Print This PostLet’s be honest, today’s article was just an excuse for me to use that line as a title. OK so that’s probably 70% true. I’ve genuinely been doing a lot of work lately that involves looking at the differences between the sexes and there’s been a glut of new literature in different diseases and models that highlights some of these differences so today we’re going to talk about sex, and why considering sex as a factor in your studies is really important. We might also brush over some light sexism whilst we’re at it but that’s by the by. It’s basically a whistle-stop tour of all the things you ever wanted to know about sex but were afraid to ask.
We can start nice and simple with some basic factoids. Women tend to live longer than men. Age is a significant risk factor for dementia and as such, women are at higher risk of developing dementia than men. According to the Alzheimer’s association there are twice as many women with Alzheimer’s as men. The picture will become more complicated as we delve deeper but we’ll stick to those basics for now. With stroke, because I have to include it, the pattern varies by age. Before the age of 30 the risk of a stroke is higher in women. After the age of 30 it’s higher in men until you reach menopause, then it evens out.
In clinical practice, understanding the basic differences between men and women and the consequences of those differences are really important. Let’s start with the really basic example of heart attack. The classic image of a heart attack victim is a man in a suit from the stock exchange in the late-80s, clutching the left side of his chest and his left arm. The message was always that heart attacks cause shooting pains down your left arm, but we now know this is relatively rare in women. They range from something that feels like heartburn, to something that feels like diffuse back pain. And missing a heart attack because you don’t know that there’s a difference between how men and women present is going to affect the clinical pathway that person goes down.
In dementia, the differences are more subtle but may be equally important. A 2021 JAMA article by Levine and colleagues took a large number of longitudinal cohort studies and extracted data from them to determine whether there was an effect of sex on cognitive decline. They found that women and men lost memory at a similar rate, but that women were more likely to have generally higher executive function and general cognitive capacity and that they lost it at a faster rate than men. This means that the first signs of cognitive decline in men and women may not be the same. And, like in the heart attack scenario, this is going to affect clinical pathways.
As you can see, sex is complicating things.
Despite having known this for some number of years, in pre-clinical research we’ve traditionally used cohorts of young male animals. Female animals have this messy thing called an oestrous cycle which just gets in the way and makes your data really noisy and why on earth would we want that?? Fortunately, attitudes have taken a big shift recently and most grant funders (at least in the UK) want you to justify yourself if you are choosing to only use males.
The reticence of most people in using both sexes is financial. They assume that if their study is powered for six males per group that suddenly to do the experiment ‘properly’ they’re going to have to use twelve animals which will double their costs. Which is simply not true in the majority of cases. Here I’m going to recommend the first paper of the day by Benjamin Phillips, Timo Haschler and Natasha Karp in PLOS Biology. The title is nicely self-explanatory: ‘Statistical simulations show that scientists need not increase overall sample size by default when including both sexes in in vivo studies’. They highlight the use of the acronym SABV, or Sex As Biological Variable, introduced by the NIH and adopted by many UK funding bodies. This basically says that you should just assume you’ll use both sexes and then include sex as a variable in your analyses, if it comes out as different, so be it.
Sex is important and complicated, and often overlooked as a factor by researchers.
In fact, the authors highlight something really important here. They use a variety of models to test different sample sizes, experimental set-ups and outcomes and they find that ‘Rarely, large interactions in the data may produce an appreciable decrease in treatment effect power. In these scenarios, we would argue the knowledge gained that the treatment has a differential impact between sexes outweighs the statistical loss of power.’ Which is an important translational point. If your treatment doesn’t work as well, or works better, in females this is something that you need to know.
But what’s driving these differences? Well, the answer to that is actually enormous and would take a very large review article to cover but in the very, very simplest terms the answer is genes and hormones.
We’ll start with some hormone stuff because it’s slightly easier. I’ll give you some examples of important differences which will give you something to chew on for your own work. We’ll stick to stroke here just because the effects are, on the face of it, a bit more obvious. Stroke risk in women significantly increases post-menopause. This has largely been attributed to the effects of oestrogen, which appears to be protective in both animal studies and in humans. I said ‘on the face of it’ because there are a lot of things which happen post-menopause which might also contribute. Your balance of oestrogen to testosterone will be different, other hormones like follicle stimulating hormone will also be different. In fact I retract my earlier statement of hormones are easier. They’re clearly not.
And chromosomes are even more complicated. I’ll be as basic and as simple as I can here and to any actual geneticists listening, I apologise. The two main sex chromosome cohorts are XX and XY (it gets too complicated with trisomy of the sex chromosomes so I’ll leave it out for now). In females who are XX, genes expressed on one of the X chromosomes are randomly inactivated. This is to stop them having twice as many genes expressed as males and to allow them to be a mix of the X chromosomes from both parents. Some genes, called escape genes, escape this inactivation and some of these can cause a bit of havoc. USP11 is involved in reversing ubiquitination, something which is important when getting rid of misfolded proteins, and this higher in females because it’s an escape gene and may be associated with Alzheimer’s. TLR7 is a toll-like receptor which is involved in sensing viral RNA and this seems to escape X-inactivation meaning the inflammatory response in the brain is going to be different between the sexes.
And if we bring all this research up to very modern standards then you can see how knowing someone’s biological sex, as well as their gender, can have an impact on the outcomes of their treatment pathway. Someone who is XY by birth but has chosen to transition is going to have a combination of increased inflammatory gene expression because of their sex chromosomes, which might be exacerbated by the generally pro-inflammatory effect of oestrogen.
To study this in animals, researchers have developed the four-core genotype model. This model exists for both mice and rats and the animals complement of sex chromosomes (XX and XY) are independent of their gonadal sex. This means you can have XX animals that have testes and associate hormones, and XY animals that have ovaries and associated hormones. These animals have been used to study stroke and it has been found that hormones seem to contribute more to stroke outcomes than chromosomes. But studies have also shown that the presence of two X chromosomes extends lifespan more than the presence of gonads, so stroke on a background of aging is more complicated.
There’s one final research niche where I think this is not being considered as much as it should and that’s cells. We’ve established, using the four-core genotype model, that chromosomal complement has an important impact on the outcome of many studies. I enthusiastically did some slightly shoddy but nevertheless important research for you. I took the first 10 primary papers from PubMed that used iPSCs in dementia research and dug around in the methods to find out how many of them reported the sex of the donor. Of the ten one had the sex in the title of the paper and two reported sex in the methods. Of the remaining eight, six used one or two iPSC lines only and of only one sex in each case (interestingly mostly female) and the last two despite much digging I could not find the sex of the original lines they used. One paper used KOLF cells, which are known as a reference iPSC line which a lot of places use. The problem with these is that they specifically chose to only use male donors because of a concern that X-inactivation could skew gene expression in female lines. So, whilst we’re definitely not using enough of a spread of animals in our studies, the same could be said of iPSCs. And when we’re using these to make organoids, the contribution of female vs male microglia or astrocytes might be quite different to the overall reactivity of the final product.
What we’ve established here is that firstly, not nearly enough X-chromosome researchers use the X-Files as a pun in their paper titles. But more fundamentally, we’ve established sex is important. And that sex is complicated. And that most people don’t think nearly enough about sex when they’re doing science.
Dr Yvonne Couch
Author
Dr Yvonne Couch is an Alzheimer’s Research UK Fellow at the University of Oxford. Yvonne studies the role of extracellular vesicles and their role in changing the function of the vasculature after stroke, aiming to discover why the prevalence of dementia after stroke is three times higher than the average. It is her passion for problem solving and love of science that drives her, in advancing our knowledge of disease. Yvonne shares her opinions, talks about science and explores different careers topics in her monthly blogs – she does a great job of narrating too.
Emily Spencer
Rebecca Williams
Dementia Researcher
Yasmin Bonsu