Steroid dynamics in myalgic encephalomyelitis / chronic fatigue syndrome: a case-control study [...], 2025, Thomas, Armstrong, Bergquist et al

[forestglip]

Steroid dynamics in myalgic encephalomyelitis / chronic fatigue syndrome: a case-control study using ultra performance supercritical fluid chromatography tandem mass spectrometry

Natalie Thomas, S. J. Kumari A. Ubhayasekera, Christopher W. Armstrong, Katherine Huang, Jonas Bergquist

[Line breaks added]

Background
Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a multisystem disorder characterised by unrelenting fatigue, post-exertional malaise, and dysfunction across immune, nervous, metabolism, and endocrine systems. Given the broad role of steroid hormones in regulating these systems, this study investigated differences in the steroid metabolome and network dynamics between ME/CFS patients and matched controls.

Methods
Blood plasma steroid levels were quantified using Ultra-Performance Supercritical Fluid Chromatography- Tandem Mass Spectrometry (UPSFC-MS/MS) in ME/CFS patients (n = 24) and age and gender matched controls (n = 24). Group comparisons of absolute steroid concentrations were performed using Mann-Whitney U tests.

Partial Spearman correlation networks were evaluated to examine direct associations between steroids within each group, and centrality metrics were used to evaluate structural differences. Steroid-steroid ratios were analysed to reflect biochemical relationships. Multivariate analysis with Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was also conducted.

Results
No significant group differences in absolute steroid concentrations were observed following FDR correction.

However, network analysis revealed a marked reduction in direct steroid-steroid relationships in ME/CFS, with controls exhibiting 52 significant partial correlations, while the ME/CFS group retained only one (cortisol - corticosterone).

Centrality analysis further revealed a shift in network structure, with cortisone emerging as highly central in ME/CFS (degree = 7, betweenness = 16.7), despite being peripheral in controls, and progesterone showing reduced integration in ME/CFS (degree = 3 vs. 12, eigenvector = 0.40 vs. 0.93).

Steroid-steroid ratio analysis revealed a higher cortisol-to-pregnanolone ratio and a lower pregnanolone-to-progesterone ratio in ME/CFS, although these findings did not remain significant after FDR correction.

OPLS-DA indicated a modest relationship between steroid levels and group classification (R²Y = 22.8%), but negative Q² values suggested poor predictive power.

Conclusions
Despite no significant differences in absolute steroid levels, network analysis revealed profound disruptions in steroid-steroid relationships in ME/CFS compared to controls, suggesting disrupted steroid homeostasis.

Collectively the results suggest dysregulation of HPA axis function and progestogen pathways, as demonstrated by altered partial correlations, centrality profiles, and steroid ratios.

These findings illustrate the importance of hormone network dynamics in ME/CFS pathophysiology and underscores the need for more research into steroid metabolism.

Web | PDF | Journal of Translational Medicine | Open Access

 

[forestglip]

Open Medicine Foundation: 'Steroid Dynamics in ME/CFS: New Publication'

The Heart of the Matter

• OMF’s research centers in Uppsala and Melbourne published a study of steroid hormones—molecules that control a lot of important systems in the body—using an advanced technique called ultra-performance supercritical fluid chromatography tandem mass spectrometry (UPSFC-MS/MS).
• The team looked at individual hormones as well as how the hormones interact with each other, or the hormone networks. They found that the networks were particularly important for finding differences between people with ME/CFS and healthy controls.
• The differences they found in the networks indicate that the hypothalamic-pituitary-adrenal (HPA) axis function and progestogen pathways aren’t working correctly in ME/CFS.
• This paper is part of a larger project tracking hormone fluctuations in ME/CFS and Long COVID (the MELLOW study), which is in the “Recruitment, Data Collection” stage of the research process.

[More at link above]

 

[Jonathan Edwards]

Not sure what these findings might mean.

 

[wigglethemouse]

Despite no significant differences in absolute steroid levels, network analysis revealed profound disruptions in steroid-steroid relationships in ME/CFS compared to controls, suggesting disrupted steroid homeostasis.

After chatting to AI I can think of no obvious reason for the profound disruptions of the links between the hormones as many of the links are based on biological processing pathways e.g. more of one hormone leads to more of another hormone in the same pathway. It seems like the feedback mechanism or signalling must be disrupted. But what would that mean in real terms? Seems a bit of gray area.

I seem to remember Klimas et al also came to the same conclusion about steroid hormones and they proposed a treatment reset plan based on silicon modelling which never got off the ground.

I think the "network analysis revealed profound disruptions" has been found elsewhere in non-hormone studies in ME/CFS such as the Cornell center exercise studies. Why would that be? I just don't understand what can disrupt these links in widespread processes.

Could something like increased heat shock proteins from oxidative stress be messing with widespread cellular systems? What else could do that.

 

[Hutan]

My starting point on this is 'rather skeptical'. The number of samples is really small for systems that we know fluctuate and change with lifestyle.

A lot of researchers have a preconceived idea that the problem with ME/CFS is in the HPA axis. Perhaps they are right, but the result seems to be a certainty that cortisol must be "wrong" in some way. We therefore have seen relentless testing of cortisol levels, and theories about in what way they might be wrong. The many findings that cortisol levels are actually pretty normal just results in more complicated theories. Maybe the researchers are right, but I think we should also consider the possibility that people are looking for answers in the wrong place.

A useful finding of this paper is that the steroid concentrations were not different between the groups. So, they have moved on to more complicated theories involving relationships between the different steroids. There seems to be a lot of null results reported in the abstract, but they speak of "profound disruptions".

I'm interested to read this paper to see how well the groups were matched for sex, age, lifestyle, contraceptive use. I would like to know how careful they were to sample at appropriate and consistent times of the day, and, for the women, at appropriate times of the menstrual cycle. And to see how much consistency the ME/CFS group showed in the reported disruptions.

 

[Hutan]

Sadly, this paper is not starting well. There are issues with the introduction, here are just some:

This condition is further characterised by cognitive impairment, unrefreshing sleep, chronic pain, autonomic dysregulation, and disruptions in both neuroendocrine and immune function [2]. The impact of ME/CFS on daily life profoundly restricts routine activities and results in a lower quality of life than that associated with cancer, multiple sclerosis, or stroke [3]. Recognised by the World Health Organization (WHO) as a neurological disorder [4], ME/CFS is driven by disruptions to neuro-immune-metabolic-endocrine homeostasis that are believed to be central to its complex pathology [1].

Among them, we don't actually know that ME/CFS is characterised by neuroendocrine disruption.

A bald statement saying that ME/CFS results in lower quality of life than cancer, multiple sclerosis or stroke is just inviting ridicule. Yes, some ME/CFS is horrific, but my ME/CFS is not worse than my mother's breast cancer - which killed her at a relatively young age. There's absolutely no need to make such problematic comparisons.

There is no need to mention the WHO categorisation - it's not based on any superior knowledge.

And that statement there "ME/CFS is driven by disruptions to neuro-immune-metabolic-endocrine homeostasis that are believed to be central to its complex pathology". Well, if ME/CFS was driven by those disruptions to homeostasis, then of course they are central to the pathology. And probably the pathology is somewhere in that wide sweep of neuro, immune, metabolic, endocrine systems, but it's hardly narrowing it down. It's such a definitive statement and we don't have evidence of those disruptions yet.

Many of these researchers have a track record in ME/CFS. Stuff like this should not still be being signed off by them. I hate being so negative, but it's just really disappointing to have to be pointing out basic stuff like this to an OMF team. OMF should have some systems to ensure that their scientists nail introductory paragraphs about ME/CFS every time.

When I see statements like that one suggesting all those disruptions are known, I lose trust that the researchers are approaching their work without bias.

 

[Hutan]

This gender disparity appears to emerge after puberty (ages 10–19), with another diagnostic incidence peak during a women’s reproductive years, often coinciding with pregnancy and the postpartum period (ages 30–40).

There isn't good evidence for that 'second incidence peak'.

Pregnancy and perimenopause have been reported as a trigger of ME/CFS symptom onset, and women have reported ME/CFS symptom fluctuation with hormonal changes, including those associated with the menstrual cycle and perimenopause [5]. Accordingly, it can be suggested that such endocrine shifts can influence susceptibility to ME/CFS, with gonadal sex steroids driving such changes [6].

Pregnancy has also been reported to cause improvements in ME/CFS too. People report all sorts of things, we don't have much real evidence. However, from memory, that Visible study seemed to find that the only symptom being materially affected by the menstrual cycle was migraines. We don't see rates of ME/CFS onset jumping with perimenopause, we don't see widespread reports of ME/CFS disappearing at perimenopause. For a disease that seems to be more common in women, there is actually very little evidence of any direct association with the major female life changes and the menstrual cycle.

The importance of the endocrine system is further highlighted by extensive research on adrenal cortisol, one of the most widely studied biomarkers in ME/CFS [7–10], in addition to limited research showing changes in TS/ T4 thyroid hormones [11], growth hormone [12], and aldosterone steroids [13].

And this goes to what I was saying in my last post. For goodness sake, you can't say that cortisol is important in ME/CFS because lots of people have studied it as a possible biomarker. The endocrine system is not important in ME/CFS just because quite a lot of people wish it were so.

 

[Hutan]

Steroids, including progestogens (progesterone (prog,P4), 17α-hydroxyprogesterone (17OHP), pregnenolone(P5), pregnanolone (PNL)), corticosteroids (aldosterone(Aldo), cortisol (F), corticosterone (B), 1-deoxycortico-sterone (DOC), 11-deoxycortisol (11DOC), cortisone(Cot)), androgens (androstenedione (AED, androsterone(ADT, testosterone (T), etiocholanolone (Etn), dehydro-epiandrosterone (DHEA)), and estrogens (estradiol (E2),estrone, (E1)) were profiled in peripheral plasma blood samples using ultra-performance supercritical fluid chromatography couple to tandem mass spectrometry (UPSFC-MS/MS), as previously reported

A total of 48 participants (n=24 ME/CFS, n=24 controls), matched by age and gender, were included in the analysis. Gender is used throughout the manuscript, as the variable was defined by self-reported gender rather than sex.

That's strange to use gender, when sex is what actually matters in this study. Table 1 suggests the two cohorts were well matched on age and sex. 16 females and 8 males in each group.

Study participants were diagnosed at the Gottfries clinic in Göteborg between years 2013–2018

All patients fulfilled the Canadian Consensus Criteria, the International Consensus Cri-teria and the IOM criteria

The two most common co-morbidities in patients were Hypothyroid-ism and Fibromyalgia

When recorded, the two most common disease-triggering infections were the Flu and Upper respiratory infection in addition to infections of Neuroborreliosis, TWAR, Epstein Barr virus, Gastroen-teritis and Meningitis.

In addition to the patient samples, EDTA plasma samples were collected from 24 age and gender-matched controls from the blood bank in Uppsala Academic Hospital.

All blood samples were collected from fasted participants in the AM.

There's nothing there about excluding women who were pregnant or post-menopausal or breast-feeding. Crucially, there's nothing there about timing of sample collection with respect to menstrual cycle or use of contraceptives (current use, years of use) that alter steroid levels. There's nothing about prior use of steroid medications, perhaps for those triggering respiratory infections. It looks as though the researchers probably knew little about the controls. How did the samples come to be in the Uppsala Hospital blood bank?

The sample sizes are too small for these sorts of omissions to be unimportant. If you only have 16 women in your sample, it's extremely possible that differences in contraceptive use and the stage of menstrual cycle could have a material effect on the relationships between hormones found. There's also the issue that at least some of the people with ME/CFS may have their peak morning cortisol at a different time than the healthy controls do.

I don't think this study is nearly careful enough for a detailed examination of steroid ratios to reliably tell us anything. It's really disappointing. I would have thought controlling for things like menstrual cycle timing and contraception would be normal practice in studies looking at sex hormones in females.

Given that, I think the claims made in this study are unwarranted and irresponsible. Authors, you have the luxury of getting paid to do your work. Please do better.

 

[wabi-sabi]

Among them, we don't actually know that ME/CFS is characterised by neuroendocrine disruption.

Dr Klimas has shown this in years of her work. I don't have citations to hand, but I remember her speaking about it.

There is no need to mention the WHO categorisation - it's not based on any superior knowledge.

This isn't about superior knowledge, but about using the ICD-10 classification of diseases. All medical people who claim to be evidence based have to at least pay lip service to ICD-10. So when the WHO say this is a neurologic disease (NOT a psychiatric disease) that should carry weight. That's extra important if you live in a country that doesn't recognize ME/CFS as a real illness, because then you can point to WHO and say- Look, you are out of step with the way the rest of the rational planet categorizes diseases!

A bald statement saying that ME/CFS results in lower quality of life than cancer, multiple sclerosis or stroke is just inviting ridicule.

Not necessarily. There are objective ways of classifying the suffering of different illnesses (as odd as this may seem) and ME/CFS is up there. This isn't about playing the suffering Olympics, but about referencing objective measures that clinicians recognize to say- this isn't just being tired! This is a real disease that causes real anguish and deserves real attention.

 

[wabi-sabi]

People report all sorts of things, we don't have much real evidence.

I think this is always the issue with patient driven research.

On the one hand, we want to listen to patients and what they say is important. But on the other hand, well patients say the darndest things. Do we follow up on everything in order to redress the years of ignoring people's lived experience? Or do we at least try to filter that lived experience through scientists and physicians that understand the most about our illness? I think the hormone and HPA access thing sound valid enough on the surface to invest research time and money. But, we always need so much more research....

 

[wigglethemouse]

This is from Dr Berquists 2019 NIH presentation. I suspect they used samples from this group for several projects hoping to see if there was any cross-project links. Maybe they have been sitting on the data a long time and only now did a deep analysis after originally not finding much difference.
Video LINK

 

[wabi-sabi]

Not sure what these findings might mean.

I'm not sure what they mean either, but I suspect it's because this paper is far above my analytical pay grade.

Over all, I like the paper. It suffers from being so terribly preliminary, and as the authors mention, small sample sizes, among other things. When you are in uncharted territory, how do you know before you set sail? They mention that only one other paper had looked at these issues this specifically before, and that paper only found differences when they took into account both sex and severity. Unfortunately, since that other paper didn't mention how they assessed severity, we cannot really do a comparison. Argh! On the positive side, they seem to use the most up to date tech to measure the smallest possible hormonal difference. Yay! That means we can use their results as a bade for future research and analysis.

I wish they had recruited more participants in the first place and they don't really discuss how they ended up with such a small number.

There seems to be something going on with cortisol levels- different results in different studies- so hopefully this will throw some light on it. most other studies haven't even used methods that are sensitive enough, compared to the methods used here and don't stratify participants on severity, which makes the results even harder to interpret.

 

[wigglethemouse]

Jonas Berquist gave a short talk on targeted steroidomics at the 2018 Stanford Symposium that used 24 ME/CFS and 24 controls. Perhaps it's the same samples as this study. The only finding was pregnanolone, same as this paper. Here is the video starting at 14.59. It is 3.5 minutes in duration, so a quick watch.



A transcript is available here.

https://www.omf.ngo/2018/11/20/second-annual-symposium-transcripts/edited-jonas-bergquist-the-neuroimmune-route-in-mecfs/

 

[wigglethemouse]

In the 2018 video presentation pregnanolone was described as a neuroseteroid that can have synaptic functioning and is made in the mitochondria.

 

[ChronicallyOverIt]

In the 2018 video presentation pregnanolone was described as a neuroseteroid that can have synaptic functioning and is made in the mitochondria.

View attachment 27295

I wonder if this is why some patients find PEA helpful, as it helps create allopregnanolone

Edit: this slide is referencing pregnenolone where I am thinking of a different molecule pregnanolone. Allopregnanolone is a metabolite of pregnanolone

 

[ChronicallyOverIt]

In the 2018 video presentation pregnanolone was described as a neuroseteroid that can have synaptic functioning and is made in the mitochondria.

View attachment 27295

This slide says pregNENolone where wigglemouse references pregNANolone. They are different molecules with similar names

 

[ME/CFS Science Blog]

I suspect they used samples from this group for several projects hoping to see if there was any cross-project links.

Yes looks like its relatively old data. Patients were diagnosed in 2013–2018 and ethical approval for the study was given in 2016.

Weird result. No difference between steroids but almost a total lack of correlations among them in the ME/CFS group. Unfortunately, the authors couldn't give an example in other diseases where something like this has been found.

 

[Utsikt]

Dr Klimas has shown this in years of her work. I don't have citations to hand, but I remember her speaking about it.

Has she shown, or does she claim? Lots of ME/CFS researchers claim lots of thing, and most of it doesn’t hold up under scrutiny.

This isn't about superior knowledge, but about using the ICD-10 classification of diseases. All medical people who claim to be evidence based have to at least pay lip service to ICD-10.

The ICD-10 is completely irrelevant in this context. Jonathan Edwards has spelled out why here. There is nothing evidence based about using a classification that isn’t based on evidence about the condition it classifies.

So when the WHO say this is a neurologic disease (NOT a psychiatric disease) that should carry weight.

It really shouldn’t when it isn’t based on any evidence.

That's extra important if you live in a country that doesn't recognize ME/CFS as a real illness, because then you can point to WHO and say- Look, you are out of step with the way the rest of the rational planet categorizes diseases!

That is an important, but still completely different consideration, and it doesn’t have anything to do with how the researches behind this paper describes the condition they are trying to study.

Not necessarily. There are objective ways of classifying the suffering of different illnesses (as odd as this may seem) and ME/CFS is up there. This isn't about playing the suffering Olympics, but about referencing objective measures that clinicians recognize to say- this isn't just being tired! This is a real disease that causes real anguish and deserves real attention.

The objective ways are not exhaustive ways of classifying the suffering of a disease. And those ways still only work at an aggregated level.

Regardless, the point still stands: We don’t need a DALY-comparison to get the point across. Stating how bad ME/CFS is on its own would be more than enough. See the factsheets for examples of how that can be done.

 

[Hutan]

(cross posted with Utsikt )

Dr Klimas has shown this in years of her work. I don't have citations to hand, but I remember her speaking about it.

Unfortunately a lot of Dr Klimas's work is rather questionable. I'm interested to see what you are remembering when you find it.

So when the WHO say this is a neurologic disease (NOT a psychiatric disease) that should carry weight.

But the WHO didn't know if this is a neurological disease when it categorised it. It still doesn't know, because no one really knows. Possibly there is some increasing evidence to support the idea, but it's fairly recent. Sure, if you want to say in advocacy materials that the WHO categorises ME/CFS as a neurological disease, go for it. But, this is a scientific paper. Given the categorisation was not based on evidence, there's no point in mentioning it.

Not necessarily. There are objective ways of classifying the suffering of different illnesses (as odd as this may seem) and ME/CFS is up there. This isn't about playing the suffering Olympics, but about referencing objective measures that clinicians recognize to say- this isn't just being tired! This is a real disease that causes real anguish and deserves real attention.

I've seen some of the ratings, and they are rather questionable. And regardless of what method was used to rate the suffering from different illnesses, the point is that there is no need to make these questionable comparisons in an introduction to a ME/CFS paper.

If someone wants to say 'causes suffering similar to that of MS and affects a substantially bigger proportion of the population', that's fine. But, saying that it is worse than cancer, MS and stroke just makes people roll their eyes. What sort of cancer, the benign sort or the one that kills you in weeks? The stroke where there is no noticeable permanent damage or the one that kills you? MS when you just have one episode, or MS when it stops you breathing?

Yes, I had noticed that ME/CFS is a real disease that causes real anguish and deserves real attention. That's exactly why I want the people working on finding the pathology, often using donations from people with ME/CFS to do it, to make sure that they do good quality research.

I think this is always the issue with patient driven research.

On the one hand, we want to listen to patients and what they say is important. But on the other hand, well patients say the darndest things. Do we follow up on everything in order to redress the years of ignoring people's lived experience? Or do we at least try to filter that lived experience through scientists and physicians that understand the most about our illness? I think the hormone and HPA access thing sound valid enough on the surface to invest research time and money. But, we always need so much more research....

I'm not disagreeing with looking at hormones @wabi-sabi, I'm just asking that it be done in way that actually gives us some useful results.

Contraceptives can lower pregnenolone. We have no idea how many women in the cohorts were on oral contraceptives or other sorts of hormone based contraceptives.

The stage of the menstrual cycle is characterised by different levels of many of the hormones in this study. We have no idea how many women were in what stages of the cycle (or indeed even if some were peri-menopausal or menopausal or pregnant or breast-feeding).

As the following quote from a paper I found shows, there are even daily fluctuations in the hormones, and these fluctuations change during the menstrual cycle.

A circadian rhythm in pregnenolone, 17-hydroxyprogesterone, testosterone, and dihydrotestosterone levels was detected only in certain phases of the cycle. All these steroids showed a circadian rhythm in the early follicular phase. The rhythm of pregnenolone and 17-hydroxyprogesterone was still present in the periovulatory period but was no longer detectable in the luteal phase, meanwhile that of testosterone and dihydrotestosterone was not demonstrable neither in the periovulatory period nor in the luteal phase. Compared to the levels of the follicular phase, an increase in pregnenolone and 17-hydroxyprogesterone levels was observed in the periovulatory period which was followed by a further rise in the luteal phase. This increase completely masked the circadian rhythm in the luteal phase.​

It probably matters if you take the sample at 8 am or 11 am. And then there is the complication of people with ME/CFS possibly having different usual wake times.

There are many factors affecting steroid levels and this study has ignored all of that. That makes the results virtually useless, at least for the women. Perhaps the results for the men might tell us something, but there were only 8 men in each group, which isn't enough.

There's also the issue of samples being quite old and from different sources, perhaps with different storage. Steroids can degrade over time.

I'm not sure what they mean either, but I suspect it's because this paper is far above my analytical pay grade.

I doubt that it is, wabi-sabi.

There seems to be something going on with cortisol levels- different results in different studies- so hopefully this will throw some light on it. most other studies haven't even used methods that are sensitive enough, compared to the methods used here and don't stratify participants on severity, which makes the results even harder to interpret.

This study found that cortisol levels were not different. That has been the case in many studies of cortisol in ME/CFS. We have a thread on the studies here. I'll add this one to the list.

 

[wabi-sabi]

I'm interested to see what you are remembering when you find it.

It was a presentation I saw on youtube years ago. There's no way I'd be able to find it again unfortunately.

I just can't agree with your objections to the introduction. The writing looks to me like the standard ways of describing disease severity, what we know about disease physiology and drawing on respected authorities like WHO. I can't see saying that the ICD-10, even on a little known disease like ours, isn't evidence based. I don't think this intro should draw more ire than the intros that band on about ME/CFS being "unexplained", subtext psychosomatic. And I think it would be better to focus on the results.

I've seen some of the ratings, and they are rather questionable.

They may seem silly to you (and hey, maybe they are), but people in healthcare take them seriously. If we are going to be understood, we need to speak the language they understand. Papers are meant to start off with the seriousness of the illness to grab the reader's attention. Typically you do that either with numbers of affected or severity of illness. It's a legit tactic for the authors to use. It really, really doesn't make us look hysterical.

However, I can't focus on the results since I don't have the expertise to evaluate them properly. But I am glad the authors are doing this research, even if it is awfully preliminary. I feel like there's some biting the hand that feeds us going on here.

The authors acknowledge all of the menstrual cycle and birth control and menopause stuff you bring up, so they do understand that it is an issue and are not accidentally ignoring it. But this is a preliminary study with a small sample size. There's just not enough people in the study to make that possible. This is really the basics- let's look for differences between men and women. I really wish they had had more people too... I also wish they had described a bot more why they chose this sample as most appropriate.

This study found that cortisol levels were not different.

I am referencing this quote here, not specific study findings. I should have made that more clear.
"Research on cortisol, one of the most studied steroid hormones and potential biomarkers in ME/CFS, generally supports the presentation of mild hypocortisolism inpatients [37] which is in contrast with the current findings. However, the evidence remains mixed and somewhat controversial, with studies showing variability in absolute cortisol levels, diurnal patterns, and responses,leading to ongoing debate about its role in the condition[38].