Elsevier

International Journal of Cardiology

Volume 179, 20 January 2015, Pages 514-535
International Journal of Cardiology

Review
Need for gender-specific pre-analytical testing: The dark side of the moon in laboratory testing

https://doi.org/10.1016/j.ijcard.2014.11.019Get rights and content

Highlights

  • Numerous methodological problems appear considering the sex–gender in experiments.

  • We discuss pre-analytical aspects of research.

  • Gender-specific pre-clinical research will produce more evidence-based medicine.

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 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 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 of research could affect the predictive value of all analyses. Furthermore, the standardisation of 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 pre-analytical aspects could produce a decrease in the time for translation from the bench to bedside. Furthermore, sex-gender-specific pre-clinical pharmacological testing will enable adequate assessment of pharmacokinetic and pharmacodynamic actions of drugs and will enable, where appropriate, an adequate gender-specific clinical development plan. Therefore, sex-gender-specific pre-clinical research will increase the gender equity of care and will produce more evidence-based medicine.

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 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, 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 sciences [2], [3], [4], [5]. In the last few years, it has emerged that sex–gender differences start in utero [6] and are organ specific [7] and cell specific [8].

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 [9], cancer, depression and brain disorders, and infectious, cardiovascular, renal, hepatic, pulmonary, inflammatory and autoimmune diseases [3].

Indeed, the biological differences between sexes should be considered across the entire range of research, starting from the pre-analytic conditions embodying research in genetics, epigenetics [10], developmental biology, biochemistry, physiology, pharmacology, toxicology, and 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], [13], [14]. In this regard, we have to highlight that the 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 indicate only female sex [15]. This viewpoint is widespread, and some studies that mention the word gender in their title incorrectly use the term as a synonym for women [16], [17]. Of course, this attitude has important consequences, and men's gender-specific needs may not be sufficiently considered.

Furthermore, there is a relevant debate regarding the stability and validity of the binaries of 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 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 instead the use of the term sex–gender. Thus, through this review, the term sex–gender will be used 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 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].

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].

Overall, existing knowledge suggests that:

  • a)

    it is inappropriate to assume that results obtained only in one sex can be applied to the other [30]:

  • b)

    sex–gender demands a lot of attention in the construction of measurements and variables, especially because of different body sizes and body composition of males and females;

  • 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 functions of the organs, cells, biomarkers and indicators [31], [32];

  • e)

    and, finally, sex–gender is not adequately considered in preclinical and clinical interventions involving diagnostics, medical devices, medications and clinical setting, and yet it is 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 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 males and females can improve the prevention, the efficacy, the safety and the appropriateness of the treatments [3], [33], [37], [38], [39].

A certain number of papers on methodological issues in sex–gender research are available [26], [28], [40], [41], [42], [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 models of diseases and issues that sex–gender research meets after sampling [26], [28], [40], [41], [42], [43], neglecting pre-analytic issues, which also include the influences of environment, with few exceptions [23].

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 [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 processing) are of critical relevance and importance, being encompassed in all sample preparations. 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, fasting, stress, restraint, analgesia and anaesthesia, dosing, diet, etc.)

  • b)

    actions performed at collection (time of collection, blood sampling, collection technique, amount of blood, etc.)

  • c)

    actions performed on the specimen such as dimension of collection tubes, sample separations, storage time, and temperature.

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 sex-gender research, we include in the pre-analytical aspect the importance of the professionals and researchers' sex–gender, 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 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-analytic variables that have a special interest in sex–gender research through the lens of gender.

Section snippets

Complexity of sex gender research

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 complex and numerous and may affect the manner in which sex–gender influences outcomes in 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].

Effects 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

Experimental design

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.

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 male mouse foetus that develops between female foetuses is more sexually active as an adult, less aggressive

Action performed at collection of the samples

One of the most critical issues in biomedical research is sample preparation [231], [232]. Blood samples are used widely, and blood is generally collected via venipuncture in order to obtain cells and cellular fragments, plasma and serum.

Formulation, administration routes, dosing and other pharmaco-toxico-kinetic considerations

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 administration issues in animals dedicates only a few words to sex–gender differences [245], 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.

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, it 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], [362]

Conclusions

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

Future perspectives

In the era of individualised medicine, it is evident that to be male or female impacts how an 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 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

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.

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