in comparison with female livers, suggesting the presence of a higher number of lysosomes in the male rat liver. However, LAMP-1 expression does not differ in the heart or in the kidney of female and male rats [7]. Overall, the previous results suggest that lysosomes might have role in sex-gender differences, but to reach a firm conclusion, more data are needed because the differences could be organ-, species- and strain-specific. Many cellular and subcellular experiments use housekeeping genes and proteins to normalise the results. The housekeeping proteins shown in Table 7 may be differently expressed in male and female cells and tissues [7, 315-317]. Interestingly, this effect appears to be protein-, celland organ-specific. Thus, pilot experiments should be performed in order to find housekeeping proteins and genes equally expressed in males and females.
ACCEPTED MANUSCRIPT In conclusion, the sex of donors of biological research regents must be known, because organelles and cells have memory of their sex and gender. Considering that changes at cellular and organelle levels can be used as end points, biomarkers and/or targets of pharmacological studies, it is urgent
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to know if sex and gender affect the functions of these cells. It is also relevant to administer a questionnaire as illustrated in Tables 5 and 6 to human donors or to professionals collecting
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samples to assess relevant information about the lifestyles and xenobiotic use of the donor. Moreover, for cells obtained from animals, information on age, sex, diet and housing cross fostering
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should be collected, and journal editors should encourage the inclusion of such information in
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“Materials and Methods” sections as standard practice.
6.10 Isolated organs
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For over 150 years, isolated tissue and isolated organ preparations have been widely used as convenient biological models [318].. Indeed, the development of methods of gene manipulation
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and protein expression allow the study of organs obtained from animals with normal or pathological
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tissues, allowing molecular biologists to quantify the physiological impact of altered gene sequences, mechanisms, physiology and cellular signalling. Moreover, isolation of organs is also
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performed to compare the weight of organs. Importantly, regulatory agencies use dose-related organ weight effects as end points to build toxicity reference values and in physiologically-based pharmacokinetic models [319]. Generally, organ weight is greater in male animal models than in female animal models [320]. Notably, the vast majority of studies employing isolated organs have used male animals. For example, only male isolated perfused rat hearts have been used to study ischemic arrhythmias [25, 26, 321]. Regarding sex-gender differences in ischemic post-conditioning, results obtained in the Langendorff perfused rat hearts have been contradictory. In particular, Lee et al [322] report no significant differences between male and female hearts, whereas Zheng et al [323] show that Langendorff perfused female rat hearts have greater resistance to ischemic-reperfusion insult than
ACCEPTED MANUSCRIPT male perfused hearts, concluding that male hearts are able to develop cardioprotection after sevoflurane post-conditioning through mechanisms that seem to involve the PI3K/Akt pathway. Sex-gender differences have also been described in Langendorff perfused mouse hearts in both
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basal conditions and after ischemia [324]. As already mentioned, cardiac contraction varies during the oestrous cycle [190]. In perfused rat hearts, there is also a sex-gender specific metabolic
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modulation [325]. For example, during ischemia and reperfusion, the metabolic state of rat hearts is sex-dependent. In fact, lactate originates mostly from [U-(13) C] glucose and from [3-(13) C]
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glucose in hearts from males and females, respectively [325].
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The sex-gender of donors influences the secretion of bile acids [326] and the uptake of long chain fatty acids from the isolated liver [327]. Notably, isolated livers from female rats, especially pregnant ones, are strikingly resistant to the effects of tetracycline [327].
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The above results strongly suggest that male and female organs can behave differently in vitro. However, the use of isolated organs requires a multistep protocol including: a) anaesthesia
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(see above); b) anticoagulant administration, which theoretically reduces the risk of thrombus
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formation; c) organ isolation; d) cannulation, which must take into account the dimension of the organ and of the vessels, which generally are smaller in the females than in the males; d) and
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perfusion. Indeed, with few exceptions including anaesthesia, the influence of sex-gender on each step of organ isolation is scarcely studied. The post-mortem period is crucial for degradation of cells, tissues and organs, and it is pivotal that samples be processed correctly to preserve quality [328]. In conclusion, the sex of donors must be known, because isolated organs, cells and cellular organelles have a sex and have a gender. It is not yet known which, if any, activities of isolated organs are influenced by the environment in a sexual dimorphic manner. Considering that changes in functions of isolated organs can be used as end points, biomarkers and/or targets of pharmacological studies, it is urgent to know if sex and gender affect their functions. To improve our knowledge on this specific point, it is critical to collect information as illustrated in Tables 5
ACCEPTED MANUSCRIPT and 6. Finally, as standard practice, these details should be published in the Methods Section of articles.
Formulation,
administration
routes,
dosing
and
considerations
other
pharmaco-toxico-kinetic
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7.
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Many relevant pharmacokinetic parameters are sexually dimorphic as described in several extensive reviews [33, 37, 38, 108, 329]. However, a detailed review focused on drug
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administration issues in animals dedicates only a few words to sex-gender differences [245],
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indicating that awareness of the importance of sex-gender in this specific topic is still neglected. Attempts have to be made to improve this situation, and consideration of the specific condition of females is required. recently , the Food and Drug Administration
has decreased the initial
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recommended dose of zolpidem in women [29].
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7.1 Formulation and routes of administration
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After selection of a molecular entity, pre-formulation activities should be performed to determine its physical and chemical properties. At this stage, the route of administration intended
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for the clinic should be identified, because each route of administration requires special formulation. In drug formulation, the active compound is mixed with other chemical ingredients to create the drug product. Recently, it has emerged that excipients may exert sex–specific effects. In particular, polyethylene glycol enhances the bioavailability of ranitidine in men (up to 63%), whereas polyethylene glycol decreases ranitidine bioavailability in women (up to 24%) [330], showing the fallacy of assuming that males and females handle inactive ingredients similarly. Furthermore, some inactive ingredients may have different safety profiles in males and females. For example, we recall that cellulose and hydroxypropyl methylcellulose exert their adverse reproductive and/or developmental effects in a manner that appears to be sex-gender–dependent, at
ACCEPTED MANUSCRIPT least in non-clinical studies [331]. In conclusion, the formulation of a drug should consider both sexes, both to improve the efficiency and effectiveness of care and to improve safety profiles.
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7.2 Dosing
The correct selection of an optimal dose is critical for establishing the efficacy of a
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potential therapeutic agent. Ideally, therapeutics should to achieve the right dose, of the right drug,
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for the right time, in the right subject. Choice of dosage regimen depends on drug pharmacokinetic and on the individual. Pharmacokinetic-pharmacodynamic (PK-PD) modelling is essential to
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evaluate the plasma concentration and effects of the compound, including the homeostatic feedback mechanisms that might modulate the response and the interaction of the drug effect with disease processes. All pharmacokinetic parameters are affected by different covariates, including sex and
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gender [332].
Drugs are generally administered according to fixed dosing. Usually, in a clinical setting,
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the recommended adult dose of a drug is based on the assumption that the subject is an adult
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Caucasian man that is 173 cm tall and weighs 70 kilograms [22], forgetting that many people, including women, do not fit into this category. Women are often significantly smaller in height and
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weight and also have a different body composition than men. Alternative weight descriptors (BOX 1) such as ideal body weight, adjusted body weight, fat-free weight, and lean body weight are used to prevent drug overexposure observed with weight-based dosing, but their benefits and limitations must be understood [333, 334]. In calculating a dose, blood and plasma volume should have a role. Although there are a few contradictory studies [277, 335], it has been generally noted that in humans and in rats, depending on various breeds and ages, as in many other animal species, females have a lower blood volume and haematocrit than males. Therefore blood and plasma volume are different in males and in females [192, 275, 278, 336, 337]. According to Probst et al. [192], it appears advisable, if the individual assessment of plasma and blood volumes is not possible, to consider that plasma volume
ACCEPTED MANUSCRIPT is at least 10% greater in female rats in comparison with male ones. Plasma and serum volume of females change as function of reproductive life stage and menstrual or oestrous cycles [338]. Finally, men have a larger total blood volume (by about 6–8%) than women [286].
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The oral route is the most common route for drug administration. Drug cans be administered by gavage, or alternatively, medications can be dissolved in drinking water or food. Water and food
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are often given ad libitum. The amount of ad libitum water and food actually consumed must be determined before any medications are added to the drinking water and food. It is necessary to
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determine whether any added compound changes fluid and food intake by altering taste, smell, or
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other factors. If we wish to know the dose administered to males and females, we should consider that females and males could have different daily intake rates of water and food. Interestingly, daily water intake is significantly higher in adult female rats compared with adult males rats, and daily
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consumption of water is indexed to body weight [339].
Obviously, the use of an oral route should consider the multiple differences between males
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and females in gastric and intestinal systems, recently described in a clear review [109], because
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these differences affect oral bioavailability, which greatly influences dosing. Less is known about sex-gender differences in bioavailability, which can be species-specific. For example, female rats
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have higher oral bioavailability than males following administration of the same dose (mg/kg) of αthujone by gavage, while this difference is not present in mice [340]. The differences in bioavailability are partially attributable to CYP enzyme levels and to specific enzymes of phase 2 metabolism, predominantly in terms of different enzyme activity [33, 37, 38, 108, 329]. Indeed, it has recently been shown that metabolism of PF-02341066, a selective c-Met/Alk tyrosine kinase inhibitor, occurs predominantly through oxidation in the male rat liver and through sulfo-conjugation in the female rat liver [341], indicating that sex-gender differences can be either qualitative or quantitative due to the major activity of a single isoform of the enzymes, or to the activity of different enzymes. Sex difference in CYP enzyme levels may be significant enough to warrant different dosing strategies, especially for drugs with a narrow therapeutic index.
ACCEPTED MANUSCRIPT Sex-gender variations in microsomal enzyme activities are of relevance in the process of drug discovery. Recently, the Ministry of Health, Labour and Welfare of Japan issued a guidance document recommending that induction studies in rats should be conducted in females rather than in
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males, as the magnitude of CYP enzyme induction tends to be greater in females [342]. Drug
during the menstrual cycle or with the use of OC [108].
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metabolism can vary in the different phases of women’s lives (pregnancy, puerperium) [343] and
Another source of variation in bioavailability is liver blood flow, which is faster in male rats
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than in female rats [344].
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Distribution of drugs is influenced by blood proteins, especially albumins, which conjugate numerous drugs. However, this bond is not constant and can be changed in numerous clinical settings such as hypoalbuminaemia observed in critically ill patients [345, 346] and pregnancy [343,
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347]. Unbound free drug has therapeutic and potentially toxic activity [348] and affects drug penetration into tissues [349, 350], drug elimination and metabolism [351, 352]. Drug distribution
PT
depends on body composition, which is essentially sexually dimorphic as described in numerous
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reviews [108, 201]; females have more fat than males. This fact affects the distribution of medications having lipophilic properties, causing a greater distribution in females than in males,
AC
while the reverse is true for hydrophilic medications. Evidence suggests that there are significant differences in renal physiology between male and female animals, including humans, due to a sexual dimorphism in vascular responsiveness and in expression and activity of renal transporters [353]. The acknowledgement of renal sex differences is of special importance, because drug elimination occurs mainly by renal route and depends on GFR, tubular secretion and tubular reabsorption. In rats, whole kidney GFR and single nephron GFR are higher in males than in females [265-267]. However, most findings show that, when the higher body weight or kidney weights of males are considered as indices of renal function, the sex differences diminish [266]. In addition, human and animal females drink more and excrete a greater volume of less concentrated urine [261-263, 354]. We recall that tubular secretion mainly occurs
ACCEPTED MANUSCRIPT through a wide variety of active transporters that could work in a sex-dependent manner [33]. Sexgender differences in the expression of Oat1 and Oat3 are present in the kidneys of rodents in that mRNA expression levels of Oat1 and Oat3 are higher in male adult rats than in female ones [355].
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These differences are enhanced by androgens and inhibited by oestrogens [356]. Notably, the renal expression of numerous cytochrome P-450 enzymes, which are involved in the metabolism of
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xenobiotics, is sex-gender-dependent in mice and rats, whereas the enzymes of the phase II system present only few sex-gender differences [357]. Finally, we remember that renal function is strongly
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influenced by age, and the age-related decline in GFR is slower in female rats than in male rats,
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irrespective of strain [358], suggesting that in old rodents, these differences may be lower than in young animals. Renal sexual differences may influence dosing of drugs, drug–drug interactions, and drug-induced nephrotoxicity. Traditionally, renal function has been measured using plasma
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creatinine and calculating creatinine clearance, but estimated creatinine clearance predominantly measures GFR. However, at least in humans, there is evidence of a poor correlation between
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estimated creatinine clearance and renal drug clearance [359], necessitating dosage adjustment not
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just based on GFR.
In conclusion, the dosing of a drug is influenced by sex at different levels, and each drug
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should be carefully evaluated in order to find the right regime for male and female animals.
8. The choice of test and animal models of diseases The use of animals in biomedical research dates back at least two millennia when Galenus dissected Barbary macaques and made physiological experiments in pigs, goats, and sheep [360]. Today, animal testing is relevant in many fields including pharmacological testing for drugs; therefore, is crucial to understand and to validate these tests for each sex. For example, in humans and in rodents, sex-related differences are found in the performance of spatial learning and memory tasks [361-364]. Furthermore, males tend to guide navigation using geometric configuration, whereas females seem to attend more to landmark cues [43, 365]. Therefore, when results obtained
ACCEPTED MANUSCRIPT in animals of both sexes are compared, the comparison should include the description of physiological differences and how these are included in the analysis of the results. This is relevant because animal models of human diseases retain a crucial role in
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understanding the biological basis of disease and are of special interest to test efficacy and toxicological profiles of drugs. In spite of this, the vast majority of studies employing models of
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diseases, including transgenic ones, use male animals [146, 366, 367]. However, numerous
therefore, both sexes should be included in studies.
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differences exist between males and females in different models of animal diseases [368-370];
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The inclusion of sex and gender as variables should occur with great caution on the validities of extrapolation to humans, and the extrapolation range should satisfy the more severe criteria of Russel [371], who demonstrated that knowledge obtained in animals is relatively
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predictive in humans. In this respect, each model in a single species should be considered separately. For instance, in mice, in contrast to humans, more atherosclerosis is seen in females than
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in age-matched males [368, 372-374], whereas female macaques are relatively resistant to
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atherosclerosis in comparison with males [375]. In contrast to humans, in numerous models of type 2 diabetes mellitus, male animals are more susceptible to diabetes and have more severe disease
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than females [40, 376]. Additionally, in some animal models, particularly in the rat, females show less ischemia-reperfusion injury; however, this is not observed in all animal studies [377]. As stated previously, psychosocial phenomena have a profound influence on the disease process, and they should also be considered in animal models of diseases if adopting a sex-gender research strategy. Again, it is not possible to have a global policy on these matters, because it is necessary to consider each model separately. The described relative resistance of female macaques to atherosclerosis [375] also depends on social status, because only dominant female animals are less susceptible to atherosclerosis in comparison with males. The female resistance to atherosclerosis seems to be linked to oestrogens, because atherosclerosis is exacerbated in ovariectomised monkeys and suppressed by pregnancy and OC [450]. Indeed, a successive study
ACCEPTED MANUSCRIPT showed that dominant macaques, under conditions of social stress, develop exacerbated coronary artery atherosclerosis, and this exacerbation is attenuated by a beta-adrenergic blocking agent [378], revealing the role of sympathetic system. Thus, in female and in male macaques, the susceptibility
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to atherosclerosis is associated with different mechanisms.
The importance of social factors is further highlighted by experiments performed in
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cynomolgus monkeys. Female cynomolgus monkeys housed individually have a more adverse plasma lipid profile and more extensive coronary atherosclerosis than those socially housed when a
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chronic lipid-rich diet is administered [379]. Interestingly, only female but not male rats born from
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mothers fed a soya diet with lard throughout pregnancy and lactation have an elevated diastolic and systolic blood pressure as adults, while endothelium-dependent relaxation by acetylcholine is blunted in both sexes [380].
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There are numerous models of heart failure [381], and some of these present sex-gender differences. In general, female mice undergo less extensive ventricular remodelling than males
PT
[382], and this is of relevance for the design of experiments. Notably, sex-gender differences have
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been found in spontaneously hypertensive heart failure-prone rats with defective leptin receptors, which, at a relatively young age, first develop obesity followed by heart failure [383]; male rats are
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more prone to heart failure than females in this model [383, 384]. Sex-gender differences in blood pressure have been described in animals as well as in humans [385-387]. Hypertensive men are also at greater risk for developing cardiovascular and renal complications than hypertensive women [386, 387]. Dahl salt sensitive rats are a mutant strain of Sprague–Dawley rats that exhibit genetic hypersensitivity to sodium intake [388]. In this model, female rats are less hypertensive after sodium intake than male rats. A greater rise in blood pressure has also been reported in spontaneously hypertensive female rats after ovariectomy [389, 390]. There are numerous animal models of depression, and most of these have been developed in male rodents and were only later applied to female rodents [366]. Despite their limitations, these models show numerous sex differences in the neurobiology of depression and antidepressant
ACCEPTED MANUSCRIPT response [146, 366]; however, some of these models may not be appropriate for female rats. Some of the differences observed depend on baseline sex-gender differences, while other differences only emerge after stress exposure or drug administration. Differences have been described in learned
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helplessness models, chronic mild stress, forced swim tests, Flinders sensitive line and lipopolysaccharide-induced sickness behaviour. Overall, the data indicate that sex differences are
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present in the phenotype (behaviour) and/or in the endophenotype (neurobiology) of depression [366].
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In conclusion, there are a number of diseases models, but not all are suitable for every
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experimental protocol in terms of sex-gender differences. In some cases, the model of disease is appropriate only for one sex-gender (as is the case for many models of type 2 diabetes mellitus), indicating that data obtained in this model cannot be extrapolated to females. The identification of
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appropriate animal models that can be used to screen gender-based differences is urgent [40, 42, 146, 292] to emphasise the value of translational or integrational research [40]. In addition, it is also
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urgent to evaluate the influence of the oestrous cycle phases, pregnancy and lactation on diseases
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9. Conclusions
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[391-397].
In the past several years, a series of papers has illustrated the wide scope of sex-gender research. They have clearly demonstrated that sex-gender differences are fundamental, complex, and long-lasting and have underlined that sex-gender research is a complex scenario that needs to consider interdisciplinary and intersectorial aspects. There is enough evidence that sex-gender affects many outcomes in life science research, and the need exists to embed the so called “gender dimension” into basic and clinical scientific research. These studies also stress the need of a broad approach to gender mainstreaming. According to Australian Bureau of Statistics sex-gender is a basic complex variable, not a confounder, starting from pre-analytical phases of research [398]. Here, it has been shown that the
ACCEPTED MANUSCRIPT pre-analytical phase is influenced by sex and gender, and data coming from males and females should not be pooled to increase statistical power. To avoid any bias in research, a team should include men and women and should
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communicate to minimise or eliminate many variables and to foster better science and animal wellbeing. The team must strive to minimise or eliminate non-protocol variables that could
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adversely affect the validity and repeatability of the experimental data. Harmonising national
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statistics on animal use patterns will allow rational priorities for reduction and refinement research to be identified internationally.
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Journal editors should encourage the inclusion of sex-gender information in “Materials and Methods” sections as standard practice. It is of importance that results be published, even if they are
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not statistically significant or if they are negative, to avoid conducting redundant studies.
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10. Future perspectives
In the era of individualised medicine, it is evident that to be male or female impacts how an
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individual will respond to or metabolise a particular drug regimen. In our opinion, it is clear that male and female animals and organs, cells and organelles from male and female animals should be
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utilised to screen drugs, devices and procedures in order to provide gender-based medicine which could lead to novel therapeutic approaches and strategies that could improve the appropriateness and the safety of therapy. Indeed, it is also plausible to open new field of research to understand the whole complexity of gender medicine. For example, maternal foetal microchimerism (the results from bi-directional transfer of cells across the placental barrier in pregnancy) [399, 400] is found in numerous species including humans [401]and should be considered when enrolling post-pregnant animals or women in studies, because these cells can contribute to female physiology. Indeed, male progenitor cells (CD34+ and CD34+ CD38+) remain in women’s blood for several decades after pregnancy [402,
ACCEPTED MANUSCRIPT 403], participating in the development of autoimmune disease in multiparous women [404-407] and in regeneration of damaged tissues [407, 408]. Considering that biological and social factors are age-dependent [409-411], sex-gender and
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age should be considered together without forgetting life in utero. In fact, the majority of female and male animals used in biomedical research analyses are young. Furthermore, females have a
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regular reproductive cycle and often have not been pregnant before use in biomedical research. These restrictions of parity and age may influence the results of the experiments, making the
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findings inapplicable to human populations in which older individuals may predominate and
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childbearing is normal. Longitudinal studies are required to understand sex-gender differences throughout the lifespan, including the different phases of female life. Furthermore, experiments should be conducted in females in each oestrus or menstrual cycle phase and, if not practical, the
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cycle phase should be indicated in the methods section.
It appears urgent to have information on pregnancy and on neonatal life, because these
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periods are fundamental for the plasticity of development in both animals and humans. Notably,
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stressors can exert effects in a sex-gender specific manner. The widespread human use of OC suggests that a female experimental group should always
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be treated with oestrogen and progestin drugs or with other hormonal blockers of ovulation. The presence of different OC formulations in the market could indicate that more than one specific formulation could be used. This point is relevant, especially considering the influence of OC on physiological parameters [32], drugs, xenobiotic metabolism and receptors [42, 108]. Finally, papers should contain a complete description of the experiments [412].
ACCEPTED MANUSCRIPT Box 1: Pharmacokinetic Size Descriptors
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Total body weight (TBW): subject’s real weight (kg).
Body mass index (BMI) or ‘Quetelet’s Index’ TBW (Kg)/height (m2) is now the international
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metric recommended to classify obesity and overweight. This formula utilises body weight and
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height. However, it cannot differentiate adipose tissue from muscle mass. Relevantly, BMI is not gender-specific, because it was not derived from women and its predictive value for morbidity has
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not been evaluated in women [413].
Lean body mass or Ideal Body Weight (IBW) considers height and sex; however, it does not take
ED
into account differences in body composition. The lean body mass, which is similar but not identical to fat-free mass, may be a better basis for drug dosage than either TBW or body surface area,
PT
although the rationale for this is not clear. Lean body mass in humans can be calculated by Devine
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estimation [414], and lean body mass can be calculated by Robinson estimation [415]. Distribution volume of relatively hydrophilic drugs has a good correlation with LBM, but for lipophilic drugs,
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the correlation is better between volume of distribution and total body weight than with lean body weight. Thus, although most drugs are dosed using body weight, some medications are best dosed using IBW or a combination of IBW and actual body weight, particularly in obese patients [416418]. Finally, lung capacity correlates best with lean body mass rather than actual body weight [416].
Body surface area is used for the dosing of the antineoplastic agents and is based on the assumption that height, TBW and a constant C are linked to body surface. Like BMI, body surface area does not consider the sex [413]. It can be also calculated by the Mosteller formula [419].
ACCEPTED MANUSCRIPT Adjusted Body Weight (ABW) is the first size descriptor developed for pharmacokinetic studies [420]. It uses a correction factor to apply to IBW (IBW + CF (TBW- IBW)), and the choice of adjustment depends upon the drug and the clinician’s judgment. ABW, however, seems a plausible
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size descriptor as it considers sex, TBW and height [413].
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Fat-free mass (FFM) relates weight and fat mass. The metric was derived in guinea pigs, where the live weight and eviscerated wet and dry weights were used to determine the total fat mass of the
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animal. This descriptor depends upon sex, TBW and height [413].
Percent total body surface area (TBSA) is calculated through different formulas. Regarding animals, most formulas are based on the original Meeh Formula (TBSA= ¼ kW 2/3) with K being a
ED
constant that is empirically determined and varies greatly by species and size. For rats, values of K constants range from 9.00 to 11.36 [421]. The constant calculated in thirty five-month-old female
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Wistar rats weighing 195–240 g is 9.83.
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Formulas are referred to humans
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ACCEPTED MANUSCRIPT
M/F
M/F
M/F
WISTAR RATS
SD RATS
C57BL/6 MICE
M/F
M/F
M/F
M/F
BALB/c
CD1 MICE
NZ RABBIT
WM GUINEA
SC RI
PARAMETER
PT
Table 1. Effect of sex on some biochemical parameters of rodents
= [422, 423];
= [426-429]
+/-[422]
+/-[424]
+/-[430]
-/+[431]
=[427]
=[439]
-/+ [425] HDL
= [423, 438]
-/+ [425]
TG
-/+[422]
=[426-428]
=[423, 438] +/-[424, 425, 440]
=[432]
-/+[435]
+/-
+/-[433]
=[436, 437]
-/+[435]
-/+[435]
-/+[437]
-/+[435]
-/+[436]
=[434]
=[437]
AC
= [423, 425] +/-[424]
=[433, 434]
CE
HDL2 LDL
+/-[439]
+/-
PT
= [425] HDL1
+/-[271, 431]
PIG
ED
+/-[424]
MA
Tot-Chol
NU
MICE
+/-*[441] +/-[430, 442]
=[422]
+/-[271]
=[432]
ACCEPTED MANUSCRIPT Bilirubin
= [422]
= [426, 428]
=[422]
-/+[430]
-/+[243]
+/-[431]
=[432]
=[436]
ALT
= [422]
+/- [426, 428]
PT
+/-[431] = [243, 422]
AST
-/+[422]
+/-[426, 428]
SC RI
= [430]
+/- $[431]
=[243, 422]
+/-[422]
+/-[426, 428,
=[422]
430]
-/+[243]
=[426, 428]
-/+[422, 431]
444, 445]
+/-*[441]
+/-[446]
+/-[424]
+/-[430]
=[423, 438, 445]
=*[441]
Urea Uric acid
-/+[422] -/+[422] =[422]
-/+[426, 428]
=[422]
=[430]
-/+[243]
-/+[426]
=[243, 422]
=[428, 430]
-/+[431]
+/-[430]
=[422]
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Creatinine
=[280]
-/+[436]
=[280, 434]
=[436]
+/-[280]
+/-[436]
=[446]
CE
-/+[444]
=[436]
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=[422, 423, 438,
+/-[424]
=[280]
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Insulin
=[436]
-/+ *[431]
+/-[431] Glucose
= [432, 443]
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AP
=[280]
-/+ [432]
NU
=[430]
= [443]
= +/-[431] -/+*[431]
+/-[436] -/+
SD=Sprague-Dawley, NZ= New Zealand, WM= Weiser-Maples, HDL = high density lipoprotein, LDL= low density lipoprotein, TG= Triglycerides, ALT= Alanine aminotransferase, AST= Aspartate aminotransferase, γ-GT= Gamma-glutamyltransferase, AP=Alkaline phosphatase, *= old animals, $ = 1-3 month old, & = 9 month old, = 6 years old
ACCEPTED MANUSCRIPT
Table 2 Effect of sex on some biochemical parameters of rodents M/F
BEAGLE DOGS
CYNOMOLGUS MONKEYS
Tot-Chol
= &[447]
=[448]
TG
= &[447]
-/+[448] =[449]
Bilirubin
-/+[448] =[280]
=[448, 449]
AST
=[280]
=[449]
AP
=[280]
+/-[449]
+/- &-[447, 450] =[280, 447]
Creatinine
=[280, 447] = &[447]
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-/+ [450]
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Glucose
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ALT
Urea
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M/F
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PARAMETER
=[449]
+/-[448]
=[448, 449]
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* = old animals, $ = 1-3 month old, & = 9 month old, = 6 years old
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Table 3 Distribution of the sex of cells in publications Unspecified sex 75%
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Female Cells 5%
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Male cells 20% Data from [24].
ACCEPTED MANUSCRIPT Table 4 Examples of sex differences in aortic VSMC obtained from male and female rats
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Protein expression of ER alphab
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Protein expression of ER betab
M/F +/+/+/+/+/+/-/+ = -/+ -/+ +/= +/+/+/-/+ -/+ = -/+ = -/+ +/+/= +/= +/-/+
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PARAMETERS Basal proliferationa Proliferation in serum-free mediuma,c Cell migrationc,d Basal apoptosis Apoptosis induced by radiationb Basal autophagy Senescence induced by radiationb Adhesion in normal serum mediuma Adhesion in low serum mediuma Adhesion in serum-free mediuma Basal cell contractiond Basal [Ca2+](i)c H2O2 production Superoxide anion production 4-HNE GSH Total proteina mRNA expression of ER alpha and betab
[451] [451] [452] [290] [290] [290] [289] [290] [451] [451] [451] [453] [290] [290] [290] [290] [454] [451] [455] [452, 456] [455] [291] [289] [455, 456] [291] [291, 455] [457] [451]
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Protein expression of GPER Tissue inhibitor of metalloproteinase-1 gene expressionb Content of alpha-actin, vimentin, alpha(v) integrin and mesh-like networks of alpha-actin microfilamentsa RLIP76 expression [458] -/+ a b c Wistar specific pathogen free rats, Sprague-Dawley rats, Spontaneously hypertensive rats, d Wistar Kyoto rats, ER: Oestrogen Receptor, ET: Endothelin, lysoPC: lysophosphatidylcholine
ACCEPTED MANUSCRIPT Table 5 Questionnaire to be administered to human donors Sex Age Actual weight Birth weight Actual height Birth height Race Place of birth Place of house
Lifestyle
Physical activity: how many times a week and type Smoker (yes/no; how long and how many cigarettes per day) Alcohol use (yes/no; type; how many cups per day)
Social status
Occupation Level of education (years) Care giver (hours/day)
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Demographic information
Blood pressure at the time of sampling Diseases at the time of sampling and in the previous 3 months Drug use at the time of sampling and in the previous 3 months (drug type, duration of treatment, generally the use of drug is an exclusion criteria ) For women: Oral contraceptives use (how long and type) Date of last menstruation and menstrual cycle phase Pregnancies (number, type) Abortions (number and which month of pregnancy)
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Health status of subjects
Blood collection
Fasting subjects * Time of sampling (hour) Site of collection, anticoagulants, In fertile women, blood should be collected in the same period of menses cycle *Ingestion of food and of some beverages can influenced the composition of blood, plasma, and serum [459]
ACCEPTED MANUSCRIPT Table 6 Questionnaire to be administered to professionals before the use of animals or animal cells Sex Age Actual weight Actual height Species
Environmental information
Diet type and parent diet Cross Fostering Maternal care and weaning Travelling, stabilisation, acclimatisation period Housing, isolation, spacing and environmental enrichment
Health status of subjects
Evaluation of health status
Blood collection
Fasting subjects * Time of sampling (hour) Site of collection, anticoagulants, blood volume, anaesthesia restraint, handling Blood should be collected in the same oestrous phase determined by vaginal examination
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Demographic information
Cells and tissues
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Table 7. Influence of sex-gender on the expression of housekeeping proteins beta tubulin, alpha actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in different cells and tissues. Beta tubulin
Alpha actin
GADPH
References
Human VSMCs from artery of umbilical cord
=
=
=
Franconi et al personal communication
Human Monocytes
=
NA
NA
Franconi et al personal communication
= + males + males + males
NA = +females =
NA = + females + males
Franconi et al personal communication [7] [7, 315-317] [7]
Human monocyte-derived macrophages Rat heart Rat liver Rat kidney NA=not available
ACCEPTED MANUSCRIPT Manuscript Title: Need for gender-specific pre-analytical testing: The dark side of the moon in
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laboratory testing
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List of all Authors: Flavia Franconi, Giuseppe Rosano, Ilaria Campesi
Corresponding Author: Flavia Franconi, Dipartimento di Scienze Biomediche Via Muroni 23,
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Sassari, Italy ; e-mail [email protected], phone +39079228717
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Highligths
Numerous methodological problems appear considering the sex-gender in experiments We discuss pre-analytical aspects of research
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Gender-specific pre-clinical research will produce more evidence-based medicine
S0167-5273(14)02161-5 doi: 10.1016/j.ijcard.2014.11.019 IJCA 19175
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
8 September 2014 27 October 2014 3 November 2014
Please cite this article as: Franconi Flavia, Rosano Giuseppe, Campesi Ilaria, Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing, International Journal of Cardiology (2014), doi: 10.1016/j.ijcard.2014.11.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing
1
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Flavia Franconi1 , Giuseppe Rosano2, Ilaria Campesi3
Department of Biomedical Sciences, University of Sassari, National Laboratory of Gender
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Medicine of the National Institute of Biostructures and Biosystems, Osilo-Sassari, Italy, , Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Italy;
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Author had the idea to indagate the effect of sex and gender on pre-analytical phase of research and
2
Cardiovascular and Cell Sciences Research Institute, St George's University of London. The
author wrote and discussed the review
Department of Biomedical Sciences, University of Sassari, National Laboratory of Gender
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3
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wrote and discussed the review.
Medicine of the National Institute of Biostructures and Biosystems, Osilo-Sassari, Italy. The author
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wrote the review and took care of bibliography
Corresponding author: Flavia Franconi, Dipartimento di Scienze Biomediche Via Muroni 23,
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Sassari, Italy ; e-mail [email protected], phone +39079228717
Conflict of Interest The authors report no relationships that could be construed as a conflict of interest.
Acknowledgement: This research was partially funded by a grant of Sardinia Region “Effetti avversi e farmaci utilizzati nel trattamento del diabete di tipo 2: un approccio di genere” and by a grant of Istituto Superiore di Sanità “Piattaforma italiana per lo studio sulla polimorbidità”. We express deep gratitude to the Italian Pharmacological Society that provided the award “Gender Innovation” to Ilaria Campesi
ACCEPTED MANUSCRIPT Abstract Many international organisations encourage studies in a sex-gender perspective. However, research with a gender perspective presents a high degree of complexity, and the inclusion of sex-gender
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variable in experiments presents many methodological questions, the majority of which are still neglected. Overcoming these issues is fundamental to avoid erroneous results. Here, pre-analytical
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aspects of the research, such as study design, choice of utilised specimens, sample collection and processing, animal models of diseases, and the observer’s role, are discussed. Artefacts in this stage
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of research could affect the predictive value of all analyses. Furthermore, the standardisation of
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research subjects according to their lifestyles and, if female, to their life phase and menses or oestrous cycle, is urgent to harmonise research worldwide. A sex-gender-specific attention to preanalytical aspects could produce a decrease in the time for translation from the bench to bedside.
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Furthermore, gender-specific pre-clinical pharmacological testing will enable adequate assessment of pharmacokinetic and pharmacodynamic actions of drugs and will enable, where appropriate, an
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adequate gender-specific clinical development plan. Therefore, gender-specific pre-clinical research
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will increase the gender equity of care and will produce more evidence-based medicine.
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Key words: sex-gender, research complexity, pre-analytical conditions
ACCEPTED MANUSCRIPT 1. Introduction Although Hippocrates of Cos (460-370 BC) evidenced that “A woman does not take the gout unless her menses has stopped,” describing a sex-gender difference in the susceptibility of the
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development of disease [1] , it is indisputable that the differences between men and women were ignored until the two last decades of the last century [2]. However, over the past 20-30 years,
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research has shown, from single cells to more complex biological systems, that biological differences (sex) between men and women are numerous and involve all branches of biomedical
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and are organ specific [7] and cell specific [8].
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sciences [2-5]. In the last few years, it has emerged that sex-gender differences starts in utero [6]
Thus, it is not surprising that remarkable sex-gender differences have been described in the prevalence, progression, treatment and outcome of numerous diseases including diabetes mellitus
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[9], cancer, depression and brain disorders, and infectious, cardiovascular, renal, hepatic, pulmonary, inflammatory and autoimmune diseases [3].
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Indeed, the biological differences between sexes should be considered across the entire
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range of research, starting from the pre-analytic conditions embodying research in genetics, epigenetics [10], developmental biology, biochemistry, physiology, pharmacology, toxicology, and
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epidemiology as well as social sciences adopting all available technologies including omics [10]. All ages should be considered, including pre-natal life, because sex differences start in utero [6]. It is also important to recall that the placenta may play a key role not only in buffering environmental effects transmitted by the mother but also in expressing and modulating effects due to preconception exposure of both the mother and the father to stressful conditions [6]. In addition, in designing experiments, epigenetic modifications should also be taken into consideration. Inevitably, the stress response may play a role . The pioneer paper of Critchlow and colleagues [11] showed that the stress response is sexually dimorphic. Consistently, social status, domestic violence and caregiver role are related to the stress response and can lead to depression, cardiovascular diseases, and diabetes mellitus [12-14]. In this regard, we have to highlight that the
ACCEPTED MANUSCRIPT definitions of sex (biological differences in male and female body) and gender (environmental and social influences) may assume different meanings in biomedical fields and in social sciences. As a result, gender is sometimes mistakenly employed as an updated version of the term sex or to
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indicate only female sex [15]. This viewpoint is widespread, and some studies that mention the word gender in their title incorrectly use the term gender as a synonym for women [16, 17]. Of
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course, this attitude has important consequences, and men’s gender-specific needs may not be sufficiently considered.
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Furthermore, there is a relevant debate regarding the stability and validity of the binaries of
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nature and culture that underpin the concepts of sex and gender [18, 19], and there are some difficulties in segregating sex and gender influences on health, given the constant and dynamic interactions between genes and environment [20]. Some authors [21, 22] reject the discourse of
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biological versus social determinism and advocate a deeper analysis of how interactions between the biological being and the social environment impact on ‘‘individual’’ capacities, suggesting
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instead the use of the term sex-gender. Thus, through this review, the term sex-gender will be used
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to give equal status to the two concepts [23] and to indicate that sex-gender is a domain of complex and highly integrated phenomena and, as consequence of sex-gender research, requires
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intersectionality and integration with other disciplines, including social sciences. It is important to recognise that gender applies to all vertebrates and humans and that sexual dimorphism varies in the species and in the strains of animals. However, in numerous biomedical fields, almost all cellular studies [24]do not differentiate between genetic male or female cells. Furthermore, the majority (68–76%) of preclinical studies use only males or do not report the sex of the animals [25, 26], and the same occurs in some clinical studies [27]. Even fewer studies have been designed to address influences of psycho-social status (gender) of the donors on physiological outcomes [28].
ACCEPTED MANUSCRIPT Randomised clinical trials are still enrolling fewer women than men, especially in phase 1 and 2 trials. Nevertheless, the U.S. Food and Drug Administration recently recommended, for the first time, the use of different dosages of zolpidem in women and in men [29].
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Overall, existing knowledge suggests that:
a) it is inappropriate to assume that results obtained only in one sex can be applied to the
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other [30]:
b) sex-gender demands a lot of attention in the construction of measurements and variables,
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especially because of different body sizes and body composition of males and females;
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c) some baseline questions such as diet, housing, breeding, and cross fostering are still without a clear response;
d) characteristics of donors should be known, because donor lifestyles could affect the
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functions of the organs, cells, biomarkers and indicators [31, 32]; e) and, finally, sex-gender is not adequately considered in preclinical and clinical
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interventions involving diagnostics, medical devices, medications and clinical setting, and yet it is
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unknown if sex-gender influences placebo and nocebo effects and drug adherence [33, 34]. To overcome the scarce enrolment of females versus males, it is necessary to perform more
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studies on females comparing directly the two sexes and to incorporate a sex-gender-focused approach in the entire process of the research, which is something more than the simple enrolment of both sexes (see below). This can facilitate the aims of tailored medicine and further translational science, elevating basic knowledge upon which to build translational approaches that shorten the time from bench to patient bed [35, 36]. Furthermore, the awareness of the differences and similarities between male and females can improve the prevention, the efficacy, the safety and the appropriateness of the treatments [3, 33, 37-39]. A certain number of papers on methodological issues in sex-gender research are available [26, 28, 40-43]. However, they are mainly focused on defining differences between males and females and the origin of these differences such as the role of hormones [44] and on discussing animal
ACCEPTED MANUSCRIPT models of diseases and issues that sex-gender research meets after sampling [26, 28, 40-43], neglecting pre-analytic issues, which also include the influences of environment, with few exceptions [23].
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Indeed, pre-analytic procedures can be the source of a multitude of errors. It has been calculated that in the clinical laboratory, about 62% of errors happen during pre-analytical phases
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[45]. Pre-analytic variations (study design, compliance of the investigated subjects, compliance of the technical staff in adherence to protocols, choice of utilised specimens, sample collection and
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processing) are of critical relevance and importance, being encompassed in all sample preparations.
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Consequentially, an artefact in this stage could affect the predictive value of all analyses. They can be classified [46] in:
a) actions performed in animals or in humans before sample collection (animal handling,
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fasting, stress, restraint, analgesia and anaesthesia, dosing, diet, etc.) b) actions performed at collection (time of collection, blood sampling, collection technique,
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amount of blood, etc.)
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c) actions performed on the specimen such as dimension of collection tubes, sample separations, storage time, temperature, etc.
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Theoretically, all pre-analytic variations may be sex-gender dependent. Thus, pre-analytic variations are of special interest in sex-gender research. Considering that we are focusing on gender research, we include in the pre-analytical aspect the importance of the professionals and researchers’ sexgender, and the reason for focusing on this variable is illustrated below. Inclusion of XX cells and female animals in experiments and analysis of data by sex may contribute to resolve, at least in part, the issue of irreproducibility seen in preclinical biomedical research. This requires a special attention to methodological questions. Therefore, we suggest that it is no longer reasonable to ignore the methodological issues in sex-gender research, because only awareness of these issues can lead to sex-gender innovations. Although many studies tend to simply compare males and females on a number of health indicators, it emerges that there is an urgency to
ACCEPTED MANUSCRIPT use more sophisticated experimental designs, to redefined old methods and to develop new ones to produce new measures to study the influence of sex-gender on health. Here we discuss the pre-
2.
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analytic variables that have a special interest in sex-gender research through the lens of gender.
Complexity of sex gender research
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There are numerous criticisms regarding the ways in which sex-gender issues are addressed in health research because the various relationships among biological sex, gender and health are
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complex and numerous and may affect the manner in which sex-gender influences outcomes in
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health research. Indeed, sex-gender research has to consider the biosocial approach of medicine, which is composed of the interactions of biology and social environments [21], and the intersectional approaches [47]. Intersectionality is defined by Davis [48] as ‘the interaction
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between gender, race, and other categories of difference in individual lives, social practices, institutional arrangements, and cultural ideologies and the outcomes of these interactions in terms
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of power’. In other words, intersectionality: a) considers simultaneously interactions among various
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aspects of social life [49-51]; b) examines how social life and structural forces interact to shape and
our lives [53].
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influence human experiences [50, 52]; c) and explains how social organisation shapes all aspects of
Importantly, sex-gender researchers must also consider the similarities between women and men [54, 55]. In this regard, a sex-and gender-based analysis has been developed in Canada [56]. This analysis prioritises examinations of similarities and differences between women and men. Furthermore, sex-gender research should also include the boundaries of gender (masculine/feminine) and sex (male/female) [57], because the perpetuation of the dichotomies between sex and gender discourages going beyond two definable sexes and genders in view of the fact that intersex and transgendered people destabilise the dichotomy [58]. In this respect, FaustoSterling’s dynamic system theory [21] and Bekker’s Multi-Facet Gender and Health Model [59] clearly evidence that sex and gender must be integrated.
ACCEPTED MANUSCRIPT However, if the relevance of intersectionality is theoretically clear, it is less clear how it could be translated into research practice, and in this regard, we agree completely with Bowleg, who wrote that researchers “often have to self-teach and learn from trial and error” [49]. Indeed, if
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it is not possible to consider all factors, it is possible to say which factors are included or excluded, explaining the reason of the choices made. Actually, no single study is able avoid these problems
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completely, but it is possible to plan different studies which address different aspects [60]. Finally, researchers and professionals should have the expertise to recognise, perceive and
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incorporate existing sex-gender differences into their decision to make experimental designs and
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actions [50]. As a consequence, a scientist with sex-gender expertise cannot be a reductionist. Reductionism has permitted a great progression of science but has failed to answer numerous questions. Most life events at the organismal and cellular levels, in fact, cannot be only understood
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by understanding molecular functions in the cells and/or in the cell-free condition. Organisms, cells and cellular organelles, before becoming objects of research, live a life characterised by specific
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events (diet, stressors, lifestyle etc.) that may affect the way they perform in individual experiments
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and may even change in the genome, by both genetic and epigenetic mechanisms [61, 62]. Thus,
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social and biomedical sciences have to work together in controlling for pre-analytical variations.
3. Effets of sex-gender of researchers and professionals on research outcomes The recognition and the awareness of the difficulties that researchers meet to capture sex and gender aspects are fundamental. Nevertheless, recommendations and guidelines to make sex-gender research and analysis ([23]and quoted literature) often do not consider the sex-gender of the researcher, forgetting that he/she is a person and every individual is sexed and gendered. The potential influences of the sex-gender of the scientist on the results of the research have recently been shown in both preclinical [63, 64] and clinical studies [65]. In particular, experimental pain in humans is better tolerated when the researcher is of the opposite sex of the examined subject, while higher pain intensity has been measured for subjects tested by female experimenters [66].
ACCEPTED MANUSCRIPT Furthermore, men report lower pain to female experimenters than to male experimenters in the absence of significant changes in automatic response (heart rate and skin conductance), suggesting that the effect of the experimenter’s sex-gender is due to psychosocial factors [67]. The previous
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results indicate the importance of the relationship between research and subjects of research. Further confirmation of this principle comes from the work of Hadjistavropoulos and co-workers
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[68], which examines patients with low back pain, that showed that an observer is significantly influenced by patient gender and by the patients’ physical attractiveness. Rats and mice responses to
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pain depend on the sex of researchers [69]. Furthermore, male and female researchers focus on
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different aspects of behaviour and interpret the very same results in a different ways [70-72]. Indeed, sex-gender modulates visual attention during listening, and this could affect the response of the subject under study [73].
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In conclusion, a research team should be constituted by women and men, and the leadership should include men and women in order to reduce or to avoid inappropriate conclusions linked to
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the sex-gender of the researchers.
4. Experimental design
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Experimental planning should define outcomes and independent measures a priori in order to explain the biological basis of sex differences. Further experimental design should develop paradigms that include a sufficient number of samples that involve social and biosocial aspects including appropriate control groups for all hypotheses. Small sample size may prevent the emergence of important biological and biosocial interactions [74]. Finally, sensitivity analyses should be performed. Experimental planning should consider, with the highest priority, the animal welfare and the relationship between researcher and subject. As illustrated below, experimental errors could occur in a sex-gender specific manner and have profound consequences on outcomes.
ACCEPTED MANUSCRIPT The experimental plan should include social and environmental situations, because these factors may influence disease vulnerability, therapeutic response and experimental outcomes [42], depending on the sexual dependence of the phenomena being studied [75, 76].
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As already mentioned, the stress response is essentially sexually dimorphic [11]. Thus, stressful events can have different features in males and females. Notably, this sexually dimorphic
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stress response seems to occur in a stressor-, species- and strain-specific manner [11, 77], and may also depend on bedding materials (see below). Thus, animal studies should take into account the
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potential role of environmental stress and sex-gender differences. In other words, studies with
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animals should consider that they have a social interaction.
The definition of primary endpoints and their variability are required for group size calculations, which also require the definition of power for detecting a difference [78]. Primary and
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secondary endpoints should be defined before starting the study; further, data should be analysed by a blinded observer and, if needed, statisticians should be consulted. If possible, studies should use
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balanced sex-gender and sex-gender X age factors. In addition, protocols should analyse data by
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sex-gender and interactions, rather than simply adjusting for gender. Power should be adequate, or lack of power (if inevitable) should be clearly mentioned. Inclusion and exclusion criteria need to
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be defined for each sex, and studies should include only individuals who match defined diagnostic criteria. Theoretically, more than one animal model should be used to represent the diversity in human patients.
5. Actions performed before sample collection 5.1 Age and sex differences The effect of age on data has been well recognised. Sexual dimorphism starts in utero and seems to occur at a pre-gonadal stage [10]. In line with the previous observations, in multiparous rodents such as mice, rats and gerbils, the position of male and female foetuses within the uterus can generate long lasting sex-gender differences in novelty seeking at puberty [79]. Furthermore, a
ACCEPTED MANUSCRIPT male mouse foetus that develops between female foetuses is more sexually active as an adult, less aggressive and has smaller seminal vesicles [80]. The intrauterine position of female mice can predict reproductive traits including genital morphology, sexual attractiveness, behaviour, and
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timing of puberty [81]. This effect seems to depend on sexual hormones, because a male mouse foetus located between males experiences greater concentrations of testosterone than a foetus of the
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same sex positioned between females. Likewise, a female foetus positioned between females experiences greater concentrations of oestradiol than a foetus of the same sex positioned between
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males [81]. These results suggest that standardisation even in experimental animals is very difficult
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to achieve, because the influence of intrauterine life is difficult to control adequately. This effect can be amplified by sex-gender aspects. Notably, prenatal and neonatal events can perpetuate into adulthood and in the next generation, and this effect seems to occur in a sex-gender-dependent
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manner [82, 83], increasing the complexity of research.
The interaction between sex and age is seen at all ages also depends on the stress effect.
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Aged male and female Fischer 344 rats, without stress and when given 21 days of restraint for 6
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h/day, present significant differences. In particular, female rats loose less weigh than male rats when stressed. Furthermore, unstressed males are more anxious than unstressed female. Interestingly,
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stress increases anxiety-related behaviour in male rats, but decreases it in female rats [84]. Previously, it has been shown that young unstressed female rats are generally less anxious than young control male rats. However, stress exerts anxiogenic and anxiolytic effects on young female rats and young male rats, respectively [85, 86]. Interestingly, the sexual dimorphism observed after stress induction appears to be reversed during aging. These results suggest that sexual dimorphism needs to be studied considering the influence of age. Remarkably, the previous observations suggest that sexual dimorphism is highly dependent on the sex and age combination, indicating that these factors should be studied together to obtain the optimisation of standardisation.
ACCEPTED MANUSCRIPT 5.2 Environmental and social factors and handling of animals Animals are widely used in toxicological and pharmaceutical research using methods developed in the last 50–60 years [87]. Animal use includes the breeding of laboratory animals as
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well as the housing of animals in the animal facilities, both of which are complex processes that can influence the results of the experiments in a significant manner. Indeed, animal tests as a surrogate
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for human research are subjected of numerous criticisms because their use involves a number of assumptions and extrapolations. In view of the fact that more male animals are used in comparison
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to female ones [25, 26, 44], more criticisms have to be made for extrapolation of results to women.
5.3 Cross fostering
The method of cross fostering, in which a litter of rodents born to one dam is suckled by
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another dam, is largely used in animal facilities and by animal producers, although cross fostering has little relevance for humans. However, it is not yet clear whether the process of fostering is
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harmless or if cross fostering itself is influenced by sex-gender. Rare findings suggest that cross
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fostering is not innocuous, because the procedure for group formation, which involves housing of unfamiliar and unisexual subjects, can be stressful [88]. For example, in mice, cross fostering is
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linked with reduced growth and altered behaviours [88-90], while in rats, cross fostering alters nociception and emotional behaviour [91, 92]. Furthermore, cross fostering per se influences cardiovascular and metabolic function in adulthood, programming a ‘thrifty’ phenotype, especially in male mice [93]. Notably, alterations induced by cross fostering are sex-gender-dependent [94]. In conclusion, the previous results suggest that cross fostering procedures may have different effects in male and female animals, indicating that cross fostering should be indicated, at least, in the method section of papers that enrol male and female animals. In our opinion, in preclinical testing of drugs, if animals are grouped through cross fostering, appropriate controls should be given, because prenatal and neonatal events including environmental changes can have long-term effects on adult phenotypes, predisposing individuals to diseases [95-99].
ACCEPTED MANUSCRIPT 5.4 Maternal care and weaning Experiments in rats, mice and monkeys show that maternal care in the first 2 weeks of life
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has critical effects on the neuroendocrine stress response [100, 101]. Offspring, deprived of maternal care, increase glucocorticoid secretion, and this is thought to be a risk factor for mental
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and cardiometabolic disorders [95, 96, 99]. Notably, in adult male mice but not in female ones, postnatal handling (maternal deprivation plus sham injection) induces hormonal and metabolic
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conditions similar to mild metabolic syndrome/type-2 diabetes, suggesting that female sex exerts
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effective protection against the hypothalamus-pituitary-adrenal homeostasis disruption induced by postnatal handling [99].
The amount of maternal care that a pup receives can be affected by litter size. Multiple
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siblings can result in a slower growth curve, lower dominance status, delayed onset of weaning and lower probabilities of survival for particular progeny [102-104]. For example, large siblings induce
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stereotypic behaviour in 2 inbred strains (C57BL/6N and C57BL/6J) and an outbred stock (CD1
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[ICR]) in female but not in male mice, indicating the saliency of the sex and gender [102]. Weaning is one of the most important events in the very early stage of life. Shorting weaning
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can produce long-lasting consequences in adulthood, such as anxiety- and aggression-augmenting effects [97, 98]. Early-weaned male rats have a higher sympathetic response to stressors [105], and early-weaned mice have elevated endocrine responses to mild stress [106]. Further, in early-weaned Balb/c mice, at 3 weeks of age, a reduction in the number of open-arm entries in an elevated plusmaze for both male and female mice is observed, but this effect is long-lasting only in males [107]. Kikusui et al [107] also show that myelin formation increases in normally weaned male and female mice and in early-weaned male mice, but not in early-weaned female mice, in which myelin formation increases strongly at week 3 and then declines at week 5. In conclusion, caution is necessary to evaluate experiments performed with animals subject to early and late weaning and maternal deprivation, because these procedures exert long lasting
ACCEPTED MANUSCRIPT effects on adult phenotypes, and this effect seems to occur in a sex-gender dependent manner. The sex-gender dependency of a phenomenon could produce artefacts that can invalidate results. At least, the modality of maternal care, litter sizes and the time of weaning should be clearly indicated
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in the method section of papers. In our opinion, in preclinical testing of experimental drugs,
5.5 Diets and gut microbiota differences
observed
between
males
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Physiological
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appropriate controls should be given.
and
females
can
influence
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pharmacokinetics as well as pharmacodynamics of endogenous and exogenous compounds such as foods, beverages [22] and drugs [108]. Regarding foods, the sex-gender differences observed at the gastrointestinal level assume a higher relevance in the bio-availability of food and drug [108-110]
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which is also influenced by food form (males eat pelleted food better than females) [111-113], suggesting that sex can play a role. Indeed, for gender studies, the materials where foods and
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beverages are held are also important, because oestrogenic compounds can be present in water
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bottles and cages and may be leaching, especially when cages and bottles are old and visibly worn [114]. However, little attention has been given to this aspect, even in studies focused on the
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investigation of endocrine disruptors [115]. Moreover, commercial diets can contain different compounds such as casein or soya protein. The soya diet contains a large amount of oestrogenic isoflavones (daidzein and genistein) [115118], which may have different pharmacodynamic effects in males and females. For example, in rabbit aortas, isoflavones inhibit formation of neointimal cells and are more active in males than in the females ([119] and quoted literature). Furthermore, genistein may afford greater cardioprotection in female guinea pigs than in males, and a soya-based diet worsens hypertrophic cardiomyopathy, especially in male mice ([119] and quoted literature). Finally, soya phytoestrogen supplementation augments cardiac growth in male mice but not in female ones ([119] and quoted literature). Regarding the importance of sex-gender on the pharmacokinetic of polyphenols, there
ACCEPTED MANUSCRIPT are no univocal data ([119] and quoted literature). After the administration of [14C] genistein, the maximal plasma concentrations of radioactivity are significantly lower in females than in males, whereas the concentration of radioactivity is greater in the liver of female rats than in male rats
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([119] and quoted literature). However, not all authors found sex-gender differences in plasma levels of genistein and daidzein and their metabolites ([119] and quoted literature). Notably,
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genistein’s oral bioavailability appears to be dependent on oestrous phase, being lower when oestrogen is higher ([119] and quoted literature). Polyphenol’s excretion as conjugated forms occurs
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by renal (7-30 %) and biliary routes (10%) ([119] and quoted literature). Considering that renal
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excretion is lower in women than in men [108], this could be of some importance in the elimination of polyphenols.
Finally, isoflavones may modify the metabolism of endogenous and exogenous compounds
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through the inhibition or induction of phase 1 and phase 2 enzymes. In particular, pregnane X receptors, which are implicated in the activation of CYP3A, CYP2B6, CYP2C9, sulfotransferase,
and inhibited by coumestrol ([119] and quoted literature). These results
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daidzein and equol
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UGT1A1, glutathione S-transferases, and multidrug resistance protein-2 , are activated by genistein,
indicate that isoflavone-rich diets, such as soya-derived diets, could alter the pharmacokinetics of
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other xenobiotics including medications, and this point is of extraordinary relevance in pharmacokinetic studies.
Additionally, the isoflavones of a dam’s diet reach the foetus through the placenta and the neonate through the dam’s milk [117, 120], and, consequentially, they could affect development of offspring in a sex-gender-dependent manner. This maternal effect could have salient consequences on predisposing one sex over the other to later adult-onset of diseases [121]. In the absence of awareness of the implication of the previous results, it is possible to unwittingly test drugs in animals that have different risks to develop diseases as a consequence of maternal diet [122, 123]. Finally, sex-gender also influences the outcomes of mild and severe food restriction, because female mice are more impulsive than male mice under mild food restriction [124]. In addition, food
ACCEPTED MANUSCRIPT restriction disrupts the oestrous cycle in female rats [125] without adversely affecting male fertility [126, 127]. Male rats maintain body weight better than females and do not have altered activity or cognitive ability after starvation [128]. Finally, mild starvation diminishes some of the sex-gender
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differences evidenced in hepatic transcript profiles and miRNAs [129].
Another important aspect regards whether interactions in overall gene expression responses
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as a result of diet occur in a sex- and tissue=specific manner. Actual information on this point is lacking. However, this information is necessary to interpret results and to generalise and extrapolate
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effects between tissues. This point is also relevant for surrogate tissues such as peripheral blood
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mononuclear cells. because they are largely used in mechanistic studies in humans [130]. Changes in dietary composition are associated with changes in the composition of gut microbial populations [131]. Recently, Markle et al. [132] suggested that the gut microbiota could
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be a major contributor in the development of sex-gender differences. In fact, the researchers showed that the pronounced sensitivity of female mice versus resistance of male mice to type 1 diabetes
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mellitus (non-obese diabetic (NOD) mouse model), could be directly attributed to the commensal
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microbiota, which differs in males and females at puberty. Indeed, the removal of microbiota increases and decreases plasma testosterone in female and male mice, respectively. Saliently,
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Markle et al. [132] also showed that the interaction between the host and microbes on disease susceptibility can be transferred to the next generation. This study revealed a new source of sexgender differences which could have unexpected consequences. Gut microbiota can be affected by foods and drugs such as antimicrobials [133, 134], and this could induce variations in body weight, metabolism and systemic inflammation in a sex-gender specific way [135-137] either in humans or in animals [132, 138, 139]. For example, olanzapine, an atypical antipsychotic agent, induces specific alterations to the gut microbiota which are associated with an increase in body weight in female but not in male rats suggesting that variations in gut microbiota can affect drug response in a sex-gender specific way [140].
ACCEPTED MANUSCRIPT In conclusion, for sex-gender studies, it appears that the use of diets with low amounts of oestrogenic compounds and oestrogen-free bedding, cages, and water bottles is critical. Additionally, one should not forget that high-fat foods or mild and low caloric intake could affects
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numerous parameters in a sex-gender specific manner. Furthermore, in view of the importance of the very early life events, it would be of relevance to know the diet of dams. This information
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should be included in the method section of papers. Of course, maximal caution should be practiced in the evaluation of endocrine disruptors and sexual hormones, and this is of extraordinary
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relevance in bioassays focused on drug action. Finally, it is necessary to also consider gut
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microbiota, because the microbiome could have heavy consequences on experimental outcomes, and this can occur in a sex-specific manner.
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5.6 Travelling, stabilisation, and acclimatisation periods Some laboratories have facilities to maintain their own breeding colony, whereas others
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purchase the animals from outside vendors, and animal transportation from the supplier to the
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laboratory induces stress responses [141, 142]. Indeed, during transportation, animals leave socially established environments and are exposed to overcrowding in a confined space, unfamiliar and loud
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noises, vibrations and jolting, variation in temperature and humidity, inhalation of gases (from urine, faeces and carburant), food and water deprivation, and change in bacterial environment among other stressors. The manner of shipping is not identical among animal producers [143]. In fact, it is not known if male and female animals require the same travelling conditions. Indeed, a recent paper indicated that shipping-induced stress responses could be scarcely affected by the sex of animals [143]. Upon their arrival into the laboratory, animals require a period of stabilisation and acclimation [144]. It is not clear whether sex-gender influences the duration of stabilisation and acclimation periods, though it is known that animals vary in these requirements according to species and strain. Considering that the acclimation period is of fundamental importance for the outcomes of experiments, it is mandatory to investigate whether these stress responses are sex-gender specific
ACCEPTED MANUSCRIPT parameters. This finding could exert a major influence on tests, because just moving a cage from
5.7 Housing, isolation, spacing and environmental enrichment
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one room or floor to another is stressful [145].
Animal welfare also depends on the housing environment. Recently, it has been shown that
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housing social context differently affects males and females [146]. Thus, in selecting a suitable social environment, sex-gender should have a role [147-150]. Social housing can induce a
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hierarchical structure, which could either be different or have different consequences in males and
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females [151-153]. In general, social housing may enhance coping with stress in female rats, whereas, in male rats, social housing may not have a positive influence on stress-sensitivity [154]. The social context also plays a role in inflammation [155]. For example, group housing exacerbates
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inflammatory responses and sickness behaviours in females, but attenuates these responses in males [156]. Isolation also modifies drug response: in particular, isolation housing reduces barbiturate-
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induced sleeping more in females than in males, and the difference is less evident in castrated males
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[157].
Over the past decade, housing of laboratory animals has largely evolved, and recently individually
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ventilated cages have been developed to maintain optimal hygienic conditions. However, recent data suggest that housing mice in individually ventilated cages impacts animal behaviours. Indeed, this could occur in a sex-gender specific manner. Female mice kept in individually ventilated cages become more anxious when compared to FILTER-housed females, while cognitive tests are not influenced by sex-gender [158]. A peculiar aspect of isolation is the use of metabolic cages: implicitly, there is the presumption that animal functions are relatively normal in animals housed in metabolic cages. However, this is not always true, because isolation in metabolic cages seems to alter, in a sexgender-dependent way, neurohumoral control [159] and renal [160] and cardiovascular function [161, 162], and may also induce significant weight loss in pregnant rats [163].
ACCEPTED MANUSCRIPT Metabolic cages are largely used in pharmacokinetic studies, but it less known that the pharmacokinetics of xenobiotics may be altered by the use of metabolic cages [164]. For example, rats acclimatised to metabolic cages for 21 days have a different response to non-acclimatised rats:
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the LD50 of ammonium diuranate is 360 and 6 mg/kg, respectively, in these mice [165]. However, it is not known whether alterations induced by metabolic cages are influenced by sex and gender.
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Nonetheless, there are some indications which suggest that living in a small space may have a different effect in male and female animals [166]. For example, female mice have a major body
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weight effect, present alterations in inflammatory response [167] and show an increase in grooming
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and sitting behaviours in comparison with male mice [168].
Environmental enrichment is a technique used to increase animal satisfaction. Nevertheless, most experimental paradigms are built on information obtained from studies performed on males.
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There are some suggestions that indicate the influence of sex-gender in the effects of environmental enrichment [169]. In particular, an enriched environment ameliorates spatial memory only in female
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mice [170]. In rats, environmental enrichment decreases baseline level of adrenocorticotropic
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hormone (ACTH) and corticosterone in a sex-gender-specific manner: the decrease in ACTH is greater in female rats than in male rats [171]. Others have found that environmental enrichment
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increases plasma testosterone and ACTH in males but not in females [172]. Furthermore, environmental enrichment raises the hippocampal brain-derived neurotrophic factor, especially in females, and attenuates oxytocin and aldosterone only in females [172]. Although they are not all in agreement, the results suggest that environmental enrichment exerts a sex-gender-specific effect. In a mouse model of Huntington's disease, environmental enrichment associated with handling significantly improved cognitive performance in the Morris water maze in female mice, but not in male mice [173]. Finally, bedding material may have a role in inducing sex differences or blunting them. For example, corncob bedding, which has estrogenic activity, blunted response to defeat in female mice but not in male mice [174].
ACCEPTED MANUSCRIPT In conclusion, experimental design must consider confounding variables such as group size, strain, age, and quality of space as well as social aspects such as environmental enrichment and isolation. In addition, reported results suggest that the response to the previous factors of
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environmental enrichment could be a sex-gender-associated phenomenon, indicating that male and female animals could have different needs and suggesting that the mutual needs of males and
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females should meet in order to arrive at a standardisation that it is optimum for the single sexgender considering the age and the strain. As also suggested by Girbovan and Plamondon [175], an
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accurate description of animal environment should be reported in the method sections of
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publications.
5.8 Timing of sample collection and drug administration
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It is well documented that some biological activities are characterised by prominent rhythms of various periods [176-179]. Biological rhythms can involve only one sex, such as oestrus or
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menstrual cycles [180-183], or both sexes [178, 184-186]. The expression of S100b, a soluble
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protein released by glial cells, changes during the day in a sex-gender specific way, as males and females have an inverse rhythm with changes larger than 30% from 5 to 21 h [187]. Moreover,
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Francavilla and colleagues [188] showed that nuclear oestrogen receptors in the liver are highest early in the morning and lowest in the middle afternoon, which parallels diurnal changes in oestradiol levels in the serum. Finally, we recall that sex-gender differences exist in the circadian rhythmicity of the CYP family of liver genes and corresponding nuclear receptors [189], suggesting that xenobiotic metabolism could be different at diverse times of the day. Oestrus cycle can also influence organ functions. In fact, it has been shown that oestrous cycle impacts myocardial electrical and contractile functions and modifies Ca2+ handling [190]. Additionally, tolerance to severe hypoxia is better in the pro-oestrus female rodent [191]. During oestrus and menses cycles, brain volume, plasma and blood volume change [192-194]. The variations in plasma and blood volume imply changing distributions volume of drugs and levels of
ACCEPTED MANUSCRIPT metabolites. Indeed, the pharmacokinetics of oral ranitidine varies with gender and stage of the menstrual cycle [195]. Biological rhythms can affect numerous pharmacological targets such as receptor number or
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conformation and signalling. For example, the antineoplastic action of IFN-β and the antiviral action of IFN-α in nocturnally active mice is higher in early light phase and is closely related to that
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of IFN receptors and ISGF expression in tumour cells or lymphocytes [196, 197]. The therapeutic-to-toxicity ratio of a drug changes predictably according to biological
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rhythm. For example, the body weight loss induced by irinotecan hydrochloride in nocturnally
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active mice is more severe in the late active phase and the early rest phase [198]. Preclinical and clinical studies show that the foetal toxicity of drugs varies not only with developmental stage but also with circadian time of administration [199]. The above results indicate
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the importance of timing in drug administration, while evidences on sex-gender differences in administration time are not yet sufficient to have clear conclusions. Nevertheless, these issues
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should be accurately investigated.
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In conclusion, special caution should be used for variations induced by menstrual and oestrous cycles, because existing data show that they affect body composition and renal,
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cardiovascular, haematological and immune systems; thus, these changes could impact pharmacokinetics and/or pharmacodynamics [200, 201]. The question of sex-gender-dependent influence on biological rhythms that involve both sexes is still obscure. However, the acknowledgement of this possibility is necessary to conduct high-quality animal and human research and to adopt more appropriate therapies for both sexes. This point is also fundamental because numerous drug delivery systems are under development to be employed for the treatment of chronotherapeutic diseases such as hypertension and asthma.
5.9 Pre-anaesthesia and anaesthesia
ACCEPTED MANUSCRIPT Anaesthesia has the aim to reduce pain relief, to induce the loss of consciousness and to induce muscle relaxation [202]. Appropriate care prior to anaesthesia is necessary [203], and drugs used in pre-anaesthesia, which present some sex-gender differences [204], are aimed to reduce fear,
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pain, oral and respiratory secretions and the quantity of other anaesthetic agents required and to block the vaso-vagal reflex associated with intubation and surgery and analgesics [205]. The central
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nervous system, the main target of anaesthesia, is sexually dimorphic [206]. In view of the huge differences in the male and female central nervous system, it is surprising to find that females are
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often excluded from preclinical and clinical investigation, even in the field of anaesthesia, and this
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bias has led to a large knowledge gap with relevant consequences. Anaesthesia is a large diffuse experimental procedure and is performed for dissecting organs, to isolate cells and to help to restrain animals.
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Anaesthesia in rodents inevitably induces autonomic nervous system depression, alters cardiovascular functions, induces respiratory depression, hormonal and metabolic alterations that
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induce hypothermia [207] and modifies hepatic and renal functions [208-210].The effects of
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anaesthetics on biochemical parameters are often molecule- and animal-strain specific. Therefore, caution is required when interpreting data from anaesthetised animals. Once again, the influence of
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sex-gender on variations induced by anaesthetics is not well known. However, sodium pentobarbital and other barbiturates have a longer ‘‘sleep time’’ in female rats than in male rats [211-213]. Plasma pentobarbital concentrations are lower in male rats than in female rats and decrease more rapidly in male rats [214], depending on different distribution and metabolism in males than in females. The difference in phenobarbital anaesthesia is not surprising, because in 1932, it was described that the hypnotic effect of hexobarbital lasts longer in female rats than in male rats [215], and this effect is dependent on sexual dimorphism in hexobarbital metabolism [216]. Notably, the differing effects of pentobarbital are not only linked to biological differences between male and a female rats (sex), but also depend on environment [157] as described above. Furthermore, the effect of phenobarbitone, at least in mice, is affected by foods, bedding materials,
ACCEPTED MANUSCRIPT and even the temperature used in the experiments [217]. Notably, Lovell [217] reported that environmental influence on pentobarbitone effect could be species-, strain- or possibly sex-genderspecific. However, no sex-gender differences with phenobarbital are found in mice [216, 218].
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The effect of ketamine, a non-competitive antagonist of the N-methyl-D-aspartate glutamate receptor, presents some sex-gender-specific differences, as liver metabolism of ketamine
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is more efficient in males than in females [219, 220]. Furthermore, male rats are less vulnerable to the neurotoxic and to some behavioural effects of ketamine in comparison with female rats [221,
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222]. Sex-gender differences in response to propofol have been reviewed elsewhere [223, 224].
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Notably, at least in women, the EC50 of propofol required to induce loss of consciousness is higher in the follicular phase than in the luteal phase. Emergence time is also longer in the follicular phase than in the luteal phase, and this seems to depend on variation in progesterone levels [225]. Some
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sex-gender differences are also described in the use of steroid anaesthetics: the male rat requires about four times more steroid anaesthetic than the female rat, and this sex-gender difference is age-
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dependent and disappears upon administration of oestrogen to the male [226]. On the other hand,
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the sex-gender differences observed with alfaxalone treatment seem to be dependent on formulation [227]. Notably, a single exposure in prenatal life to either the inhalant isoflurane or the injectable
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phenobarbital leads to significant decrements in cognitive abilities and a reduction in volume and neuron number in the hippocampus in adulthood, and this reduction is significantly greater in males than in females [228].
Finally, opioid analgesics behave differently in males and females. Different efficacy and safety profiles are observed with opioid analgesics in males and females. However, animal and human studies show that the direction and magnitude of sex-gender differences in response to a single compound can depend on numerous variables such as age, dose, route and time of administrations, type of pain, hormonal status, and species [229]. In conclusion, sex-gender differences in pre-anaesthetic and anaesthetic effects should be considered in all protocols requiring anaesthesia. Moreover, it is of value to know the recovery time
ACCEPTED MANUSCRIPT of anaesthesia for ethical considerations as well as for the possibility of drug interactions that might alter the results. The details are essential to understand what influence this may have on either the
post mortem, as also stated in the ARRIVE guidelines [230].
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6. Action performed at collection of the samples
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data obtained from in vivo experiments or from in vitro experiments performed on tissue obtained
One of the most critical issues in biomedical research is sample preparation [231, 232]. Blood
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and cellular fragments, plasma and serum.
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samples are used widely, and blood is generally collected via venipuncture in order to obtain cells
6.1 Blood collections
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Blood collection, handling, processing, and storage can impact final results, and this could occur in a sex-gender-specific manner [233]. Therefore, knowledge of these effects is a prerequisite
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to control their impact on research findings in order to reduce variability and decreasing the
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possibility of errors. There are numerous variables (the selection of blood collection tubes, which are much more complex devices than is commonly appreciated, anticoagulants, lag time before
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centrifugation, anticoagulants, temperature, etc.) that impact sampling procedures of plasma/serum and blood cells [234, 235].
6.2 Anticoagulants Various salts of heparin, EDTA, and sodium citrate are widely used in the clinical laboratory. Heparin salts are intended for determination of electrolyte levels and other routine chemistry values. The choice of anticoagulants is important, because the compound chosen can impact biochemical results. Numerous peptides differ in amount, not only between serum and plasma specimens, but also among plasma specimens prepared with different anticoagulants [236].
ACCEPTED MANUSCRIPT 6.3 Handling and processing from blood collection to specimen analysis Beyond anticoagulants, the time and temperature of storage of the specimen and the
temperature) can introduce pre-analytic variables [237-241].
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preparation of serum/plasma and cell separation (for istance centrifugation time, speed,
It is relevant to establish a standard protocol to monitor the stability of plasma/serum
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samples upon storage, and storage temperature should at least be considered. Storage temperature can induce pre-analytical variability depending on the specific analyte [237]. A recent paper
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indicates that proteins are sensitive to storage temperature in a protein-specific manner [242],
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though at the moment it is not known if this process is sex-gender-dependent. In this context, it is very important to establish standard protocols to monitor the stability of plasma/serum samples upon storage. It is not well known if these pre-analytical procedures
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performed on the specimen are associated with donor sex and age.
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6.4 Volume and frequency of blood collections
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Rodents are widely used in biomedical research. However, due to the animals’ relatively small body size, blood collection can be difficult. Males weigh significantly more than females and
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have more available blood volume than females. Consequentially, blood collection is more difficult in females than in males, and this can produce more artefacts in females. For example, blood cell damage occurs more often in blood obtained from females [243]. Obviously, repeated blood collections are technically difficult in small animals, especially in females that have smaller amounts of blood; indeed the volume and frequency of blood collections should be carefully considered in the female, because excessive blood withdrawal can negatively affect animal wellbeing [244, 245] . In particular, the volume of blood removed and the frequency of sampling will be based on the purpose of the scientific procedure and the total blood volume of the animal. Total blood volume is generally estimated as 55-70 ml/kg body weight. Thus, total blood volume is lower in females than in male, because female animals weigh less than male animals [246].
ACCEPTED MANUSCRIPT In conclusion, minor changes in procedures can cause large changes in biologically relevant variables, highlighting the importance of establishing uniform and validated procedures to
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improve reproducibility of animal phenotypic data. This should occur in both sexes.
6.5 Sampling of blood
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The manoeuvres (handling, restraint, and anaesthesia) needed for collection of blood from animals are stressful, and the operator should keep stress at the lowest possible level in order to
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preserve animal welfare and to produce good results. For example, stress associated with the
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handling of mice during bleeding increases glucose serum concentrations proportionally to the handling time [247]. As already mentioned, anaesthetics (enflurane and halothane) and animal discomfort can modify serum biochemical parameters [247, 248].
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Notably, biochemical parameters also depend on the site of blood sampling [249-252], and this phenomenon seems to occur in a sex-gender-specific manner [243]. Additionally, in rodents,
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significant differences in leukocyte, red cell and platelet counts have been described in blood
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samples collected from different sites [243, 253-255]. Indeed, a study performed in male and female mice and rats using the techniques of cardiac puncture and venous puncture of the tail, foot and
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saphenous veins indicates that total peripheral leukocyte counts are lowest in blood collected from the heart in both male and female rodents [253-259], and this effect seems to occur in a sex-genderdependent manner [259]. The above results suggest that the phlebotomy method and phlebotomy site are important factors affecting haematological and biochemical outcomes, and the practice of utilising different sites of sampling should be avoided, because the introduction of different techniques in a single study introduces a source of error. The cannulation technique can be used to remove blood. Cannulation decreases the stress of multiple samplings, reducing the stress linked to repeated restraint and needle sticks . However, this technique can cause pain, and therefore it may require analgesic medications and appropriate monitoring for the duration of the cannulation period. Furthermore, infections may arise despite the
ACCEPTED MANUSCRIPT use of sterile equipment [260]. Cannulation also permits access to deep vessels, ambulatory infusions and administration of drugs [245]. The influence of sex-gender on cannulation is not well studied, but considering the small diameter of female animal blood vessels, sex-gender differences
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are plausible.
In conclusion, the available data suggest that numerous pre-analytical variables could affect
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haematological and biochemical results, and this seems to depend on the analyte being studied and possibly on the sex of the animal. Thus, in experimental design, it is necessary to establish the
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normal range of analytes for both males and females based upon the procedure adopted for
6.6 Urine collection and processing
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preparation of the samples.
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Urine collection and processing remains a major in vitro diagnostic screening test and has a special role in pharmacokinetics. The pre-analytical phase of planning should include sample
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collection, specimen transport and preparation of samples for testing, and it is important to consider
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potential sources of error. It is relevant to recall that female and male animals may have different urine volumes. Rodent females drink more and have a larger urinary volume which is less
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concentrated [261-263]. Recently, it has been shown that spiny female mouse excrete more urine than male mice, but the urine excretion of sodium and proteins does not differ in the two sexes [264]. It has also been also reported that glomerular filtration rate (GFR) is higher in males than in females due to higher renal plasma flow and lower renal vascular resistance [265-267]. The differences between males and females are lessened when correcting for body weight, which is lower in females [264-266].
6.7 Clinical chemistry, haematological data, and reference values In clinical or pre-clinical research, the normal range of haematological components and biochemical parameters of blood are used for diagnosis and as target for pharmacological response.
ACCEPTED MANUSCRIPT Thus, acknowledgment of reference values is of great relevance. Many sources of variation can affect the results of clinical biochemistry assays in both animals and humans [233, 247, 268-270]. These sources of variation are significantly influenced by environmental factors and characteristics
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of the individual, including sex. Although, in the past 20 years, awareness of the influence of sexgender on blood, plasma, serum and urinary constituent levels is growing, these factors are still
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neglected in clinical chemistry and haematology [248, 271, 272]. Rats, mice, dog, and primates present sexual dimorphism in serum, urine, cerebrospinal and bronchoalveolar lavage fluid
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proteomes in both control situations and during some diseases (Table 1, 2) [273]. Biomarkers of
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lipid metabolism vary in Meishan pigs in a sex-gender- and an age-dependent manner [271]. In 4 months old pigs, total and free cholesterol, LDL and triglycerides are higher in females than in males [271]. On the contrary, in multiple strains of mice, total cholesterol and triglycerides are
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higher in males than in females [274]. A Japanese study [272], which analyses historical control data of clinical pathology provided by sixty-seven companies of the Japan Pharmaceutical
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Manufacturers, notes that differences for haematocrit, haemoglobin concentration, white blood cell
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count, glucose, cholesterol, triglycerides, total protein, albumin/globulin ratio, urea nitrogen, and inorganic phosphorus in rats and in mice are sex-dependent. Indeed, sex-gender differences have
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been found by many authors [275-278]. Notably, some sex-gender differences and parameters are age- [279], species- and strain-specific [280, 281]. For example lionesses have higher haemoglobin values than those found in the lion [282]. However, reference values of haemoglobin in Old World primates, which have pre-menses, breeding and menopausal phases [283, 284]and menstruation frequencies which are very similar to humans [284], do not present significant differences between females and age-matched males [283]. Notably, in studying sex-gender differences, it is also necessary to study the distribution of data, because different distributions could be present in male and female cohorts. Generally, normally hydrated, conscious female rats and women have lower haematocrit levels than their male counterparts, indicating that the volume of red cells and plasma may differ
ACCEPTED MANUSCRIPT between sexes [192, 285]. When comparing the values obtained in males and females, it is relevant to recall that men have a larger total blood volume (about 6–8% greater) than women [286]. Thus, metabolite concentration in blood is diluted more in men than in women, resulting in even greater
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differences between sexes than if this factor is not considered. Considering that reference range values in animals and humans have great value as biomarkers in testing xenobiotic efficacy and
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safety beyond their value in prevention in the natural course of diseases and in outcomes, it is fundamental to reduce errors by determining the normal range, which should be obtained for each
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sex at a single age, taking also into consideration the oestrous and menses cycles and reproductive
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phase of females. Additionally, a number of drugs and their metabolite interfere with analytes. This interference may cause false-positive and false-negative values in clinical patient material, and this could occur in a sex-gender-dependent manner, because drug distribution and metabolism can be
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sex-dependent [108].
In conclusion, changes in blood values are used as end points and/or biomarkers in
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pharmacological studies. Thus, a laboratory must set up and maintain a reference range database to
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minimise environmental interferences. Databases could help to firmly establish which parameters
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are different between sexes and to obtain an international harmonisation.
6.8 Collection and isolation of the cells It is difficult to determine the sex of cells used in scientific papers (Table 3), and within those publications that do indicate the sex of the cells used, some do not perform sex-gender analysis. However, sex-gender should be considered in all cell studies, because cells obtained from males and females can behave differently [8, 31, 32, 287-293]. Many of the differences observed could depend on genetic, epigenetic and hormonal processes [294, 295]. As example of the complexity of these phenomena, we recall that CYP3A4 activity is greater in female cryopreserved human hepatocytes than in male counterparts, whereas, in freshly cultured hepatocytes, induction of CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 does not appear to be influenced by sex-
ACCEPTED MANUSCRIPT gender or by the age of the donor [296], suggesting that freezing exerts a sex-dependent effect. Primary osteoblasts isolated from pre- and post-menopausal women show age-dependent biochemical changes that are not observed in the same bone cells from younger or older men [297].
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Macrophages from female arthritis-susceptible B10.RIII or B10.G mice synthesise more prostaglandins and thromboxane and have higher circulating anti-type II collagen antibodies than
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male counterparts [287]. Sex-gender-dependent differences in vascular smooth muscular cells (VSMC) from the rat aorta are summarised in Table 4. In particular, VSMC from rat aortas have
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different cell migration phenotypes and expression of oestrogen receptors. The differences in steroid
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receptor expression disappear after 14 passages post-isolation [291], indicating the importance of cell passage for studying sex-gender differences and suggesting that these differences could be better-studied in primary culture rather than in cell lines. Furthermore, XX and XY VSMC from the
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aortas of rats present a sexual dimorphism in cell fat; male cells are more prone to apoptosis, anoikis, and autophagy than female cells [289, 290]. Furthermore, XY neurons are more susceptible
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to glutamate exposure than are XX cells, whereas XX cells are more sensitive to pro-apoptotic
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stimuli [298].
Jog and Caricchio [299] demonstrate that bone marrow-derived macrophages obtained from
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male mice are more prone to necrosis through a PARP-1 dependent mechanism than female mice. Conversely, female cell the death is PARP-1-independent. Notably, oestrogens increase survival only in female bone marrow-derived macrophages, independently from oestrogen receptor expression. Human macrophages derived from monocytes isolated from men and women retain the effects of smoking in a sex-gender specific manner [31], and female human macrophages derived from monocytes retain the effects of oral contraceptive (OC) use [32], indicating that the lifestyles of donors can affect cell function. Thus, cells not only have a sex also have a gender, either in basal conditions or after stimulation. Indeed, it is not yet well known whether cessation of OC and hormonal replacement therapy affects isolated cells. However, it has been reported that past OC users have lower (about 5%-10%) levels of endogenous estradiol, estrone, androstenedione,
ACCEPTED MANUSCRIPT testosterone, and sex hormone-binding globulin compared with never-users independently from age, body mass index, smoking, physical activity, and reproductive factors [300]. Past users of hormonal replacement therapy
have lower levels of testosterone and 17alpha-hydroxyprogesterone,
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suggesting that the use of exogenous hormones produces long-lasting effects that persist for years after the cessation of treatments. These changes could also affect the function of blood cells isolated
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to perform ex vivo experiments.
Additionally, samples need to be lysed to measure some components localised inside cells.
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Thus, it is necessary to determine the type of lysis buffer to be used, because this choice could have
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consequences on the biochemical parameters to be determined. The influence of sex-gender on the previous parameters has not been studied. Obviously, it is time to overcome this lack of knowledge to improve research and to increase its translational value.
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In conclusion, fresh isolated cells retain memory of the sex and the gender of the donor, and thus it is crucial to use cells obtained from males and females to characterise mechanisms and to
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avoid erroneous conclusions. Furthermore, it is of relevance to administer a questionnaire (Table 5)
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to human donors to collect more relevant information about lifestyle and xenobiotic use, while, for cells obtained from animals, information on age, sex, diet and housing cross fostering should be
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collected in order to verify the influence of environmental factors in generating sexual differences (Table 6). Journal editors should encourage the publication of questionnaires in “Materials and Methods” sections as standard practice, and scientific societies should participate in the validation process of questionnaires.
6.9 Collection and isolation of cell organelles Sex-gender differences are described in subcellular organelles and their enzyme activities. In particular, mitochondria obtained from rat, mouse and rabbit brain, cardiac tissue, liver, and skeletal muscle present sexual dimorphism [301-308]. Sex-gender differences, particularly regarding calcium handling, oxygen consumption and reactive oxygen species production, have
ACCEPTED MANUSCRIPT been reported. However, mitochondria obtained from B6 (C57Bl/6J) mice of both sexes do not present significant variations in heart, skeletal muscle and liver mitochondrial oxygen consumption, ATP content, H2O2 generation, oxidative stress levels or apoptosis [309]. Previous data suggest that
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mitochondria can have a role in sex-gender differences, but more data are needed to reach a firm conclusion.
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Sex-gender differences have been also observed in lysosomes, which are involved in multiple physiological and pathological cell processes. In particular, sex-and strain-based
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differences in the size of lysosomes in the proximal tubular epithelium have been identified in mice
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[310, 311]. For example, the DBA/2Cr strain possesses giant lysosomes that predominate in the proximal convoluted tubules and in the proximal straight tubules of males and females, respectively [312]. The appearance of giant lysosomes in males and females is controlled by testosterone and
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oestrogen, respectively [312]. Whereas liver cholesterol ester hydrolase activity is decreased by progesterone concentrations >100 µM, the activity of this enzyme is not changed by estradiol in
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vivo or in vitro [313]. Furthermore, beta-galactosidase, beta-glucosidase and acid phosphatase
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acidity are higher in female rat cardiac lysosomes in comparison with male counterparts [314]. A recent study performed in rats revealed that male livers are enriched in LAMP-1, a constitutive
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protein of lysosomes, in comparison with female livers, suggesting the presence of a higher number of lysosomes in the male rat liver. However, LAMP-1 expression does not differ in the heart or in the kidney of female and male rats [7]. Overall, the previous results suggest that lysosomes might have role in sex-gender differences, but to reach a firm conclusion, more data are needed because the differences could be organ-, species- and strain-specific. Many cellular and subcellular experiments use housekeeping genes and proteins to normalise the results. The housekeeping proteins shown in Table 7 may be differently expressed in male and female cells and tissues [7, 315-317]. Interestingly, this effect appears to be protein-, celland organ-specific. Thus, pilot experiments should be performed in order to find housekeeping proteins and genes equally expressed in males and females.
ACCEPTED MANUSCRIPT In conclusion, the sex of donors of biological research regents must be known, because organelles and cells have memory of their sex and gender. Considering that changes at cellular and organelle levels can be used as end points, biomarkers and/or targets of pharmacological studies, it is urgent
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to know if sex and gender affect the functions of these cells. It is also relevant to administer a questionnaire as illustrated in Tables 5 and 6 to human donors or to professionals collecting
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samples to assess relevant information about the lifestyles and xenobiotic use of the donor. Moreover, for cells obtained from animals, information on age, sex, diet and housing cross fostering
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should be collected, and journal editors should encourage the inclusion of such information in
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“Materials and Methods” sections as standard practice.
6.10 Isolated organs
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For over 150 years, isolated tissue and isolated organ preparations have been widely used as convenient biological models [318].. Indeed, the development of methods of gene manipulation
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and protein expression allow the study of organs obtained from animals with normal or pathological
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tissues, allowing molecular biologists to quantify the physiological impact of altered gene sequences, mechanisms, physiology and cellular signalling. Moreover, isolation of organs is also
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performed to compare the weight of organs. Importantly, regulatory agencies use dose-related organ weight effects as end points to build toxicity reference values and in physiologically-based pharmacokinetic models [319]. Generally, organ weight is greater in male animal models than in female animal models [320]. Notably, the vast majority of studies employing isolated organs have used male animals. For example, only male isolated perfused rat hearts have been used to study ischemic arrhythmias [25, 26, 321]. Regarding sex-gender differences in ischemic post-conditioning, results obtained in the Langendorff perfused rat hearts have been contradictory. In particular, Lee et al [322] report no significant differences between male and female hearts, whereas Zheng et al [323] show that Langendorff perfused female rat hearts have greater resistance to ischemic-reperfusion insult than
ACCEPTED MANUSCRIPT male perfused hearts, concluding that male hearts are able to develop cardioprotection after sevoflurane post-conditioning through mechanisms that seem to involve the PI3K/Akt pathway. Sex-gender differences have also been described in Langendorff perfused mouse hearts in both
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basal conditions and after ischemia [324]. As already mentioned, cardiac contraction varies during the oestrous cycle [190]. In perfused rat hearts, there is also a sex-gender specific metabolic
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modulation [325]. For example, during ischemia and reperfusion, the metabolic state of rat hearts is sex-dependent. In fact, lactate originates mostly from [U-(13) C] glucose and from [3-(13) C]
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glucose in hearts from males and females, respectively [325].
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The sex-gender of donors influences the secretion of bile acids [326] and the uptake of long chain fatty acids from the isolated liver [327]. Notably, isolated livers from female rats, especially pregnant ones, are strikingly resistant to the effects of tetracycline [327].
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The above results strongly suggest that male and female organs can behave differently in vitro. However, the use of isolated organs requires a multistep protocol including: a) anaesthesia
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(see above); b) anticoagulant administration, which theoretically reduces the risk of thrombus
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formation; c) organ isolation; d) cannulation, which must take into account the dimension of the organ and of the vessels, which generally are smaller in the females than in the males; d) and
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perfusion. Indeed, with few exceptions including anaesthesia, the influence of sex-gender on each step of organ isolation is scarcely studied. The post-mortem period is crucial for degradation of cells, tissues and organs, and it is pivotal that samples be processed correctly to preserve quality [328]. In conclusion, the sex of donors must be known, because isolated organs, cells and cellular organelles have a sex and have a gender. It is not yet known which, if any, activities of isolated organs are influenced by the environment in a sexual dimorphic manner. Considering that changes in functions of isolated organs can be used as end points, biomarkers and/or targets of pharmacological studies, it is urgent to know if sex and gender affect their functions. To improve our knowledge on this specific point, it is critical to collect information as illustrated in Tables 5
ACCEPTED MANUSCRIPT and 6. Finally, as standard practice, these details should be published in the Methods Section of articles.
Formulation,
administration
routes,
dosing
and
considerations
other
pharmaco-toxico-kinetic
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7.
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Many relevant pharmacokinetic parameters are sexually dimorphic as described in several extensive reviews [33, 37, 38, 108, 329]. However, a detailed review focused on drug
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administration issues in animals dedicates only a few words to sex-gender differences [245],
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indicating that awareness of the importance of sex-gender in this specific topic is still neglected. Attempts have to be made to improve this situation, and consideration of the specific condition of females is required. recently , the Food and Drug Administration
has decreased the initial
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recommended dose of zolpidem in women [29].
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7.1 Formulation and routes of administration
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After selection of a molecular entity, pre-formulation activities should be performed to determine its physical and chemical properties. At this stage, the route of administration intended
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for the clinic should be identified, because each route of administration requires special formulation. In drug formulation, the active compound is mixed with other chemical ingredients to create the drug product. Recently, it has emerged that excipients may exert sex–specific effects. In particular, polyethylene glycol enhances the bioavailability of ranitidine in men (up to 63%), whereas polyethylene glycol decreases ranitidine bioavailability in women (up to 24%) [330], showing the fallacy of assuming that males and females handle inactive ingredients similarly. Furthermore, some inactive ingredients may have different safety profiles in males and females. For example, we recall that cellulose and hydroxypropyl methylcellulose exert their adverse reproductive and/or developmental effects in a manner that appears to be sex-gender–dependent, at
ACCEPTED MANUSCRIPT least in non-clinical studies [331]. In conclusion, the formulation of a drug should consider both sexes, both to improve the efficiency and effectiveness of care and to improve safety profiles.
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7.2 Dosing
The correct selection of an optimal dose is critical for establishing the efficacy of a
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potential therapeutic agent. Ideally, therapeutics should to achieve the right dose, of the right drug,
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for the right time, in the right subject. Choice of dosage regimen depends on drug pharmacokinetic and on the individual. Pharmacokinetic-pharmacodynamic (PK-PD) modelling is essential to
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evaluate the plasma concentration and effects of the compound, including the homeostatic feedback mechanisms that might modulate the response and the interaction of the drug effect with disease processes. All pharmacokinetic parameters are affected by different covariates, including sex and
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gender [332].
Drugs are generally administered according to fixed dosing. Usually, in a clinical setting,
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the recommended adult dose of a drug is based on the assumption that the subject is an adult
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Caucasian man that is 173 cm tall and weighs 70 kilograms [22], forgetting that many people, including women, do not fit into this category. Women are often significantly smaller in height and
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weight and also have a different body composition than men. Alternative weight descriptors (BOX 1) such as ideal body weight, adjusted body weight, fat-free weight, and lean body weight are used to prevent drug overexposure observed with weight-based dosing, but their benefits and limitations must be understood [333, 334]. In calculating a dose, blood and plasma volume should have a role. Although there are a few contradictory studies [277, 335], it has been generally noted that in humans and in rats, depending on various breeds and ages, as in many other animal species, females have a lower blood volume and haematocrit than males. Therefore blood and plasma volume are different in males and in females [192, 275, 278, 336, 337]. According to Probst et al. [192], it appears advisable, if the individual assessment of plasma and blood volumes is not possible, to consider that plasma volume
ACCEPTED MANUSCRIPT is at least 10% greater in female rats in comparison with male ones. Plasma and serum volume of females change as function of reproductive life stage and menstrual or oestrous cycles [338]. Finally, men have a larger total blood volume (by about 6–8%) than women [286].
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The oral route is the most common route for drug administration. Drug cans be administered by gavage, or alternatively, medications can be dissolved in drinking water or food. Water and food
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are often given ad libitum. The amount of ad libitum water and food actually consumed must be determined before any medications are added to the drinking water and food. It is necessary to
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determine whether any added compound changes fluid and food intake by altering taste, smell, or
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other factors. If we wish to know the dose administered to males and females, we should consider that females and males could have different daily intake rates of water and food. Interestingly, daily water intake is significantly higher in adult female rats compared with adult males rats, and daily
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consumption of water is indexed to body weight [339].
Obviously, the use of an oral route should consider the multiple differences between males
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and females in gastric and intestinal systems, recently described in a clear review [109], because
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these differences affect oral bioavailability, which greatly influences dosing. Less is known about sex-gender differences in bioavailability, which can be species-specific. For example, female rats
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have higher oral bioavailability than males following administration of the same dose (mg/kg) of αthujone by gavage, while this difference is not present in mice [340]. The differences in bioavailability are partially attributable to CYP enzyme levels and to specific enzymes of phase 2 metabolism, predominantly in terms of different enzyme activity [33, 37, 38, 108, 329]. Indeed, it has recently been shown that metabolism of PF-02341066, a selective c-Met/Alk tyrosine kinase inhibitor, occurs predominantly through oxidation in the male rat liver and through sulfo-conjugation in the female rat liver [341], indicating that sex-gender differences can be either qualitative or quantitative due to the major activity of a single isoform of the enzymes, or to the activity of different enzymes. Sex difference in CYP enzyme levels may be significant enough to warrant different dosing strategies, especially for drugs with a narrow therapeutic index.
ACCEPTED MANUSCRIPT Sex-gender variations in microsomal enzyme activities are of relevance in the process of drug discovery. Recently, the Ministry of Health, Labour and Welfare of Japan issued a guidance document recommending that induction studies in rats should be conducted in females rather than in
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males, as the magnitude of CYP enzyme induction tends to be greater in females [342]. Drug
during the menstrual cycle or with the use of OC [108].
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metabolism can vary in the different phases of women’s lives (pregnancy, puerperium) [343] and
Another source of variation in bioavailability is liver blood flow, which is faster in male rats
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than in female rats [344].
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Distribution of drugs is influenced by blood proteins, especially albumins, which conjugate numerous drugs. However, this bond is not constant and can be changed in numerous clinical settings such as hypoalbuminaemia observed in critically ill patients [345, 346] and pregnancy [343,
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347]. Unbound free drug has therapeutic and potentially toxic activity [348] and affects drug penetration into tissues [349, 350], drug elimination and metabolism [351, 352]. Drug distribution
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depends on body composition, which is essentially sexually dimorphic as described in numerous
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reviews [108, 201]; females have more fat than males. This fact affects the distribution of medications having lipophilic properties, causing a greater distribution in females than in males,
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while the reverse is true for hydrophilic medications. Evidence suggests that there are significant differences in renal physiology between male and female animals, including humans, due to a sexual dimorphism in vascular responsiveness and in expression and activity of renal transporters [353]. The acknowledgement of renal sex differences is of special importance, because drug elimination occurs mainly by renal route and depends on GFR, tubular secretion and tubular reabsorption. In rats, whole kidney GFR and single nephron GFR are higher in males than in females [265-267]. However, most findings show that, when the higher body weight or kidney weights of males are considered as indices of renal function, the sex differences diminish [266]. In addition, human and animal females drink more and excrete a greater volume of less concentrated urine [261-263, 354]. We recall that tubular secretion mainly occurs
ACCEPTED MANUSCRIPT through a wide variety of active transporters that could work in a sex-dependent manner [33]. Sexgender differences in the expression of Oat1 and Oat3 are present in the kidneys of rodents in that mRNA expression levels of Oat1 and Oat3 are higher in male adult rats than in female ones [355].
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These differences are enhanced by androgens and inhibited by oestrogens [356]. Notably, the renal expression of numerous cytochrome P-450 enzymes, which are involved in the metabolism of
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xenobiotics, is sex-gender-dependent in mice and rats, whereas the enzymes of the phase II system present only few sex-gender differences [357]. Finally, we remember that renal function is strongly
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influenced by age, and the age-related decline in GFR is slower in female rats than in male rats,
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irrespective of strain [358], suggesting that in old rodents, these differences may be lower than in young animals. Renal sexual differences may influence dosing of drugs, drug–drug interactions, and drug-induced nephrotoxicity. Traditionally, renal function has been measured using plasma
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creatinine and calculating creatinine clearance, but estimated creatinine clearance predominantly measures GFR. However, at least in humans, there is evidence of a poor correlation between
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estimated creatinine clearance and renal drug clearance [359], necessitating dosage adjustment not
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just based on GFR.
In conclusion, the dosing of a drug is influenced by sex at different levels, and each drug
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should be carefully evaluated in order to find the right regime for male and female animals.
8. The choice of test and animal models of diseases The use of animals in biomedical research dates back at least two millennia when Galenus dissected Barbary macaques and made physiological experiments in pigs, goats, and sheep [360]. Today, animal testing is relevant in many fields including pharmacological testing for drugs; therefore, is crucial to understand and to validate these tests for each sex. For example, in humans and in rodents, sex-related differences are found in the performance of spatial learning and memory tasks [361-364]. Furthermore, males tend to guide navigation using geometric configuration, whereas females seem to attend more to landmark cues [43, 365]. Therefore, when results obtained
ACCEPTED MANUSCRIPT in animals of both sexes are compared, the comparison should include the description of physiological differences and how these are included in the analysis of the results. This is relevant because animal models of human diseases retain a crucial role in
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understanding the biological basis of disease and are of special interest to test efficacy and toxicological profiles of drugs. In spite of this, the vast majority of studies employing models of
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diseases, including transgenic ones, use male animals [146, 366, 367]. However, numerous
therefore, both sexes should be included in studies.
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differences exist between males and females in different models of animal diseases [368-370];
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The inclusion of sex and gender as variables should occur with great caution on the validities of extrapolation to humans, and the extrapolation range should satisfy the more severe criteria of Russel [371], who demonstrated that knowledge obtained in animals is relatively
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predictive in humans. In this respect, each model in a single species should be considered separately. For instance, in mice, in contrast to humans, more atherosclerosis is seen in females than
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in age-matched males [368, 372-374], whereas female macaques are relatively resistant to
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atherosclerosis in comparison with males [375]. In contrast to humans, in numerous models of type 2 diabetes mellitus, male animals are more susceptible to diabetes and have more severe disease
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than females [40, 376]. Additionally, in some animal models, particularly in the rat, females show less ischemia-reperfusion injury; however, this is not observed in all animal studies [377]. As stated previously, psychosocial phenomena have a profound influence on the disease process, and they should also be considered in animal models of diseases if adopting a sex-gender research strategy. Again, it is not possible to have a global policy on these matters, because it is necessary to consider each model separately. The described relative resistance of female macaques to atherosclerosis [375] also depends on social status, because only dominant female animals are less susceptible to atherosclerosis in comparison with males. The female resistance to atherosclerosis seems to be linked to oestrogens, because atherosclerosis is exacerbated in ovariectomised monkeys and suppressed by pregnancy and OC [450]. Indeed, a successive study
ACCEPTED MANUSCRIPT showed that dominant macaques, under conditions of social stress, develop exacerbated coronary artery atherosclerosis, and this exacerbation is attenuated by a beta-adrenergic blocking agent [378], revealing the role of sympathetic system. Thus, in female and in male macaques, the susceptibility
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to atherosclerosis is associated with different mechanisms.
The importance of social factors is further highlighted by experiments performed in
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cynomolgus monkeys. Female cynomolgus monkeys housed individually have a more adverse plasma lipid profile and more extensive coronary atherosclerosis than those socially housed when a
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chronic lipid-rich diet is administered [379]. Interestingly, only female but not male rats born from
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mothers fed a soya diet with lard throughout pregnancy and lactation have an elevated diastolic and systolic blood pressure as adults, while endothelium-dependent relaxation by acetylcholine is blunted in both sexes [380].
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There are numerous models of heart failure [381], and some of these present sex-gender differences. In general, female mice undergo less extensive ventricular remodelling than males
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[382], and this is of relevance for the design of experiments. Notably, sex-gender differences have
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been found in spontaneously hypertensive heart failure-prone rats with defective leptin receptors, which, at a relatively young age, first develop obesity followed by heart failure [383]; male rats are
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more prone to heart failure than females in this model [383, 384]. Sex-gender differences in blood pressure have been described in animals as well as in humans [385-387]. Hypertensive men are also at greater risk for developing cardiovascular and renal complications than hypertensive women [386, 387]. Dahl salt sensitive rats are a mutant strain of Sprague–Dawley rats that exhibit genetic hypersensitivity to sodium intake [388]. In this model, female rats are less hypertensive after sodium intake than male rats. A greater rise in blood pressure has also been reported in spontaneously hypertensive female rats after ovariectomy [389, 390]. There are numerous animal models of depression, and most of these have been developed in male rodents and were only later applied to female rodents [366]. Despite their limitations, these models show numerous sex differences in the neurobiology of depression and antidepressant
ACCEPTED MANUSCRIPT response [146, 366]; however, some of these models may not be appropriate for female rats. Some of the differences observed depend on baseline sex-gender differences, while other differences only emerge after stress exposure or drug administration. Differences have been described in learned
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helplessness models, chronic mild stress, forced swim tests, Flinders sensitive line and lipopolysaccharide-induced sickness behaviour. Overall, the data indicate that sex differences are
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present in the phenotype (behaviour) and/or in the endophenotype (neurobiology) of depression [366].
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In conclusion, there are a number of diseases models, but not all are suitable for every
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experimental protocol in terms of sex-gender differences. In some cases, the model of disease is appropriate only for one sex-gender (as is the case for many models of type 2 diabetes mellitus), indicating that data obtained in this model cannot be extrapolated to females. The identification of
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appropriate animal models that can be used to screen gender-based differences is urgent [40, 42, 146, 292] to emphasise the value of translational or integrational research [40]. In addition, it is also
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urgent to evaluate the influence of the oestrous cycle phases, pregnancy and lactation on diseases
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9. Conclusions
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[391-397].
In the past several years, a series of papers has illustrated the wide scope of sex-gender research. They have clearly demonstrated that sex-gender differences are fundamental, complex, and long-lasting and have underlined that sex-gender research is a complex scenario that needs to consider interdisciplinary and intersectorial aspects. There is enough evidence that sex-gender affects many outcomes in life science research, and the need exists to embed the so called “gender dimension” into basic and clinical scientific research. These studies also stress the need of a broad approach to gender mainstreaming. According to Australian Bureau of Statistics sex-gender is a basic complex variable, not a confounder, starting from pre-analytical phases of research [398]. Here, it has been shown that the
ACCEPTED MANUSCRIPT pre-analytical phase is influenced by sex and gender, and data coming from males and females should not be pooled to increase statistical power. To avoid any bias in research, a team should include men and women and should
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communicate to minimise or eliminate many variables and to foster better science and animal wellbeing. The team must strive to minimise or eliminate non-protocol variables that could
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adversely affect the validity and repeatability of the experimental data. Harmonising national
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statistics on animal use patterns will allow rational priorities for reduction and refinement research to be identified internationally.
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Journal editors should encourage the inclusion of sex-gender information in “Materials and Methods” sections as standard practice. It is of importance that results be published, even if they are
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not statistically significant or if they are negative, to avoid conducting redundant studies.
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10. Future perspectives
In the era of individualised medicine, it is evident that to be male or female impacts how an
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individual will respond to or metabolise a particular drug regimen. In our opinion, it is clear that male and female animals and organs, cells and organelles from male and female animals should be
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utilised to screen drugs, devices and procedures in order to provide gender-based medicine which could lead to novel therapeutic approaches and strategies that could improve the appropriateness and the safety of therapy. Indeed, it is also plausible to open new field of research to understand the whole complexity of gender medicine. For example, maternal foetal microchimerism (the results from bi-directional transfer of cells across the placental barrier in pregnancy) [399, 400] is found in numerous species including humans [401]and should be considered when enrolling post-pregnant animals or women in studies, because these cells can contribute to female physiology. Indeed, male progenitor cells (CD34+ and CD34+ CD38+) remain in women’s blood for several decades after pregnancy [402,
ACCEPTED MANUSCRIPT 403], participating in the development of autoimmune disease in multiparous women [404-407] and in regeneration of damaged tissues [407, 408]. Considering that biological and social factors are age-dependent [409-411], sex-gender and
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age should be considered together without forgetting life in utero. In fact, the majority of female and male animals used in biomedical research analyses are young. Furthermore, females have a
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regular reproductive cycle and often have not been pregnant before use in biomedical research. These restrictions of parity and age may influence the results of the experiments, making the
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findings inapplicable to human populations in which older individuals may predominate and
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childbearing is normal. Longitudinal studies are required to understand sex-gender differences throughout the lifespan, including the different phases of female life. Furthermore, experiments should be conducted in females in each oestrus or menstrual cycle phase and, if not practical, the
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cycle phase should be indicated in the methods section.
It appears urgent to have information on pregnancy and on neonatal life, because these
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periods are fundamental for the plasticity of development in both animals and humans. Notably,
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stressors can exert effects in a sex-gender specific manner. The widespread human use of OC suggests that a female experimental group should always
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be treated with oestrogen and progestin drugs or with other hormonal blockers of ovulation. The presence of different OC formulations in the market could indicate that more than one specific formulation could be used. This point is relevant, especially considering the influence of OC on physiological parameters [32], drugs, xenobiotic metabolism and receptors [42, 108]. Finally, papers should contain a complete description of the experiments [412].
ACCEPTED MANUSCRIPT Box 1: Pharmacokinetic Size Descriptors
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Total body weight (TBW): subject’s real weight (kg).
Body mass index (BMI) or ‘Quetelet’s Index’ TBW (Kg)/height (m2) is now the international
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metric recommended to classify obesity and overweight. This formula utilises body weight and
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height. However, it cannot differentiate adipose tissue from muscle mass. Relevantly, BMI is not gender-specific, because it was not derived from women and its predictive value for morbidity has
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not been evaluated in women [413].
Lean body mass or Ideal Body Weight (IBW) considers height and sex; however, it does not take
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into account differences in body composition. The lean body mass, which is similar but not identical to fat-free mass, may be a better basis for drug dosage than either TBW or body surface area,
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although the rationale for this is not clear. Lean body mass in humans can be calculated by Devine
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estimation [414], and lean body mass can be calculated by Robinson estimation [415]. Distribution volume of relatively hydrophilic drugs has a good correlation with LBM, but for lipophilic drugs,
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the correlation is better between volume of distribution and total body weight than with lean body weight. Thus, although most drugs are dosed using body weight, some medications are best dosed using IBW or a combination of IBW and actual body weight, particularly in obese patients [416418]. Finally, lung capacity correlates best with lean body mass rather than actual body weight [416].
Body surface area is used for the dosing of the antineoplastic agents and is based on the assumption that height, TBW and a constant C are linked to body surface. Like BMI, body surface area does not consider the sex [413]. It can be also calculated by the Mosteller formula [419].
ACCEPTED MANUSCRIPT Adjusted Body Weight (ABW) is the first size descriptor developed for pharmacokinetic studies [420]. It uses a correction factor to apply to IBW (IBW + CF (TBW- IBW)), and the choice of adjustment depends upon the drug and the clinician’s judgment. ABW, however, seems a plausible
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size descriptor as it considers sex, TBW and height [413].
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Fat-free mass (FFM) relates weight and fat mass. The metric was derived in guinea pigs, where the live weight and eviscerated wet and dry weights were used to determine the total fat mass of the
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animal. This descriptor depends upon sex, TBW and height [413].
Percent total body surface area (TBSA) is calculated through different formulas. Regarding animals, most formulas are based on the original Meeh Formula (TBSA= ¼ kW 2/3) with K being a
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constant that is empirically determined and varies greatly by species and size. For rats, values of K constants range from 9.00 to 11.36 [421]. The constant calculated in thirty five-month-old female
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Wistar rats weighing 195–240 g is 9.83.
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Formulas are referred to humans
ACCEPTED MANUSCRIPT 11. References [1] Enomoto A, Endou H. Roles of organic anion transporters (OATs) and a urate transporter (URAT1) in the pathophysiology of human disease. Clin Exp Nephrol. 2005;9:195-205.
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[2] Wizemann T, Pardue M. Exploring the biological contributions to human health: does sex matter? . Washington: National Academy Press; 2001.
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[3] Legato MJ. Principles of gender-specific medicine. 2 ed. Amsterdam; Boston: Elsevier Academic Press; 2009.
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[4] Regitz-Zagrosek V. Sex and gender differences in pharmacology: Springer; 2012.
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hepatic cholesterol 7alpha-hydroxylase activity and mRNA abundance by dietary soluble fiber in the guinea pig. Atherosclerosis. 2002;163:29-37. [438] Lamota L, Bermudez-Silva FJ, Marco EM, Llorente R, Gallego A, Rodriguez de Fonseca F, et al. Effects of adolescent nicotine and SR 147778 (Surinabant) administration on food intake, somatic growth and metabolic parameters in rats. Neuropharmacology. 2008;54:194-205. [439] Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerotic lesions and HDLlipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H. Atherosclerosis. 1987;64:215-21. [440] Onyebuagu PC. Xylopia aethiopica lowers plasma lipid precursors of reproductive hormones in Wistar rats. IJHPR. 2013:48-53.
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metabolism in analbuminemic rats. Kidney Int. 2000;57:2268-74.
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[442] Shearer GC, Joles JA, Jones H, Walzem RL, Kaysen GA. Estrogen effects on triglyceride
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[443] Bennett CF, Kornbrust D, Henry S, Stecker K, Howard R, Cooper S, et al. An ICAM-1
Pharmacol Exp Ther. 1997;280:988-1000.
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antisense oligonucleotide prevents and reverses dextran sulfate sodium-induced colitis in mice. J
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[444] Vital P, Larrieta E, Hiriart M. Sexual dimorphism in insulin sensitivity and susceptibility to develop diabetes in rats. J Endocrinol. 2006;190:425-32. [445] Ribot J, Rodriguez AM, Rodriguez E, Palou A. Adiponectin and resistin response in the onset
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of obesity in male and female rats. Obesity (Silver Spring). 2008;16:723-30. [446] Noonan WT, Banks RO. Renal function and glucose transport in male and female mice with
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diet-induced type II diabetes mellitus. Proc Soc Exp Biol Med. 2000;225:221-30.
Sci. 1991;16:49-59.
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[447] Ikeuchi J, Yoshizaki T, Hirata M. Plasma biochemistry values of young beagle dogs. J Toxicol
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[448] Schuurman HJ, Smith HT. Reference values for clinical chemistry and clinical hematology parameters in cynomolgus monkeys. Xenotransplantation. 2005;12:72-5. [449] Matsuzawa TN, Y. Comparative haematological and plasma chemistry values in purpose-bred Squirrel, Cynomolgus and Rhesus Monkeys. Comparative Haematology International. 1994;4:43-8. [450] Diez M, Michaux C, Jeusette I, Baldwin P, Istasse L, Biourge V. Evolution of blood parameters during weight loss in experimental obese Beagle dogs. J Anim Physiol Anim Nutr (Berl). 2004;88:166-71. [451] Bacakova L, Mares V, Lisa V. Gender-related differences in adhesion, growth and differentiation of vascular smooth muscle cells are enhanced in serum-deprived cultures. Cell Biol Int. 1999;23:643-8.
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[453] Murphy JG, Khalil RA. Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat. Am J Physiol Cell Physiol. 2000;278:C834-44.
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contraction is associated with increased estrogen receptor expression in single vascular smooth muscle cells of female rat. Cell Physiol Biochem. 2010;26:457-70. [456] Hogg ME, Vavra AK, Banerjee MN, Martinez J, Jiang Q, Keefer LK, et al. The role of
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[457] Woodrum DT, Ford JW, Ailawadi G, Pearce CG, Sinha I, Eagleton MJ, et al. Gender
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differences in rat aortic smooth muscle cell matrix metalloproteinase-9. J Am Coll Surg.
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[458] Matarrese P, Colasanti T, Ascione B, Margutti P, Franconi F, Alessandri C, et al. Gender disparity in susceptibility to oxidative stress and apoptosis induced by autoantibodies specific to RLIP76 in vascular cells. Antioxid Redox Signal. 2011;15:2825-36. [459] Osborn CA BJ. Influence of fasting and eating on laboratory values, in Bonagura JD. In: W.B. Saunders Co., editor. Kirk’s Current Veterinary Therapy. Philadelphia, PA1992. p. 20-3.
ACCEPTED MANUSCRIPT
M/F
M/F
M/F
WISTAR RATS
SD RATS
C57BL/6 MICE
M/F
M/F
M/F
M/F
BALB/c
CD1 MICE
NZ RABBIT
WM GUINEA
SC RI
PARAMETER
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Table 1. Effect of sex on some biochemical parameters of rodents
= [422, 423];
= [426-429]
+/-[422]
+/-[424]
+/-[430]
-/+[431]
=[427]
=[439]
-/+ [425] HDL
= [423, 438]
-/+ [425]
TG
-/+[422]
=[426-428]
=[423, 438] +/-[424, 425, 440]
=[432]
-/+[435]
+/-
+/-[433]
=[436, 437]
-/+[435]
-/+[435]
-/+[437]
-/+[435]
-/+[436]
=[434]
=[437]
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= [423, 425] +/-[424]
=[433, 434]
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HDL2 LDL
+/-[439]
+/-
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= [425] HDL1
+/-[271, 431]
PIG
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+/-[424]
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Tot-Chol
NU
MICE
+/-*[441] +/-[430, 442]
=[422]
+/-[271]
=[432]
ACCEPTED MANUSCRIPT Bilirubin
= [422]
= [426, 428]
=[422]
-/+[430]
-/+[243]
+/-[431]
=[432]
=[436]
ALT
= [422]
+/- [426, 428]
PT
+/-[431] = [243, 422]
AST
-/+[422]
+/-[426, 428]
SC RI
= [430]
+/- $[431]
=[243, 422]
+/-[422]
+/-[426, 428,
=[422]
430]
-/+[243]
=[426, 428]
-/+[422, 431]
444, 445]
+/-*[441]
+/-[446]
+/-[424]
+/-[430]
=[423, 438, 445]
=*[441]
Urea Uric acid
-/+[422] -/+[422] =[422]
-/+[426, 428]
=[422]
=[430]
-/+[243]
-/+[426]
=[243, 422]
=[428, 430]
-/+[431]
+/-[430]
=[422]
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Creatinine
=[280]
-/+[436]
=[280, 434]
=[436]
+/-[280]
+/-[436]
=[446]
CE
-/+[444]
=[436]
ED
=[422, 423, 438,
+/-[424]
=[280]
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Insulin
=[436]
-/+ *[431]
+/-[431] Glucose
= [432, 443]
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AP
=[280]
-/+ [432]
NU
=[430]
= [443]
= +/-[431] -/+*[431]
+/-[436] -/+
SD=Sprague-Dawley, NZ= New Zealand, WM= Weiser-Maples, HDL = high density lipoprotein, LDL= low density lipoprotein, TG= Triglycerides, ALT= Alanine aminotransferase, AST= Aspartate aminotransferase, γ-GT= Gamma-glutamyltransferase, AP=Alkaline phosphatase, *= old animals, $ = 1-3 month old, & = 9 month old, = 6 years old
ACCEPTED MANUSCRIPT
Table 2 Effect of sex on some biochemical parameters of rodents M/F
BEAGLE DOGS
CYNOMOLGUS MONKEYS
Tot-Chol
= &[447]
=[448]
TG
= &[447]
-/+[448] =[449]
Bilirubin
-/+[448] =[280]
=[448, 449]
AST
=[280]
=[449]
AP
=[280]
+/-[449]
+/- &-[447, 450] =[280, 447]
Creatinine
=[280, 447] = &[447]
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-/+ [450]
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Glucose
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ALT
Urea
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M/F
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PARAMETER
=[449]
+/-[448]
=[448, 449]
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* = old animals, $ = 1-3 month old, & = 9 month old, = 6 years old
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Table 3 Distribution of the sex of cells in publications Unspecified sex 75%
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Female Cells 5%
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Male cells 20% Data from [24].
ACCEPTED MANUSCRIPT Table 4 Examples of sex differences in aortic VSMC obtained from male and female rats
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Protein expression of ER alphab
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Protein expression of ER betab
M/F +/+/+/+/+/+/-/+ = -/+ -/+ +/= +/+/+/-/+ -/+ = -/+ = -/+ +/+/= +/= +/-/+
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PARAMETERS Basal proliferationa Proliferation in serum-free mediuma,c Cell migrationc,d Basal apoptosis Apoptosis induced by radiationb Basal autophagy Senescence induced by radiationb Adhesion in normal serum mediuma Adhesion in low serum mediuma Adhesion in serum-free mediuma Basal cell contractiond Basal [Ca2+](i)c H2O2 production Superoxide anion production 4-HNE GSH Total proteina mRNA expression of ER alpha and betab
[451] [451] [452] [290] [290] [290] [289] [290] [451] [451] [451] [453] [290] [290] [290] [290] [454] [451] [455] [452, 456] [455] [291] [289] [455, 456] [291] [291, 455] [457] [451]
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Protein expression of GPER Tissue inhibitor of metalloproteinase-1 gene expressionb Content of alpha-actin, vimentin, alpha(v) integrin and mesh-like networks of alpha-actin microfilamentsa RLIP76 expression [458] -/+ a b c Wistar specific pathogen free rats, Sprague-Dawley rats, Spontaneously hypertensive rats, d Wistar Kyoto rats, ER: Oestrogen Receptor, ET: Endothelin, lysoPC: lysophosphatidylcholine
ACCEPTED MANUSCRIPT Table 5 Questionnaire to be administered to human donors Sex Age Actual weight Birth weight Actual height Birth height Race Place of birth Place of house
Lifestyle
Physical activity: how many times a week and type Smoker (yes/no; how long and how many cigarettes per day) Alcohol use (yes/no; type; how many cups per day)
Social status
Occupation Level of education (years) Care giver (hours/day)
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Demographic information
Blood pressure at the time of sampling Diseases at the time of sampling and in the previous 3 months Drug use at the time of sampling and in the previous 3 months (drug type, duration of treatment, generally the use of drug is an exclusion criteria ) For women: Oral contraceptives use (how long and type) Date of last menstruation and menstrual cycle phase Pregnancies (number, type) Abortions (number and which month of pregnancy)
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Health status of subjects
Blood collection
Fasting subjects * Time of sampling (hour) Site of collection, anticoagulants, In fertile women, blood should be collected in the same period of menses cycle *Ingestion of food and of some beverages can influenced the composition of blood, plasma, and serum [459]
ACCEPTED MANUSCRIPT Table 6 Questionnaire to be administered to professionals before the use of animals or animal cells Sex Age Actual weight Actual height Species
Environmental information
Diet type and parent diet Cross Fostering Maternal care and weaning Travelling, stabilisation, acclimatisation period Housing, isolation, spacing and environmental enrichment
Health status of subjects
Evaluation of health status
Blood collection
Fasting subjects * Time of sampling (hour) Site of collection, anticoagulants, blood volume, anaesthesia restraint, handling Blood should be collected in the same oestrous phase determined by vaginal examination
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Demographic information
Cells and tissues
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Table 7. Influence of sex-gender on the expression of housekeeping proteins beta tubulin, alpha actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in different cells and tissues. Beta tubulin
Alpha actin
GADPH
References
Human VSMCs from artery of umbilical cord
=
=
=
Franconi et al personal communication
Human Monocytes
=
NA
NA
Franconi et al personal communication
= + males + males + males
NA = +females =
NA = + females + males
Franconi et al personal communication [7] [7, 315-317] [7]
Human monocyte-derived macrophages Rat heart Rat liver Rat kidney NA=not available
ACCEPTED MANUSCRIPT Manuscript Title: Need for gender-specific pre-analytical testing: The dark side of the moon in
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laboratory testing
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List of all Authors: Flavia Franconi, Giuseppe Rosano, Ilaria Campesi
Corresponding Author: Flavia Franconi, Dipartimento di Scienze Biomediche Via Muroni 23,
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Sassari, Italy ; e-mail [email protected], phone +39079228717
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Highligths
Numerous methodological problems appear considering the sex-gender in experiments We discuss pre-analytical aspects of research
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Gender-specific pre-clinical research will produce more evidence-based medicine