# Multiple sclerosis lesions exhibit sex-specific steroid changes

Sabina Luchetti, Matthew R J Mason, Philippe Liere, Antoine Pianos, Sara Cossu, Marloes Hofstee, Arja Sluiter, Jackelien van Scheppingen, Eleonora Aronica, Michael Schumacher, et al.

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Sabina Luchetti, Matthew R J Mason, Philippe Liere, Antoine Pianos, Sara Cossu, et al. Multiple sclerosis lesions exhibit sex-specific steroid changes. Neurobiology of Disease, 2025, 214, pp.107040. 10.1016/j.nbd.2025.107040. inserm-05354135

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Multiple sclerosis lesions exhibit sex-specific steroid changes

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Sabina Luchetti<sup>a,1</sup>, Matthew R.J. Mason<sup>a,*,1,2</sup>, Philippe Liere<sup>b,3</sup>, Antoine Pianos<sup>b</sup>, Sara Cossu<sup>a</sup>, Marloes Hofstee<sup>a</sup>, Arja Sluiter<sup>a</sup>, Adelia<sup>a</sup>, Jackelien van Scheppingen<sup>a</sup>, Eleonora Aronica<sup>c</sup>, Michael Schumacher<sup>b</sup>, Inge Huitinga<sup>a,d</sup>

ARTICLE INFO

Keywords:

Neurosteroids
Allopregnanolone
Multiple sclerosis
GC/MS
Microglia
Astrocytes

ABSTRACT

Sex differences in disease susceptibility and progression in multiple sclerosis (MS) indicate hormonal influences. We previously found sex differences in steroidogenic enzyme expression in mixed active/inactive (mixed) and inactive lesions, suggesting increased synthesis and signalling of progesterone in females and of estradiol (E2) in males. Here we measured steroid levels in mixed and inactive MS lesions (14 males, 16 females) and control donor white matter (10 males, 10 females) by gas chromatography-mass spectrometry (GC/MS). In mixed and inactive lesions, we found a female-specific increase in the production of the neuroactive steroid allopregnanolone, and a reduction in testosterone metabolites and the precursor androgen dehydroepiandrosterone (DHEA). In males, however, most steroids remained unchanged, although the weakly neuroactive 5α-DHDOC was increased. No significant differences were found in steroid levels in remyelinating MS lesions (6 males, 6 females) and control donor white matter (8 males, 7 females). However, higher gene expression of steroid receptors and of the steroidogenic enzyme AKR1C3 persisted in these remyelinated lesions in females. In vitro, we found that E2, and to a lesser degree progesterone and testosterone, had mostly beneficial effects on cytokine expression in primary microglia isolated from MS donors but not from controls; and on neuroprotective gene (APOE, BDNF, GLT1, IGF1, LIF) expression in human fetal astrocytes, but only under inflammatory conditions. Sex-specific changes in the levels of steroids, their metabolizing enzymes, their receptors, and the actions of steroids on glial neuroinflammatory responses may contribute to the sex differences in MS disease progression and lesion resolution.

1. Introduction

Multiple sclerosis (MS) is a debilitating neurological disease with an unknown cause, characterized by focal demyelinating lesions in white matter and grey matter, and an accompanying progressive neurodegenerative process. Lesions are characterized by an inflammatory cellular content consisting mainly of activated macrophages/microglia, T-cells and sometimes B-cells (Fransen et al., 2021; Fransen et al., 2020; Kuhlmann et al., 2017; Luchetti et al., 2018). Sex differences in the risk

of developing MS and disease progression suggest an influence of steroid
hormones (reviewed in (Gilli et al., 2020)). Females are approximately
2-4 times more likely to develop MS (Harbo et al., 2013), but progres-
sion in females occurs more slowly (Alvarez-Sanchez and Dunn, 2023;
Confavreux et al., 2003). White matter lesions of males are more likely
to appear as 'black holes' on MRI, suggesting females have better lesion
resolution (Pozzilli et al., 2003). Furthermore, we and others recently
showed that males are more likely to develop cortical grey matter le-
sions and have more mixed active/inactive lesions, previously referred

<footnote><sup>a</sup> Neuroimmunology Research Group, Netherlands Institute for Neuroscience, Meibergdreef 47, Amsterdam 1105BA, the Netherlands</footnote>

<footnote><sup>b</sup> U1195 "Diseases and Hormones of the Nervous System", Inserm and University Paris-Saclay, 80, Rue Du Général Leclerc, 94276 Kremlin-Bicêtre, France</footnote>

<footnote><sup>c</sup> Department of (Neuro) Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam Neuroscience, Meibergdreef 9, Amsterdam 1105AZ, the Netherlands</footnote>

<footnote><sup>d</sup> Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: m.mason@nin.knaw.nl (M.R.J. Mason), philippe.liere@inserm.fr (P. Liere), antoine.pianos@inserm.fr (A. Pianos), a.sluiter@nin.knaw.nl (A. Sluiter), j.g.m.mooijman-van_scheppingen@lumc.nl (J. van Scheppingen), e.aronica@amsterdamumc.nl (E. Aronica), michael.schumacher@inserm.fr (M. Schumacher), i.huitinga@nin.knaw.nl (I. Huitinga).</footnote>

<footnote n="1">¹ These authors contributed equally.</footnote>

<footnote n="2">² Current address: Amsterdam UMC location Vrije Universiteit Amsterdam, Department of Molecular Cell Biology and Immunology, Amsterdam, The Netherlands.</footnote>

<footnote n="3">³ Current address: UMR-S 1124 Healthfex INSERM-Université Paris-Cité, 45 rue des Saints-Pères, 75006 Paris, France.</footnote>
to as chronic active lesions (Kuhlmann et al., 2017; Luchetti et al., 2018).

High levels of progesterone (P) and estrogen in pregnancy are associated with a reduction in relapses though this is followed by a postpartum rebound effect (Confavreux et al., 1998). In clinical MS trials, oral estriol (E3) reduced lesion size and volume in females, while testosterone (T) applied as transdermal gel reduced brain atrophy in males and modulated peripheral immune responses (reviewed in (Gold and Voskuhl, 2009). Steroid hormones may thus influence susceptibility, disease course and lesion development or resolution. In addition to their effects on gene expression via the nuclear receptors, some steroids, known as neuroactive steroids, modulate neurotransmitter receptors (Belelli and Lambert, 2005; Schumacher et al., 2014). These neuroactive steroids include the progesterone derivatives allopregnanolone (ALLO), a positive modulator of GABA<sub>A</sub> receptors and 3β,5α-THP (isallopregnanolone) which is a negative regulator (Reddy and Rogawski, 2002). We previously showed that ERα mRNA was increased in MS lesions in a primarily female set of donors, suggesting hormonal influences on lesion development and resolution (Koning et al., 2007). We also showed sexually dimorphic changes in the mRNA expression of genes encoding steroidogenic enzymes in female and male MS lesions and perilesional normal appearing white matter (NAWM) when compared to control donors (Luchetti et al., 2014). These findings indicated a local formation and sex-specific regulation of neuroactive steroids and are consistent with a role for these in the evolution of MS lesions. In particular they suggested a shift towards production of progesterone (PROG) and its derivatives in females and towards estrogen production in males (Luchetti et al., 2014). Of note, PROG, ALLO, T, 5α-dihydrotestosterone (5α-DHT) and estradiol (E2) are all neuroprotective (Bialek et al., 2004; Brann et al., 2007; Guennoun et al., 2015) and reduce neuroinflammation (De Nicola et al., 2018; Giatti et al., 2015; Giatti et al., 2012; Noorbakhsh et al., 2011; Spence and Voskuhl, 2012; Zahaf et al., 2023).

However, it is not known how differences in enzyme gene expression translate into changes in steroid levels within MS lesions. Steroids present in the brain originate not only from local synthesis (neurosteroids), but also from the circulation after their release by the steroidogenic endocrine glands (Robel and Baulieu, 1995). They easily cross the blood-brain-barrier and can rapidly diffuse throughout the entire brain (Banks, 2012). Quantitation of actual steroid levels in the brain is therefore necessary to understand how the steroid metabolome may influence the disease. Previous reports have indicated reductions in the concentrations of ALLO in in MS normal appearing white matter next to lesions (Noorbakhsh et al., 2011) and of dehydroepiandrosterone (DHEA) in MS white matter (Boghozian et al., 2017) when compared to controls. Sex differences were not investigated in these two studies. ALLO and its precursor 5α-dihydroprogesterone (DHP) was also reduced in CSF and plasma of male MS patients compared to controls, along with changes in some other steroids (Caruso et al., 2014). Higher plasma levels of ALLO were found in relapsing-remitting MS patients compared to patients with clinically isolated syndrome, but were not different to controls (Cheng et al., 2021). Thus, while some data is available, extended steroid profiling in MS lesions and their surrounding NAWM of males and females has not been carried out.

Here, we used the precision and sensitivity of gas chromatography coupled to mass spectrometry (GC/MS) for the extended profiling of 47 steroids (Gonzalez Deniselle et al., 2016; Hertig et al., 2010). Steroid metabolomes were analysed in MS mixed active/inactive (hereafter also referred to as mixed) lesions, inactive lesions, in male and female MS patients and compared to control donor white matter. In mixed and inactive lesions, we identified a number of sex differences in hormonal responses to lesion formation, in particular a female-specific decrease in androgens and increase in ALLO and a decrease in isoallopregnanolone, consistent with an enhanced protective GABA tonus.

Previously, semi-quantitative immunostaining of MS lesions and NAWM revealed that steroidogenic enzymes and steroid receptors were

mainly expressed in reactive microglial cells and astrocytes (Luchetti et al., 2014). Steroids are known to regulate the inflammatory responses of these glial cells to neurodegenerative processes (reviewed by (Yilmaz et al., 2019) and their actions on these cells also play an important role in neuroprotection and axon remyelination (Asbelaoui et al., 2024; Spence et al., 2013; Zahaf et al., 2023). Astrocytes are also an important source and target of neurosteroids (Arbo et al., 2016; Arevalo et al., 2015; Zwain and Yen, 1999).

To uncover potential mechanisms of MS-related actions of steroids in these cells, we tested their effects in vitro on pro- and anti-inflammatory as well as neuroprotective gene expression in primary human microglia derived from MS lesions or control donors and in fetal human astrocytes exposed or not to inflammatory conditions. We found mainly beneficial effects of estrogens on the balance of pro- and anti-inflammatory cytokine production in microglia, but only when isolated from MS brains, and on neuroprotective gene expression in astrocytes exposed to inflammatory conditions.

Remyelination is an important aspect of MS and steroids also influence this process. Estrogen receptor (ER) α in astrocytes and ERβ in oligodendrocytes, as well as androgen receptor (AR) in microglia and astrocytes have been shown to play important roles in remyelination (Asbelaoui et al., 2024; Khalaj et al., 2013; Spence et al., 2013; Zahaf et al., 2023). Progesterone has pro-myelinating effects via the intracellular progesterone receptors (PR) and via modulation of GABA<sub>A</sub> receptors after conversion to ALLO (El-Etr et al., 2011; Ghoumari et al., 2020). To investigate whether sex specific changes in steroid synthetic enzymes and steroid levels are still present following remyelination, we also carried out gene expression profiling and steroid profiling for a panel of 21 steroids in remyelinated lesions.

We found a persistent female-specific upregulation of the main intracellular steroid hormone receptors, namely the AR, ERα and PR. Among the enzymes we found a continued upregulation of the ald-o-keto reductase AKR1C3 in females. This enzyme preferentially converts weak androgens and estrogens to potent ones, and may thus yield pro-androgenic and pro-estrogenic states (Penning et al., 2015; Penning et al., 2000).

Overall our findings reveal sex differences in steroid metabolomes in MS lesions, in particular suggesting a more potent restorative response in females.

## 2. Methods

### 2.1. Subjects

Post-mortem material and clinical information were obtained from the Netherlands Brain Bank (NBB). All donors gave permission to the NBB for brain autopsy and the use of tissue and clinical information for research purposes. Characteristics of patients and control donors used for steroid measurements in inactive and mixed active/inactive lesions are shown in Table 1 (mixed lesions: 8 males and 8 females; inactive lesions: 8 males and 9 females; control donors: 10 males and 10 females). Characteristics of patients and control donors used for steroid measurements and gene expression measurements in remyelinated lesions are shown in Table 2 (remyelinated lesions: 6 males and 6 females; control white matter: 8 males and 7 females). Characteristics of patients and control donors used for in vitro studies in primary microglia are shown in Supplemental Table 1 (MS patients: 2 males and 8 females; control donors: 4 males and 5 females).

### 2.2. MS lesions

Fresh frozen blocks were selected based on lesion characterization carried out in paraffin mirror blocks immunostained by the NBB for MHC class II Human Leukocyte Antigen (HLA) and Proteolipid Protein (PLP). Lesion characterization was carried out as described (Luchetti et al., 2018). Fresh frozen blocks containing mixed active/inactive
<caption>**Table 1**</caption>

Characteristics of MS patients and control donors used for steroid measurements by GC/MS in inactive and mixed active/inactive lesions. Disease duration given in years. Abbreviations: MAI = mixed active/inactive; MS: Multiple sclerosis; PMD = post-mortem delay (hours:minutes); PP = primary progressive; PR = progressive relapsing; SP = secondary progressive. pH = pH of cerebrospinal fluid post-mortem. Summary statistics are given in the form of mean ± standard deviation.

<table><thead><tr><th>Group</th><th>MS type</th><th>Sex</th><th>Age</th><th>PMD</th><th>pH</th><th>Disease duration</th><th>Cause of death</th><th>Lesion type</th></tr></thead><tbody><tr><td rowspan="20">MS</td><td>SP</td><td>F</td><td>49</td><td>05:45</td><td>6.43</td><td>49</td><td>Metastasized breast carcinoma.</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>68</td><td>07:30</td><td>6.3</td><td>38</td><td>Aspiration pneumonia</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>43</td><td>10:45</td><td></td><td>21</td><td>Subdural haematoma, pneumonia</td><td>MAI/ Inactive</td></tr><tr><td>PP</td><td>F</td><td>66</td><td>08:30</td><td>6.53</td><td>66</td><td>Euthanasia</td><td>Inactive</td></tr><tr><td>PP</td><td>F</td><td>44</td><td>10:15</td><td>6.34</td><td>8</td><td>Decompensation</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>61</td><td>09:55</td><td>5.99</td><td>29</td><td>Peri-renal abscess</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>66</td><td>06:00</td><td>6.18</td><td>23</td><td>Unknown</td><td>MAI</td></tr><tr><td>SP</td><td>F</td><td>68</td><td>10:40</td><td>6.4</td><td>41</td><td>Euthanasia</td><td>MAI</td></tr><tr><td>PP</td><td>F</td><td>57</td><td>08:40</td><td>6.44</td><td>4</td><td>Euthanasia</td><td>Inactive</td></tr><tr><td>PR</td><td>F</td><td>84</td><td>07:35</td><td></td><td>49</td><td>Euthanasia</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>60</td><td>10:40</td><td>6.48</td><td>61</td><td>Euthanasia</td><td>MAI</td></tr><tr><td>SP</td><td>F</td><td>50</td><td>07:35</td><td>6.45</td><td>51</td><td>Euthanasia</td><td>MAI</td></tr><tr><td>SP</td><td>F</td><td>56</td><td>08:25</td><td>6.16</td><td>34</td><td>Pneumonia</td><td>MAI</td></tr><tr><td>PP</td><td>F</td><td>62</td><td>12:35</td><td></td><td>24</td><td>Cachexia and pulmonary insufficiency with MS</td><td>MAI</td></tr><tr><td>SP</td><td>F</td><td>71</td><td>07:05</td><td>6.32</td><td>34</td><td>Cachexia (MS and metastatic breast cancer)</td><td>Inactive</td></tr><tr><td>SP</td><td>F</td><td>48</td><td>11:50</td><td>6.1</td><td>23</td><td>Respiratory failure with end stage MS.</td><td>MAI</td></tr><tr><td>SP</td><td>M</td><td>43</td><td>08:30</td><td>6.48</td><td>11</td><td>Pneumonia</td><td>MAI/ Inactive</td></tr><tr><td>SP</td><td>M</td><td>47</td><td>07:15</td><td>6.2</td><td>7</td><td>Urosepsis with organ failure</td><td>MAI</td></tr><tr><td>SP</td><td>M</td><td>55</td><td>06:20</td><td>6.7</td><td>31</td><td>Respiratory insufficiency from pneumonia and urosepsis.</td><td>Inactive</td></tr><tr><td>PP</td><td>M</td><td>50</td><td>09:30</td><td>6.2</td><td>24</td><td>Unknown</td><td>MAI</td></tr><tr><td>SP</td><td>M</td><td>44</td><td>10:15</td><td>5.92</td><td>21</td><td>Cachexia by end stage MS</td><td>MAI</td></tr><tr><td>PR</td><td>M</td><td>51</td><td>11:00</td><td>6.23</td><td>52</td><td>Unknown</td><td>MAI</td></tr><tr><td>SP</td><td>M</td><td>56</td><td>09:50</td><td>5.98</td><td>14</td><td>Cachexia and exhaustion by end stage MS</td><td>MAI/ Inactive</td></tr><tr><td>PP</td><td>M</td><td>54</td><td>08:15</td><td>6.39</td><td>14</td><td>Euthanasia</td><td>MAI</td></tr><tr><td>PR</td><td>M</td><td>67</td><td>11:00</td><td></td><td>38</td><td>Sudden death with multiple sclerosis</td><td>Inactive</td></tr><tr><td>SP</td><td>M</td><td>73</td><td>08:45</td><td>6.4</td><td>51</td><td>Urosepsis</td><td>Inactive</td></tr><tr><td>SP</td><td>M</td><td>80</td><td>09:45</td><td>6.42</td><td>36</td><td>Cachexia and pneumonia in advanced MS</td><td>MAI</td></tr><tr><td>SP</td><td>M</td><td>58</td><td>09:15</td><td></td><td>30</td><td>Pneumonia by terminal renal insufficiency</td><td>Inactive</td></tr><tr><td>SP</td><td>M</td><td>63</td><td>08:15</td><td>6.41</td><td>27</td><td>Pneumonia, cachexia and dehydration</td><td>Inactive</td></tr><tr><td>PP</td><td>M</td><td>59</td><td>10:45</td><td>6.5</td><td>12</td><td>Euthanasia</td><td>Inactive</td></tr><tr><td rowspan="15">Non-demented controls</td><td>F</td><td>51</td><td>07:40</td><td>7.05</td><td></td><td>Sepsis</td><td></td></tr><tr><td>F</td><td>60</td><td>08:25</td><td>6.6</td><td></td><td>Euthanasia</td><td></td></tr><tr><td>F</td><td>65</td><td>12:50</td><td>6.94</td><td></td><td>Cardiac arrest</td><td></td></tr><tr><td>F</td><td>78</td><td>04:15</td><td>6.2</td><td></td><td>Pneumonia, CVA, seizures.</td><td></td></tr><tr><td>F</td><td>52</td><td>06:50</td><td>7.16</td><td></td><td>Euthanasia</td><td></td></tr><tr><td>F</td><td>46</td><td>10:25</td><td>5.8</td><td></td><td>Multi-organ failure</td><td></td></tr><tr><td>F</td><td>50</td><td>04:10</td><td>6.98</td><td></td><td>Metastasized large cell bronchocarcinoma</td><td></td></tr><tr><td>F</td><td>70</td><td>06:15</td><td>6.55</td><td></td><td>Pneumonia (pulmonary carcinoma)</td><td></td></tr><tr><td>F</td><td>64</td><td>05:40</td><td>6.35</td><td></td><td>Palliative sedation</td><td></td></tr><tr><td>F</td><td>57</td><td>07:40</td><td>6.47</td><td></td><td>Euthanasia with metastatic urothelial cancer</td><td></td></tr><tr><td>M</td><td>45</td><td>08:50</td><td>6.71</td><td></td><td>Hypovolemic shock (melaena from intestinal metastasis).</td><td></td></tr><tr><td>M</td><td>51</td><td>06:00</td><td>6.5</td><td></td><td>Ileus (liposarcoma)</td><td></td></tr><tr><td>M</td><td>75</td><td>07:20</td><td>6.16</td><td></td><td>Sepsis (adenocarcinoma)</td><td></td></tr><tr><td>M</td><td>49</td><td>09:50</td><td></td><td></td><td>Sudden death</td><td></td></tr><tr><td>M</td><td>53</td><td>14:25</td><td>6.58</td><td></td><td>Heart failure in Diabetes Mellitus type II</td><td></td></tr><tr><td>M</td><td>51</td><td>07:44</td><td></td><td></td><td>Ventricle fibrillation</td><td></td></tr><tr><td>M</td><td>51</td><td>07:45</td><td>7.05</td><td></td><td>Suicide by refusing food and water.</td><td></td></tr><tr><td>M</td><td>49</td><td>06:15</td><td>6.23</td><td></td><td>Euthanasia</td><td></td></tr><tr><td>M</td><td>55</td><td>07:30</td><td>6.88</td><td></td><td>Euthanasia with esophageal cancer</td><td></td></tr><tr><td>M</td><td>55</td><td>07:15</td><td></td><td></td><td>Intestinal ischemia</td><td></td></tr><tr><td>Summary statistics</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">Multiple sclerosis</td><td>F</td><td>59.6 ±</td><td>9.0 ±</td><td>6.3 ±</td><td>34.7 ± 17.6</td><td></td><td></td><td></td></tr><tr><td>F</td><td>11.1</td><td>2.0</td><td>0.16</td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>57.1 ±</td><td>9.2 ±</td><td>6.3 ±</td><td>26.3 ± 14.4</td><td></td><td></td><td></td></tr><tr><td>M</td><td>10.7</td><td>1.4</td><td>0.22</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">Non-demented control</td><td>F</td><td>59.3 ±</td><td>7.4 ±</td><td>6.6 ±</td><td></td><td></td><td></td><td></td></tr><tr><td>F</td><td>10.1</td><td>2.7</td><td>0.43</td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>53.4 ± 8.2</td><td>8.3 ±</td><td>6.6 ±</td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td></td><td>2.4</td><td>0.32</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<caption>**Table 2**</caption>

Characteristics of MS patients and control donors used for steroid measurements by GC/MS and gene expression measurements in remyelinated lesion. Disease duration given in years. Abbreviations: CVA = cerebrovascular accident; MS = multiple sclerosis; PMD = post-mortem delay (hours:minutes); PP = primary progressive; SP = secondary progressive. pH = pH of cerebrospinal fluid post-mortem. The last two columns indicate which patients were used for qPCR of gene expression and GC/MS respectively. Summary statistics are given in the form of mean ± standard deviation.

<table><thead><tr><th>Group</th><th>MS type</th><th>Sex</th><th>Age</th><th>PMD</th><th>pH</th><th>Disease duration</th><th>Cause.of.death</th><th>qPCR</th><th>GC/MS</th></tr></thead><tbody><tr><td rowspan="20">MS</td><td>SP</td><td>F</td><td>49</td><td>05:45</td><td>6.43</td><td>49</td><td>Metastasized breast carcinoma.</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>F</td><td>80</td><td>09:35</td><td>6.2</td><td>59</td><td>Heart failure or acute leukaemia</td><td>✓</td><td>✓</td></tr><tr><td>PP</td><td>F</td><td>77</td><td>10:00</td><td>6.5</td><td>29</td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>F</td><td>59</td><td>04:45</td><td>7.08</td><td>25</td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>F</td><td>61</td><td>09:55</td><td>5.99</td><td>29</td><td>Peri-renal abscess</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>F</td><td>66</td><td>06:00</td><td>6.18</td><td>23</td><td>Unknown</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>F</td><td>48</td><td>11:40</td><td>6.21</td><td>23</td><td>Hepatic encephalopathy</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>47</td><td>07:15</td><td>6.2</td><td>7</td><td>Urosepsis with organ failure</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>55</td><td>06:20</td><td>6.7</td><td>31</td><td>Respiratory insufficiency by pneumonia and urosepsis.</td><td>✓</td><td>✓</td></tr><tr><td>PP</td><td>M</td><td>82</td><td>05:35</td><td>6.42</td><td>32</td><td>Pneumonia</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>63</td><td>07:05</td><td>6.48</td><td>24</td><td>Abdominal aorta aneurysm</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>57</td><td>07:55</td><td>6.5</td><td>57</td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>75</td><td>07:45</td><td>6.53</td><td>51</td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>SP</td><td>M</td><td>73</td><td>06:45</td><td>6.41</td><td>22</td><td>Shock, ileus</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>60</td><td>08:25</td><td>6.6</td><td></td><td></td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>78</td><td>07:30</td><td>6.6</td><td></td><td></td><td>Terminal pulmonary emphysema/heart failure</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>76</td><td>04:50</td><td>7.2</td><td></td><td></td><td>ischemic CVA (tumour-induced)</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>61</td><td>06:50</td><td>6.5</td><td></td><td></td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>81</td><td>06:40</td><td>7.16</td><td></td><td></td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>65</td><td>12:50</td><td>6.94</td><td></td><td></td><td>Cardiac arrest</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>52</td><td>06:50</td><td>7.16</td><td></td><td></td><td>Euthanasia</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>61</td><td>10:15</td><td>7.18</td><td></td><td></td><td>Pneumonia and cachexia; metastasized ovarium carcinoma</td><td>✓</td><td>✓</td></tr><tr><td>F</td><td>50</td><td>04:10</td><td>6.98</td><td></td><td></td><td>Metastasized large cell bronchocarcinoma</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>49</td><td>09:50</td><td></td><td></td><td></td><td>Sudden death</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>51</td><td>07:44</td><td></td><td></td><td></td><td>Ventricle fibrillation</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>59</td><td>04:25</td><td>7.23</td><td></td><td></td><td>Myocardial infarct and cardiac decompensation.</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>70</td><td>07:30</td><td>6.4</td><td></td><td></td><td>Pancreas carcinoma</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>82</td><td>07:40</td><td>6.07</td><td></td><td></td><td>Heart attack</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>56</td><td>14:00</td><td>7.03</td><td></td><td></td><td>Unknown</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>56</td><td>09:15</td><td>6.54</td><td></td><td></td><td>Myocardial infarction</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>62</td><td>07:20</td><td>6.36</td><td></td><td></td><td>Unknown</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>75</td><td>07:20</td><td>6.16</td><td></td><td></td><td>Sepsis (adenocarcinoma)</td><td>✓</td><td>✓</td></tr><tr><td>M</td><td>82</td><td>05:10</td><td>6.75</td><td></td><td></td><td>Pneumonia</td><td>✓</td><td>✓</td></tr><tr><td colspan="10">Summary statistics (qPCR)</td></tr><tr><td rowspan="2">MS</td><td>F</td><td>62.9 ± 12.5</td><td>8.2 ± 2.7</td><td>6.4 ± 0.36</td><td>33.9 ± 14.3</td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>64.6 ± 12.5</td><td>7.0 ± 0.81</td><td>6.5 ± 0.15</td><td>32.0 ± 17.2</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Non-demented control</td><td>F</td><td>63.9 ± 11.9</td><td>8.1 ± 2.8</td><td>6.9 ± 0.25</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>64.3 ± 11.8</td><td>8.6 ± 2.8</td><td>6.5 ± 0.31</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="10">Summary statistics (GC/MS)</td></tr><tr><td rowspan="2">MS</td><td>F</td><td>65.3 ± 11.6</td><td>7.7 ± 2.4</td><td>6.4 ± 0.38</td><td>35.7 ± 14.7</td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>63.2 ± 13.1</td><td>7.0 ± 0.89</td><td>6.5 ± 0.16</td><td>33.7 ± 18.2</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Non-demented control</td><td>F</td><td>67.6 ± 10.9</td><td>7.7 ± 2.5</td><td>6.9 ± 0.31</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>M</td><td>60.6 ± 10.8</td><td>8.5 ± 2.7</td><td>6.6 ± 0.44</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

lesions or inactive lesions were used for steroid measurements. Mixed active/inactive lesions are demyelinating lesions with a sclerotic scar and HLA-positive microglia at the border. Mixed lesions were chosen which had amoeboid and foamy microglial morphologies. Inactive lesions contain a demyelinated hypocellular sclerotic scar and few or no microglia. Blocks were trimmed with a scalpel to remove as much surrounding normal appearing white or grey matter as possible, guided by the aforementioned HLA/PLP staining. Trimmed blocks were then processed for steroid measurements. White matter from control patients was similarly trimmed of grey matter and processed.

From fresh-frozen blocks containing remyelinated lesions, lesion areas were excised for gene expression analysis as follows. Briefly, 5–10 cryostat sections of 60 µm thickness were taken and the lesion area dissected from the sections at –20 °C with a scalpel. Sudan black staining was carried out on 10 µm sections taken at the beginning and after every 3–4 sections to visualize the lesions. The lesion itself and nearby normal-appearing white matter (NAWM) were taken separately. A detailed description of the dissection procedure was given previously (Luchetti et al., 2014). The remainder of these blocks were then trimmed as described above and processed for steroid measurements. Control
donor white matter was also processed for RNA extraction and steroid measurement for direct comparison. All lesions and control white matter were from subcortical locations.

## 2.3. Primary microglia

White matter containing a lesion, or normal appearing white matter, was obtained as fresh subcortical white matter tissue at autopsy from MS donors, and white matter obtained similarly from control donors. Lesions were not characterized for this experiment. Microglia were isolated from these tissue samples using density gradient centrifugation and magnetic beads coupled to CD11b as described (Mizee et al., 2017). Isolated microglia were seeded in 96 well plates at a density of 150.000 cells per well in 200 µl DMEM/F12 medium with L-glutamine and without phenol red, with 10 % charcoal-stripped fetal bovine serum (FBS) and 1 % penicillin/streptomycin (PS), and incubated for 1 day before steroid treatment.

## 2.4. Fetal astrocytes

Primary fetal astrocyte-enriched cell cultures were obtained from human fetal brain tissue (cortex, 14–19 gestational weeks) obtained from medically induced abortions (n = 3) with 2–4 replications per donor. All material was collected from donors from whom a written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic, The Netherlands. Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code provided by the Medical Ethics Committee of the Amsterdam UMC. Isolation of astrocytes was carried out as described previously (van Scheppingen et al., 2016). Cells from passage 1–4 were taken and passaged in to phenol-red free DMEM/F10 medium with 10 % charcoal stripped FBS and 1 % PS.

## 2.5. Steroid measurements by gas chromatography/ mass spectrometry (GC/MS)

Mixed active/inactive, inactive, and remyelinated lesions, and control donor WM were extracted with methanol. After adding precise amounts of stable isotope-labelled internal standards for quantification purpose, the extracts were purified and fractionated by solid-phase extraction with the recycling procedure (Liere et al., 2004), by high performance liquid chromatography, derivatized and analysed by GC/MS as described previously (Gonzalez Deniselle et al., 2016; Hertig et al., 2010; Luchetti et al., 2023) using an AS 3000 autosampler (ThermoFisher Scientific, USA), and a Focus gas chromatograph (GC) coupled with a DSQII mass spectrometer (ThermoFisher Scientific). The full list of steroids targeted for measurement and their abbreviations are given in Table 3. The detailed GC/MS methodology is given in Supplemental Methods. The diagnostic ions and the validation parameters (limit of quantification, intra- and inter-assay precision, and accuracy) for our analytical methodology are given in Supplemental Table 2. The final numbers of samples are also given in Tables 4 and 5, along with the numbers of samples below the limit of quantitation.

5α-DHADIONE measurements in control samples for the mixed and active lesion cohort and all but 2 cortisone measurements in the remyelinated cohort were lost due to sample degradation or interference. Values are presented for successfully measured samples but these were not included in further analyses.

## 2.6. RNA extraction and qPCR

Remyelinated lesions excised from frozen sections as described above were dissolved in Trisure and RNA extracted as previously described (Luchetti et al., 2014). RNA purity was determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). The RNA integrity number was measured on the Agilent

2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All tissue RNA samples used in this study had RNA integrity values of at least 6. RNA was extracted from Trisure-lysed cultured primary microglia and fetal astrocytes as described (Luchetti et al., 2014). cDNA was generated using QuantiTect reverse transcription kits (Qiagen). A dilution of 1:10 of the total cDNA yield was used for the qPCR experiments.

Expression of the following genes were measured for each experiment. Primary microglia: BDNF, CCL18, CD163, CD206, HLA, IL10, IL1B, IL4, IL6, LIF, TNF, with reference genes TUBA and GAPDH; fetal astrocytes: APOE, BDNF, GFAP, GLAST1, GLT1, IGF1, IL10, IL1B, IL6, LIF, with reference genes EF1A and GAPDH; remyelinated lesions: AKR1C2, AKR1C3, AR, AROM, BDNF, ESR1 (ERα), ESR2 (ERβ), HSD17B10, HSD3B, PR, SRD5A, TSPO, with reference genes AARS and GAPDH. Primers for qPCR are given in Supplemental Table 3.

Cycle threshold (CT) values were normalized to the reference gene values by subtracting them from the average CT of the reference genes. Primer efficiencies were taken as 2, thus this calculation gives a relative expression level on a log₂ scale. These values were used for statistical analysis.

## 2.7. Steroid treatment of cultured cells

The following steroids were used: E2 (Sigma-Aldrich; E2758), PROG (Tocris, Abingdon, UK; #2835), T (Sigma-Aldrich; T1500) and DHT (Sigma-Aldrich; A8380). Stock concentrations of each steroid at 1 µM, 10 µM, 100 µM and 1 mM were dissolved in vehicle (ethanol) to make 10,000× stocks. For addition to cultures, these, or vehicle, were diluted 1:100 in culture medium. These dilutions were then added to the medium in the culture wells in a further 1:100 dilution. Cells ready for steroid treatment were cultured in medium without phenol red since this can mimic steroid activity (Ernst et al., 1989). We also used fetal bovine serum (FBS) that was charcoal stripped to remove steroids therein. In human primary microglia, E2, PROG, T or vehicle dilution were added one day after seeding to a final concentration of 0.1 nM, 1 nM, 10 nM or 100 nM along with fresh medium. Cells were incubated for 2 days and then medium was removed and the cells harvested by resuspending them in 800 µl Trisure (Bioline, Taunton, MA, USA). In fetal astrocytes, steroids (E2, DHT, PROG) or vehicle were added to the cultures (E2 at 1 nM, 10 nM or 100 nM; DHT and PROG at 100 nM only). After 3 h, TNFα (50 ng/ml) (Peprotech, 300-01 A), IL1β (10 ng/ml) (Peprotech, 200-01B), or vehicle (PBS/0.1 % bovine serum albumin) were added and the cells incubated for a further 3 h. Then, medium was removed and the cells were harvested in 800 µl Trisure.

## 2.8. Statistics

All statistical analysis was carried out in the statistical programming environment R (R Core Team, 2019). Steroid levels in lesions as measured by GC/MS were analysed as follows. Normality of the data was assessed by plotting the residuals of a linear model and it was seen that the data were not normally distributed, even after log transformation, mainly due to a high frequency of extreme values. Within genders, MS and control patients were compared with Wilcoxon tests. To determine if male and females respond differently to disease state, robust linear regression on log-transformed data was used, and the interaction of disease state with sex was tested. This was done using package 'robustbase' (Maechler et al., 2020). Values that were below the detection limit were taken to be at this limit. Extreme outlier values were excluded from the measurement of the remyelinated lesions. P-values for Wilcoxon tests and sex differences in response to MS were adjusted for multiple testing with the Benjamini-Hochberg method.

Effects of storage time on steroid levels were assessed by inclusion of storage time in years as a co-factor in the robust linear model and comparison to the model without (Wald test). Storage-time corrected values were calculated using the co-efficient obtained from this model and used for Wilcoxon tests for within sex comparisons.
<caption>**Table 3**</caption>

List of steroids targeted for measurement by GC/MS in inactive and mixed active/inactive lesions, and in remyelinated lesions. The table also indicates which steroids were detected. 35 steroids were detected in inactive and mixed active/inactive lesions, and 20 steroids were detected in remyelinated lesions.

<table><thead><tr><th>Steroid</th><th>Abbreviation</th><th>Targeted for measurement in inactive and mixed lesions</th><th>Detected in inactive and mixed lesions</th><th>Targeted for measurement in remyelinated lesions</th><th>Detected in remyelinated lesions</th></tr></thead><tbody><tr><td>Pregnenolone and metabolites</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pregnenolone</td><td>PREG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>20α-dihydro-pregnenolone</td><td>20α-DHPREG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>17α-hydroxy-pregnenolone</td><td>17α-OH-PREG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>pregnenolone sulphate</td><td>PREGS</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>Progestagens and metabolites</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>progesterone</td><td>PROG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α-dihydroprogesterone</td><td>5α-DHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5β-dihydroprogesterone</td><td>5β-DHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5α-tetrahydro-progesterone</td><td>ALLO</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5β-tetrahydro-progesterone</td><td>3α5β-THP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3β5α-tetrahydro-progesterone</td><td>3β5α-THP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>20α-dihydroprogesterone</td><td>20α-DHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α20α-tetrahydro-progesterone</td><td>5α20α-THP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5α20α-hexahydro-progesterone</td><td>3α5α20α-HHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5β20α-hexahydro-progesterone</td><td>3α5β20α-HHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3β5α20α-hexahydro-progesterone</td><td>3β5α20α-HHP</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>16α-hydroxy-progesterone</td><td>16α-OH-PROG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>17α-hydroxy-progesterone</td><td>17α-OH-PROG</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>Corticosteroids and metabolites</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>11-deoxycorticosterone</td><td>DOC</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α-dihydro-deoxycorticosterone</td><td>5α-DHDOC</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5α-tetrahydro-deoxycorticosterone</td><td>3α5α-THDOC</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>corticosterone</td><td>B</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α-dihydro-corticosterone</td><td>5α-DHB</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5α-tetrahydro-corticosterone</td><td>3α5α-THB</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5β-tetrahydro-corticosterone</td><td>3α5β-THB</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3β5α-tetrahydro-corticosterone</td><td>3β5α-THB</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>aldosterone</td><td>Aldosterone</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>11-deoxycortisol</td><td>11-deoxycortisol</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>cortisol</td><td>Cortisol</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>cortisone</td><td>Cortisone</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>Androgens and metabolites</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>androstenediol</td><td>ADIOL</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>androstenedione</td><td>ADIONE</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α-dihydroandrostenedione</td><td>5α-DHADIONE</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5β-dihydroandrostenedione</td><td>5β-DHADIONE</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>dehydroepiandrosterone</td><td>DHEA</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>dehydroepiandrosterone sulphate</td><td>DHEAS</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>testosterone</td><td>T</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5α-dihydrotestosterone</td><td>5α-DHT</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3α5α-tetrahydrotestosterone</td><td>3α5α-THT</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>3β5α-tetrahydrotestosterone</td><td>3β5α-THT</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>epiandrosterone</td><td>epiandrosterone</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>etiocholanolone</td><td>etiocholanolone</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>Estrogens</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>estrone</td><td>E1</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>17β-estradiol</td><td>E2</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>2-methoxyestradiol</td><td>2-ME2</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>2-hydroxy-estradiol</td><td>2-OH-E2</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>estriol</td><td>E3</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>estetrol</td><td>E4</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr></tbody></table>
<caption>**Table 4**</caption>

Measurements of steroids by GC/MS in male and female mixed active/inactive lesions and inactive lesions of MS patients and control donors. All values are given as mean ± standard deviation (SD) in ng/g, with numbers of samples below the limit of quantification given in brackets (when this was not zero). N for each measurement is given to the right of the mean and SD. For the steroid abbreviations, see Table 3.

<table><thead><tr><th rowspan="2">Steroid</th><th colspan="2">Control donors, female</th><th colspan="2">Control donors, male</th><th colspan="2">MS donors, female</th><th colspan="2">MS donors, male</th></tr><tr><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th></tr></thead><tbody><tr><td>PREG</td><td>28.7 ± 24.2</td><td>10</td><td>14.7 ± 13.9</td><td>10</td><td>59.0 ± 75.6</td><td>17</td><td>19.7 ± 20.0</td><td>16</td></tr><tr><td>20α-DHPREG</td><td>1.3 ± 1.2</td><td>10</td><td>0.74 ± 0.31</td><td>10</td><td>0.083 ± 0.022</td><td>17</td><td>1.2 ± 0.31</td><td>16</td></tr><tr><td>17α-OH-PREG</td><td>6.5 ± 8.1 (1)</td><td>10</td><td>2.9 ± 4.4 (3)</td><td>10</td><td>6.7 ± 7.8</td><td>17</td><td>3.2 ± 2.6</td><td>16</td></tr><tr><td>PREGS</td><td>0.72 ± 0.55</td><td>10</td><td>0.86 ± 0.75</td><td>10</td><td>1.0 ± 1.0</td><td>16</td><td>0.56 ± 0.22</td><td>16</td></tr><tr><td>PROG</td><td>0.10 ± 0.15</td><td>10</td><td>0.12 ± 0.12</td><td>10</td><td>0.30 ± 0.75 (2)</td><td>16</td><td>0.028 ± 0.024</td><td>16</td></tr><tr><td>5α-DHP</td><td>0.82 ± 0.72</td><td>10</td><td>0.55 ± 0.49 (2)</td><td>10</td><td>1.7 ± 2.0</td><td>16</td><td>0.75 ± 0.93</td><td>16</td></tr><tr><td>5β-DHP</td><td>&lt; 0.05 (10)</td><td>10</td><td>&lt; 0.05 (10)</td><td>10</td><td>&lt; 0.05 (17)</td><td>17</td><td>&lt; 0.05 (16)</td><td>16</td></tr><tr><td>ALLO</td><td>0.071 ± 0.057 (2)</td><td>10</td><td>0.061 ± 0.085</td><td>10</td><td>0.69 ± 0.48</td><td>17</td><td>0.11 ± 0.13</td><td>16</td></tr><tr><td>3α5β-THP</td><td>0.066 ± 0.076 (2)</td><td>10</td><td>0.060 ± 0.13 (3)</td><td>10</td><td>0.18 ± 0.27</td><td>17</td><td>0.078 ± 0.051</td><td>16</td></tr><tr><td>3β5α-THP</td><td>0.0087 ± 0.0075</td><td>10</td><td>0.0075 ± 0.0092</td><td>10</td><td>0.0026 ± 0.0032 (5)</td><td>17</td><td>0.0095 ± 0.0079</td><td>16</td></tr><tr><td>20α-DHP</td><td>1.0 ± 0.87</td><td>10</td><td>0.86 ± 1.0</td><td>10</td><td>2.4 ± 2.8</td><td>17</td><td>0.84 ± 1.0</td><td>16</td></tr><tr><td>5α20α-THP</td><td>0.084 ± 0.064</td><td>10</td><td>0.076 ± 0.10</td><td>10</td><td>0.21 ± 0.22</td><td>17</td><td>0.12 ± 0.13</td><td>16</td></tr><tr><td>3α5α20α-HHP</td><td>0.19 ± 0.14</td><td>10</td><td>0.12 ± 0.16</td><td>10</td><td>0.47 ± 0.57</td><td>17</td><td>0.17 ± 0.20</td><td>16</td></tr><tr><td>3α5β20α-HHP</td><td>0.19 ± 0.23</td><td>10</td><td>0.19 ± 0.49 (1)</td><td>10</td><td>0.41 ± 0.45</td><td>17</td><td>0.089 ± 0.052</td><td>16</td></tr><tr><td>3β5α20α-HHP</td><td>0.024 ± 0.021</td><td>10</td><td>0.016 ± 0.018 (2)</td><td>10</td><td>0.047 ± 0.057</td><td>17</td><td>0.015 ± 0.018 (1)</td><td>16</td></tr><tr><td>16α-OH-PROG</td><td>0.48 ± 0.90 (6)</td><td>10</td><td>0.56 ± 0.59 (4)</td><td>10</td><td>0.50 ± 0.53 (3)</td><td>17</td><td>2.3 ± 2.8 (2)</td><td>16</td></tr><tr><td>17α-OH-PROG</td><td>4.5 ± 8.4 (1)</td><td>10</td><td>1.4 ± 1.5 (1)</td><td>10</td><td>3.0 ± 3.7</td><td>17</td><td>1.8 ± 1.8</td><td>16</td></tr><tr><td>DOC</td><td>0.43 ± 0.63</td><td>10</td><td>0.22 ± 0.21</td><td>10</td><td>0.50 ± 0.50 (1)</td><td>17</td><td>0.44 ± 0.96</td><td>16</td></tr><tr><td>5α-DHDOC</td><td>0.023 ± 0.053 (8)</td><td>10</td><td>0.017 ± 0.020 (6)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>0.10 ± 0.15</td><td>16</td></tr><tr><td>3α5α-THDOC</td><td>0.0061 ± 0.0041 (2)</td><td>10</td><td>0.0042 ± 0.0028 (1)</td><td>10</td><td>0.016 ± 0.017 (6)</td><td>17</td><td>0.012 ± 0.034 (12)</td><td>16</td></tr><tr><td>B</td><td>10.3 ± 7.2</td><td>10</td><td>16.5 ± 13.3</td><td>10</td><td>48.3 ± 24.7</td><td>17</td><td>21.8 ± 14.2</td><td>16</td></tr><tr><td>5α-DHB</td><td>&lt; 0.02 (10)</td><td>10</td><td>&lt; 0.02 (10)</td><td>10</td><td>&lt; 0.02 (17)</td><td>17</td><td>&lt; 0.02 (16)</td><td>16</td></tr><tr><td>3α5α-THB</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>&lt; 0.005 (16)</td><td>16</td></tr><tr><td>3α5β-THB</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>&lt; 0.005 (16)</td><td>16</td></tr><tr><td>3β5α-THB</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>&lt; 0.005 (16)</td><td>16</td></tr><tr><td>Aldosterone</td><td>&lt; 1 (10)</td><td>10</td><td>&lt; 1 (10)</td><td>10</td><td>&lt; 1 (17)</td><td>17</td><td>&lt; 1 (16)</td><td>16</td></tr><tr><td>11-deoxycortisol</td><td>&lt; 0.5 (10)</td><td>10</td><td>&lt; 0.5 (10)</td><td>10</td><td>&lt; 0.5 (17)</td><td>17</td><td>&lt; 0.5 (16)</td><td>16</td></tr><tr><td>Cortisol</td><td>119.6 ± 114.4</td><td>10</td><td>81.0 ± 95.1</td><td>10</td><td>88.4 ± 72.6</td><td>17</td><td>47.9 ± 33.8</td><td>16</td></tr><tr><td>Cortisone</td><td>13.8 ± 8.9</td><td>10</td><td>12.0 ± 8.5</td><td>9</td><td>6.5 ± 4.8</td><td>15</td><td>11.8 ± 6.1</td><td>15</td></tr><tr><td>ADIOL</td><td>0.59 ± 0.55</td><td>10</td><td>0.39 ± 0.32</td><td>10</td><td>0.087 ± 0.058</td><td>17</td><td>0.46 ± 0.16</td><td>16</td></tr><tr><td>ADIONE</td><td>0.31 ± 0.35</td><td>10</td><td>0.27 ± 0.23</td><td>10</td><td>0.27 ± 0.17</td><td>17</td><td>0.11 ± 0.078 (2)</td><td>16</td></tr><tr><td>5α-DHADIONE</td><td>no data</td><td>10</td><td>no data</td><td>10</td><td>&lt; 0.02 (17)</td><td>17</td><td>0.70 ± 0.93</td><td>16</td></tr><tr><td>5β-DHADIONE</td><td>&lt; 0.02 (10)</td><td>10</td><td>&lt; 0.02 (10)</td><td>10</td><td>&lt; 0.02 (17)</td><td>17</td><td>&lt; 0.02 (16)</td><td>16</td></tr><tr><td>DHEA</td><td>5.6 ± 6.1</td><td>10</td><td>5.5 ± 6.7</td><td>10</td><td>1.3 ± 1.1</td><td>17</td><td>6.6 ± 5.8</td><td>16</td></tr><tr><td>DHEAS</td><td>5.2 ± 5.5</td><td>10</td><td>5.4 ± 5.8</td><td>10</td><td>10.4 ± 14.2</td><td>17</td><td>3.2 ± 2.0</td><td>16</td></tr><tr><td>T</td><td>0.22 ± 0.35</td><td>10</td><td>0.38 ± 0.28</td><td>10</td><td>0.23 ± 0.13</td><td>17</td><td>0.17 ± 0.16</td><td>16</td></tr><tr><td>5α-DHT</td><td>0.0066 ± 0.0080 (3)</td><td>10</td><td>0.012 ± 0.0066</td><td>10</td><td>0.0011 ± 6.3 × 10<sup>-4</sup> (6)</td><td>17</td><td>0.034 ± 0.084 (1)</td><td>16</td></tr><tr><td>3α5α-THT</td><td>0.019 ± 0.013 (2)</td><td>10</td><td>0.023 ± 0.020 (2)</td><td>10</td><td>0.0014 ± 7.8 × 10<sup>-4</sup> (2)</td><td>17</td><td>0.019 ± 0.012</td><td>16</td></tr><tr><td>3β5α-THT</td><td>0.019 ± 0.013 (2)</td><td>10</td><td>0.030 ± 0.024</td><td>10</td><td>0.0010 ± 5.1 × 10<sup>-4</sup> (5)</td><td>17</td><td>0.0066 ± 0.0053 (4)</td><td>16</td></tr><tr><td>Epiandrosterone</td><td>0.022 ± 0.027 (1)</td><td>10</td><td>0.031 ± 0.027</td><td>10</td><td>0.0051 ± 0.0041 (1)</td><td>17</td><td>0.022 ± 0.0089</td><td>16</td></tr><tr><td>Etiocholanolone</td><td>0.18 ± 0.22</td><td>10</td><td>0.17 ± 0.31</td><td>10</td><td>0.23 ± 0.20</td><td>17</td><td>0.11 ± 0.086</td><td>16</td></tr><tr><td>E1</td><td>0.44 ± 0.60</td><td>10</td><td>0.78 ± 1.4</td><td>10</td><td>0.19 ± 0.21</td><td>17</td><td>0.15 ± 0.22</td><td>16</td></tr><tr><td>E2</td><td>0.12 ± 0.16</td><td>10</td><td>0.21 ± 0.34</td><td>10</td><td>0.055 ± 0.050</td><td>17</td><td>0.049 ± 0.058</td><td>16</td></tr><tr><td>2-ME2</td><td>0.0052 ± 0.013 (8)</td><td>10</td><td>0.0092 ± 0.017 (6)</td><td>10</td><td>0.0025 ± 0.0019 (9)</td><td>17</td><td>0.0013 ± 4.5 × 10<sup>-4</sup> (12)</td><td>16</td></tr><tr><td>2-OH-E2</td><td>&lt; 0.05 (10)</td><td>10</td><td>&lt; 0.05 (10)</td><td>10</td><td>&lt; 0.05 (17)</td><td>17</td><td>&lt; 0.05 (16)</td><td>16</td></tr><tr><td>E3</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>&lt; 0.005 (16)</td><td>16</td></tr><tr><td>E4</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (10)</td><td>10</td><td>&lt; 0.005 (17)</td><td>17</td><td>&lt; 0.005 (16)</td><td>16</td></tr></tbody></table>

Gene expression changes in primary microglia in response to steroids were analysed with linear mixed models (LMMs), with patient ID as a random effect, and *p*-values were calculated with a likelihood-ratio test. Gene expression changes in fetal astrocytes were analysed with a linear model with an F-test for each combination of steroid and inflammatory cytokine added (TNFA, IL1B or none). For each cell type, the *p*-values reported for F-tests were adjusted for multiple testing correction using the Benjamini-Hochberg method. Where these were significant, Dunnett post-hoc tests were carried out using package 'multcomp' (Hothorn et al., 2008) with vehicle designated as the control.

Gene expression changes in remyelinated lesions were assessed with linear models (F-test). *p*-values for the F-test were corrected for multiple testing with the Benjamini-Hochberg method, and Tukey's post-hoc test was used where these were significant (*p* < 0.05).

## 3. Results

### 3.1. Steroid metabolome changes in MS mixed active/inactive and inactive lesions

We first examined whether steroid levels in MS lesions were different to control donor white matter in males and females. The donors used for steroid measurements had an average age of 58 ± 10 years, average post-mortem delay 8.6 ± 2.2 h, and disease duration was 31 ± 16 years. Among the 30 MS cases, 20 were of secondary progressive (SP) type, 7 of primary progressive (PP) type and 3 of progressive relapsing (PR) type. There were no significant differences in age, post-mortem delay (PMD), pH of CSF MS and control donor groups, or between males and females. Donor characteristics and summary statistics per group are given in Table 1.

Among the 46 targeted steroids, we detected and measured 35 steroids in inactive and mixed active/inactive lesions (Table 3). We found that steroid levels were similar in the two lesion types with no significant differences in either males or females (see Supp. Fig. S1). Therefore, to
<caption>**Table 5**</caption>

Measurements of steroids by GC/MS in male and female remyelinated lesions of MS patients and control donors. All values are given as mean ± standard deviation (SD) in ng/g, with numbers of samples below the limit of quantification given in brackets (when this was not zero). N for each measurement is given to the right of the mean and SD. For the steroid abbreviations, see Table 3.

<table><caption>**Table 5 (continued)**</caption><thead><tr><th rowspan="2">Steroid</th><th colspan="2">Control donors, female</th><th colspan="2">Control donors, male</th><th colspan="2">MS donors, female</th><th colspan="2">MS donors, male</th></tr><tr><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th></tr></thead><tbody><tr><td rowspan="3">PREG</td><td>48.6 ±</td><td>7</td><td>13.9 ±</td><td>6</td><td>12.3 ±</td><td>5</td><td>12.9 ±</td><td>5</td></tr><tr><td>52.1 ±</td><td>7</td><td>8.1 ±</td><td>6</td><td>7.1 ±</td><td>5</td><td>16.9 ±</td><td>5</td></tr><tr><td>1.5 ±</td><td>6</td><td>5.1 ±</td><td>7</td><td>0.88 ±</td><td>5</td><td>2.7 ±</td><td>6</td></tr><tr><td rowspan="2">20α-DHPREG</td><td>0.49 ±</td><td>6</td><td>3.1 ±</td><td>7</td><td>0.47 ±</td><td>5</td><td>2.3 ±</td><td>6</td></tr><tr><td>0.34 ±</td><td>6</td><td>0.34 ±</td><td>7</td><td>0.21 ±</td><td>5</td><td>0.34 ±</td><td>6</td></tr><tr><td rowspan="2">PREGS</td><td>(1) ±</td><td>6</td><td>0.63 ±</td><td>7</td><td>0.12 ±</td><td>5</td><td>0.15 ±</td><td>6</td></tr><tr><td>0.66 ±</td><td>6</td><td>0.54 ±</td><td>7</td><td>0.23 ±</td><td>5</td><td>0.43 ±</td><td>5</td></tr><tr><td rowspan="2">PROG</td><td>0.63 ±</td><td>6</td><td>0.76 ±</td><td>6</td><td>0.19 ±</td><td>5</td><td>0.45 ±</td><td>5</td></tr><tr><td>0.27 ±</td><td>6</td><td>0.27 ±</td><td>6</td><td>0.033 ±</td><td>5</td><td>0.17 ±</td><td>5</td></tr><tr><td rowspan="3">5α-DHP</td><td>2.5 ±</td><td>7</td><td>0.22 ±</td><td>7</td><td>1.3 ±</td><td>6</td><td>0.096 ±</td><td>5</td></tr><tr><td>1.9 ±</td><td>7</td><td>0.11 ±</td><td>7</td><td>1.2 ±</td><td>6</td><td>0.10 (4) ±</td><td>5</td></tr><tr><td>3.3 ±</td><td>7</td><td>0.15 ±</td><td>7</td><td>2.1 ±</td><td>6</td><td>2.1 ±</td><td>6</td></tr><tr><td rowspan="2">ALLO</td><td>2.4 ±</td><td>7</td><td>1.5 (2) ±</td><td>7</td><td>1.0 ±</td><td>6</td><td>1.6 ±</td><td>6</td></tr><tr><td>0.098 ±</td><td>7</td><td>0.12 ±</td><td>7</td><td>0.033 ±</td><td>6</td><td>0.17 ±</td><td>6</td></tr><tr><td rowspan="2">3β5α-THP</td><td>(2) ±</td><td>6</td><td>0.047 ±</td><td>7</td><td>0.012 ±</td><td>5</td><td>0.10 ±</td><td>6</td></tr><tr><td>2.5 ±</td><td>6</td><td>1.5 ±</td><td>7</td><td>0.83 ±</td><td>5</td><td>0.79 ±</td><td>6</td></tr><tr><td rowspan="2">20α-DHP</td><td>2.8 ±</td><td>5</td><td>0.45 ±</td><td>6</td><td>0.44 ±</td><td>5</td><td>0.55 ±</td><td>5</td></tr><tr><td>0.005 ±</td><td>6</td><td>0.005 ±</td><td>7</td><td>0.005 ±</td><td>6</td><td>&lt; 0.005 ±</td><td>6</td></tr><tr><td rowspan="2">3α5α20α-HHP</td><td>(6) ±</td><td>6</td><td>0.075 ±</td><td>7</td><td>0.075 ±</td><td>6</td><td>0.041 ±</td><td>6</td></tr><tr><td>1.2 ±</td><td>6</td><td>0.075 ±</td><td>7</td><td>0.20 ±</td><td>6</td><td>0.098 ±</td><td>6</td></tr><tr><td rowspan="2">3β5α20α-HHP</td><td>0.54 ±</td><td>6</td><td>0.061 ±</td><td>7</td><td>0.063 ±</td><td>6</td><td>0.084 ±</td><td>6</td></tr><tr><td>0.20 ±</td><td>6</td><td>0.052 ±</td><td>7</td><td>0.060 ±</td><td>6</td><td>0.038 ±</td><td>6</td></tr><tr><td rowspan="2">DOC</td><td>0.25 ±</td><td>5</td><td>0.001 ±</td><td>6</td><td>0.001 ±</td><td>5</td><td>0.022 ±</td><td>5</td></tr><tr><td>0.001 ±</td><td>6</td><td>0.001 ±</td><td>7</td><td>0.001 ±</td><td>6</td><td>&lt; 0.001 ±</td><td>6</td></tr><tr><td rowspan="2">3α5α-THDOC</td><td>(6) ±</td><td>6</td><td>9.8 ±</td><td>7</td><td>0.001 ±</td><td>6</td><td>1.6 ±</td><td>5</td></tr><tr><td>21.9 ±</td><td>7</td><td>11.7 ±</td><td>6</td><td>44.7 ±</td><td>6</td><td>2.1 (1) ±</td><td>5</td></tr><tr><td rowspan="2">B</td><td>55.9 ±</td><td>5</td><td>97.3 ±</td><td>7</td><td>149.4 ±</td><td>6</td><td>48.9 ±</td><td>6</td></tr><tr><td>61.8 ±</td><td>5</td><td>99.1 ±</td><td>7</td><td>152.7 ±</td><td>6</td><td>50.5 ±</td><td>6</td></tr><tr><td rowspan="2">Cortisol</td><td>4.4 ±</td><td>1</td><td>no data</td><td>7</td><td>4.0 ±</td><td>1</td><td>no data</td><td>6</td></tr><tr><td>0.22 ±</td><td>6</td><td>0.81 ±</td><td>7</td><td>0.32 ±</td><td>6</td><td>0.32 ±</td><td>6</td></tr><tr><td rowspan="2">Cortisone</td><td>(2) ±</td><td>6</td><td>0.56 ±</td><td>7</td><td>0.29 ±</td><td>6</td><td>0.32 ±</td><td>5</td></tr><tr><td>0.085 ±</td><td>6</td><td>0.052 ±</td><td>7</td><td>0.030 ±</td><td>6</td><td>0.028 ±</td><td>5</td></tr><tr><td rowspan="2">ADIONE</td><td>(2) ±</td><td>6</td><td>0.052 ±</td><td>7</td><td>0.024 ±</td><td>6</td><td>0.0084 ±</td><td>5</td></tr><tr><td>0.085 ±</td><td>6</td><td>0.050 ±</td><td>7</td><td>0.024 ±</td><td>6</td><td>0.0084 ±</td><td>5</td></tr><tr><td rowspan="2">5α-DHADIONE</td><td>0.062 ±</td><td>6</td><td>(3) ±</td><td>6</td><td>(5) ±</td><td>6</td><td>(2) ±</td><td>5</td></tr><tr><td>6.1 ±</td><td>6</td><td>6.8 ±</td><td>6</td><td>4.4 ±</td><td>6</td><td>11.1 ±</td><td>6</td></tr><tr><td rowspan="2">DHEA</td><td>6.5 ±</td><td>7</td><td>5.4 (1) ±</td><td>6</td><td>2.9 ±</td><td>5</td><td>14.1 (1) ±</td><td>6</td></tr><tr><td>0.82 ±</td><td>6</td><td>0.81 ±</td><td>7</td><td>0.32 ±</td><td>6</td><td>0.32 ±</td><td>6</td></tr><tr><td rowspan="2">DHEAS</td><td>0.72 ±</td><td>6</td><td>4.9 ±</td><td>7</td><td>1.7 ±</td><td>6</td><td>1.3 ±</td><td>5</td></tr><tr><td>0.45 ±</td><td>6</td><td>0.68 ±</td><td>7</td><td>0.24 ±</td><td>6</td><td>0.28 ±</td><td>6</td></tr><tr><td rowspan="2">T</td><td>0.59 ±</td><td>6</td><td>0.35 ±</td><td>7</td><td>0.18 ±</td><td>6</td><td>0.28 ±</td><td>6</td></tr><tr><td>&lt; 5e-04 ±</td><td>6</td><td>0.17 ±</td><td>6</td><td>&lt; 5e-04 ±</td><td>6</td><td>0.17 ±</td><td>6</td></tr><tr><td rowspan="2">5α-DHT</td><td>&lt; 5e-04 ±</td><td>6</td><td>0.14 ±</td><td>6</td><td>0.04 (6) ±</td><td>6</td><td>0.17 (2) ±</td><td>6</td></tr><tr><td>&lt; 5e-04 ±</td><td>6</td><td>&lt; 5e-04 ±</td><td>6</td><td>&lt; 5e-04 ±</td><td>6</td><td>&lt; 5e-04 ±</td><td>6</td></tr><tr><td rowspan="2">3α5α-THT</td><td>&lt; 0.001 ±</td><td>6</td><td>0.001 ±</td><td>7</td><td>0.001 ±</td><td>6</td><td>&lt; 0.001 ±</td><td>6</td></tr><tr><td>0.001 ±</td><td>6</td><td>0.001 ±</td><td>7</td><td>0.001 ±</td><td>6</td><td>0.001 ±</td><td>6</td></tr><tr><td rowspan="2">Epiandrosterone</td><td>(7) ±</td><td>7</td><td>0.001 ±</td><td>7</td><td>0.001 ±</td><td>6</td><td>(6) ±</td><td>6</td></tr><tr><td>0.005 ±</td><td>7</td><td>0.005 ±</td><td>7</td><td>0.005 ±</td><td>6</td><td>&lt; 0.005 ±</td><td>6</td></tr><tr><td rowspan="2">Etiocholanolone</td><td>(7) ±</td><td>7</td><td>0.005 ±</td><td>7</td><td>0.005 ±</td><td>6</td><td>(6) ±</td><td>6</td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Steroid</th><th colspan="2">Control donors, female</th><th colspan="2">Control donors, male</th><th colspan="2">MS donors, female</th><th colspan="2">MS donors, male</th></tr><tr><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th><th>ng/g</th><th>n</th></tr></thead><tbody><tr><td rowspan="4">E1</td><td>0.56 ±</td><td>5</td><td>0.005 ±</td><td>6</td><td>1.1 ±</td><td>6</td><td>&lt; 0.005 ±</td><td>5</td></tr><tr><td>0.15 ±</td><td>5</td><td>(6) ±</td><td>6</td><td>0.24 ±</td><td>6</td><td>(5) ±</td><td>5</td></tr><tr><td>0.038 ±</td><td>5</td><td>0.022 ±</td><td>6</td><td>0.037 ±</td><td>6</td><td>0.042 ±</td><td>5</td></tr><tr><td>0.027 ±</td><td>5</td><td>0.017 ±</td><td>6</td><td>0.021 ±</td><td>6</td><td>0.043 ±</td><td>5</td></tr><tr><td>E2</td><td>(1) ±</td><td>5</td><td>(3) ±</td><td>6</td><td>(1) ±</td><td>6</td><td>(3) ±</td><td>6</td></tr></tbody></table>

increase statistical power, the two lesion types were combined, giving for females 16 MS donors vs 10 controls and for males 14 MS donors vs 10 controls. We looked for differences between lesions and control WM in males and females separately, and we also looked for sex differences in the response to MS. This was calculated as the interaction between sex and disease state in a linear model. Steroids showing significant differences between MS donors and controls in either males or females, or a sex difference in response to MS, are shown in Fig. 1, while the measurements of the remaining steroids are shown in Supp. Fig. S2. Steroid measurements by GC/MS are also summarized as mean and standard deviation in Table 4, and the full dataset is provided in Supplemental Data.

Overall, 10 steroids showed significant differences between MS lesions and control WM, and 12 steroids showed a sex difference in response to MS. The reduced metabolite of pregnenolone, 20α-dihydropregnenolone (20α-DHPREG) was markedly reduced in female MS lesions (p = 1 × 10⁻⁵) and slightly increased in male MS lesions (p = 0.02) (sex difference: p < 10⁻⁶) (Fig. 1A). Among progesterone (PROG) and its metabolites (Fig. 1B), levels of PROG itself showed a sex difference in response to MS (p = 0.01), with a tendency to increase in females and decrease in males, though neither change was significant. Levels of allopregnanolone (ALLO), which positively modulates GABAA-R, showed a clear female-specific response, being higher in MS lesions of females compared to control WM (p = 0.0002). The sex difference in response was also significant (p = 0.045). On the contrary, concentrations of 3β5α-THP, which exerts inhibitory influences on GABAA-R, were decreased in females only (p = 0.01), again with a significant sex difference (p = 0.0001) and consistent with increased GABAA-R activity in female MS lesions. Of the corticoids, 5α-DHDOC levels were increased in MS lesions of males (p = 0.01), while corticosterone levels were markedly increased in females (p = 2 × 10⁻⁵; sex difference p = 0.01) (Fig. 1C). Among the androgens (Fig. 1D), we found female-specific reductions in the levels of DHEA (p = 0.02), androstenediol (ADIOL) (p = 0.0004), 3α5α-tetrahydrotestosterone (3α5α-THT) (p = 0.005) and epiandrosterone (p = 0.01), and the difference between sexes was significant in all these cases. Furthermore, 3β5α-THT levels were reduced in both sexes, but to a significantly greater degree in females (males: p = 0.001, females: p = 0.002, sex difference p = 0.003). Lastly, the levels of testosterone and its metabolite 5α-DHT showed sex differences, with a tendency to increased T and reduced 5α-DHT in females when compared to males, although these changes were not significant. Thus, the conversion of T to its reduced metabolites is decreased in female MS lesions in a sex-specific manner. No differences were found among the estrogens.

## 3.2. Confounding factors

Six patients in the control cohort used for steroid measurement in mixed and active lesions had been receiving glucocorticoid treatment, and two female patients in the MS group had been receiving treatment
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Changes in steroid content, measured by GC/MS, in multiple sclerosis lesions (MS) compared to control donor white matter (CTR), in males and females. Mixed active/inactive and inactive lesions were combined. Differences were determined between MS lesions and control WM for each gender separately (asterisks). A sex difference in the response to MS is indicated by a hash symbol. (A). The pregnenolone derivative 20α-DHPREG is increased in male and decreased in female lesions. (B) Progesterone and its neuroactive derivatives show sex differences in response to MS. The concentration of allopregnanolone (ALLO), which positively modulates GABA<sub>A</sub>-R, shows an increase in MS lesions only in females, while 3β5α-THP, which exerts inhibitory influences on GABA<sub>A</sub>-R, is decreased in females. (C). The corticosteroid 5α-DHDOC is increased in male MS lesions only, while corticosterone is increased in female lesions only. (D). In females, a general reduction in androgens is seen in MS lesions, mainly for the reduced metabolites of testosterone. Females show reduced content of DHEA, ADIOL, 3α5α-THT and epiandrosterone in lesions, with clear sex differences in the response to MS. Furthermore, sex differences are seen for 3β5α-THT, T and 5α-DHT. In particular 3β5α-THT levels are reduced in lesions in both sexes, but to a greater extent in females. Steroid concentrations are shown on a logarithmic scale. Limits of quantification are indicated by horizontal dashed lines. For steroid abbreviations, see Table 3. \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001, for comparisons of control white matter (control) vs MS lesions (MS) (Wilcoxon-test); \# p < 0.05, \## p < 0.01, \#\#\# p < 0.001 for sex difference in response to MS (interaction of sex and disease in a robust linear model).</figcaption></figure>

with an aromatase inhibitor in the 3-month period prior to death. (All other patients had no such treatment for at least 6 months prior to death). Both these treatments could have an effect on steroid levels. Therefore we examined the effect of glucocorticoid treatment on steroid

levels. Three steroids showed a significant effect of steroid treatment: 20α-DHPREG (in females and in both sexes together); ADIOL (in males and in both sexes together) and PROG (in males). Although none of these retained significance after multiple testing correction, nonetheless we
repeated the statistical tests without glucocorticoid- treated patients for completeness. When these patients were excluded, the increase in 20α-DHPREG in male MS lesion tissue lost significance (p = 0.06 after multiple testing correction) due to loss of power) whereas PROG in males now showed a significant increase (p = 0.02 after multiple testing correction). Other significant results were unchanged for these steroids. In general glucocorticoid-treated patients' values were within the range of untreated patients although a tendency for lower values was seen in several steroids in the two male treated donors (Supp. Fig. S3). We conclude that glucocorticoid treatment did not cause spurious significant results.

Aromatase inhibitor treatment had no discernible effect, although statistical testing was not possible. Versions of Fig. 1 and Supp. Fig. S2 with glucocorticoid- and aromatase inhibitor-treated patients indicated are given in Supp. Fig. S3.

Storage time post-mortem (time between freezing of tissue and utilization) could also have an effect on steroid levels, and in fact the control group had significantly greater storage time compared to the MS group (12.2 ± 7.2 years for controls, 6.3 ± 3.2 years for MS). Therefore we also tested the effect of storage time in our linear model. A significant effect was found for 5 steroids after multiple testing correction 20α-DHPREG, ADIOL, DHEAS, 3β5α20α-HHP and etiocholanone. Re-testing of the sex:disease interaction for these 5 steroids in a linear model with storage time included gave results that were essentially unchanged. Storage time-corrected values were calculated and MS vs control comparisons within sex were also repeated for these 5 steroids. This gave almost identical results to the original analysis with the exception that ADIOL was now significantly increased in male MS lesion white matter. Storage time, therefore, may have a small influence on some steroid levels but had a minimal impact the results in this study.

### 3.3. Effects of steroids on primary human microglia from MS and control donors

Given that steroids have anti-inflammatory and neuroprotective properties and modulate gene expression in multiple glial cell types (Yilmaz et al., 2019), we carried out in vitro experiments to characterize their effects. We first tested them on primary microglia isolated from MS lesions (of all types) (n = 10 donors), from NAWM of the same MS patients and from white matter of control donors (n = 9). The donors used for primary microglia culture had an average age of 71 ± 17 years, post-mortem delay 7.8 ± 2.1 h, and disease duration was 40 ± 25 years. Donor characteristics for these experiments are presented in Supplementary Table 1. Although it was not possible to match the two groups on age or post-mortem delay, the pH of CSF was similar and PMD was below 11 h in all cases. MS patients were predominantly females (8/10) with a secondary progressive (SP) disease course (6/10). No donors used for microglia isolation had received glucocorticoids or aromatase inhibitors prior to death.

Microglia were treated with 1, 10 or 100 nm of PROG, T, E2 or ethanol vehicle. These doses encompass concentrations ranging from low to high physiological concentrations when referred to reference plasma values in men and women (Berkane et al., 2018; Bhasin et al., 2011; Frederiksen et al., 2024; Schumacher et al., 2014). Gene expression was measured by quantitative RT-PCR for neuroprotective and anti-inflammatory genes (BDNF, IL4, IL10, LIF, CCL18), pro-inflammatory genes (IL6, IL1β, TNFα) and markers of microglial activation (HLA) or anti-inflammatory phenotype (CD163, CD206). Our initial separate analysis of microglia from MS lesions and MS NAWM did not reveal any changes in gene expression in response to the steroid treatments. However, when microglia from lesions and NAWM of MS patients was analysed together, some steroid-induced changes in gene expression emerged, likely as a result of increased statistical power, and possibly also reflecting steroid effects on microglia not restricted to the lesions. In microglia isolated from control donors, steroids did not induce any significant changes in gene expression. In contrast, steroids induced

expression changes of several genes in microglia isolated from MS patients (Fig. 2). P-values for F-tests and post-hoc tests for these experiments are given in Supplementary Table 4. E2 induced upregulation of the anti-inflammatory cytokines IL4 at all the tested concentrations (1–100 nM) and LIF at 10 and 100 nM in an apparent dose-dependent manner. However, E2 caused a significant down-regulation of CD206 at 1 nM and 10 nM. PROG also induced IL4 up-regulation at higher concentrations, while T induced LIF expression at 10 nM. Notably, in the absence of steroid treatment, levels of IL4 and LIF tended to be lower in MS microglia when compared to control microglia, and steroids restored their expression to levels similar to the controls. No other significant gene expression changes were induced by steroid treatments (Supp. Fig. S4).

Because we had only 2 male donors with MS in this experiment we could not analyse the effects of steroids on gene expression changes separately by sex, although the responses appear broadly similar (Supp. Fig. S5).

### 3.4. Effects of steroids on cultured human fetal astrocytes

Astrocytes also participate in neuroinflammation and neuroprotection by expressing pro- or anti-inflammatory cytokines and neurotrophins, and by removing glutamate from the environment (Liberto et al., 2004). To determine whether steroids modulate astrocytic neuroinflammatory or neuroprotective activity, we tested the effects of steroids on gene expression in fetal human astrocytes. Cells were treated with PROG, 5α-DHT, E2 (1, 10 or 100 nM) or vehicle, followed by one of the inflammatory cytokines IL1B (red) or TNFA (blue) or vehicle (grey). Expression of anti-inflammatory cytokines (IL10, LIF), neuroprotective genes (BDNF, APOE, IGF1), glutamate reuptake effectors (GLAST1, GLT1), GFAP, and pro-inflammatory cytokines (IL1β, IL6) were measured. As shown in Fig. 3, E2 treatment resulted in increased expression of neuroprotective and both pro-inflammatory and anti-inflammatory genes, but only under inflammatory conditions (p-values for F-tests and post-hoc tests for these experiments are given in Supplementary Table 4). Several neuroprotective genes were induced by E2. APOE was induced by 10 nM E2 in the presence of IL1B. BDNF was induced by 1 and 10 nM E2 and GLT1 by 10 nM E2, but only in the presence of IL1B or TNFA. IGF1 expression was also increased by 1 nM E2 with IL1B and by 10 nM E2 with TNFA. The anti-inflammatory cytokine LIF was also induced by E2 under both inflammatory conditions (1 nM and 10 nM E2 with IL1B and 1 nM E2 with TNFA). However, the pro-inflammatory IL1B and IL6 were also induced by E2 when TNFA was present. Notably, baseline expression of IL1B, IL6 and LIF was substantially increased by inflammatory conditions.

Thus, the neuroprotective, pro- and anti-inflammatory effects of E2 were only found under inflammatory conditions. In all gene expression profiles, E2 was most effective at concentrations of 1-10 nM. PROG and 5α-DHT treatment did not affect fetal astrocyte gene expression. Relative expression levels of the remaining gene/steroid combinations, which did not show any significant differences, are shown in Supp. Fig. S6.

### 3.5. Steroidogenic enzyme and steroid receptor gene expression in remyelinated lesions

Considering that neurosteroids, in particular PROG and ALLO, have been shown to promote axonal remyelination, we also looked at remyelinated lesions to determine if expression of steroid metabolizing enzymes and steroid content are up- or down-regulated in these lesions, and if there are sex differences. The donors had an average age of 64 ± 11 years, post-mortem delay 7.8 ± 2.2 h, and disease duration of the MS cases was 35 ± 16 years (see Table 2). Among the 12 MS patients, 10 were SP type and 2 were PP type. Remyelinated lesions from 12 MS donors (6 females, 6 males) were compared to control donor WM (7 females, 8 males). No significant differences between groups were present in age and post-mortem delay. pH of CSF was significantly lower in
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Gene expression changes in primary microglia derived from MS patients (n = 10) and control donors (n = 9), induced by steroid treatment and measured by qPCR. In control microglia (CTR, red), steroids induced no changes in the expression of genes analysed. In MS microglia (MS, blue), E2 caused increases in expression of the anti-inflammatory cytokines IL4 and LIF, restoring them to the levels of control microglia. Paradoxically, at a concentration of 1 nM, E2 caused a reduction in CD206, indicating a move away from an anti-inflammatory phenotype. PROG and T also increased IL4 and LIF, respectively. No significant changes were induced in the expression of BDNF, CCL18, CD163, HLA, TNFA, IL10, IL1B or IL6 after progesterone (PROG), testosterone (T) and E2 treatment (see Supp. Fig. S5). \# *p* < 0.05, F-test of linear model over all concentrations. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 post-hoc test of specific concentrations (Dunnett's test, vs 0 nM as control). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

MS females compared to control females but the difference was small (6.4 vs 6.9). Donor characteristics and summary statistics are given in Table 2.

First, we measured gene expression in remyelinated lesions, nearby NAWM, and control donor WM, for the following genes: the steroid receptors AR, ERα, ERβ and PR, the steroid synthesizing and metabolizing enzymes AKR1C2, AKR1C3, AROM, HSD17B10, HSD3B, SRD5A and TSPO and the neurosteroid-regulated neurotrophin BDNF. The expression of AKR1C3 (also known as 17β-hydroxysteroid dehydrogenase type 5), which converts androstenedione to T, was regulated in both sexes (males: *p* = 0.026; females *p* = 0.014; F-tests, corrected for multiple testing). This enzyme was increased in remyelinated lesions (*p* = 0.0011) and NAWM (*p* = 0.012) of females, and slightly augmented in remyelinated lesions (*p* = 0.013) of males when compared to NAWM (Tukey post-hoc tests). RNA expression of the other genes was unchanged. Furthermore, the expression of AR, PR and ERα were all upregulated in female remyelinated lesions (AR: *p* = 0.006; PR: *p* = 0.003; ERα: 5.3 × 10⁻⁴) and NAWM (AR: *p* = 0.02; PR: *p* = 0.014; ERα: *p* = 0.014) while in males only ERα expression was regulated, being higher in remyelinated lesions than control WM (*p* = 0.02) and also than NAWM (*p* = 0.008) (Fig. 4A). (*p*-values from Tukey-s post-hoc tests following significant F-test after multiple testing correction).

### 3.6. Steroid metabolome changes in remyelinated lesions

We also measured steroid content in remyelinated lesions and control WM by GC/MS from the same patients (one extra MS donor was added for each sex; some donors in the control group were substituted, see Table 2). A total of 25 steroids were successfully targeted for measurement, 20 of which were detected (see Tables 3 and 5). Five steroids that were present in inactive and mixed lesions were not detected in

remyelinated lesions (3α5α-THDOC, 3α5α-THT, 3α5α20α-HHP, epiandrosterone and etiocholanolone).

No significant differences were found between remyelinated lesions and control donor WM in steroid content after multiple testing correction. However, before multiple testing corrections, the increase in the level of estrone (E1) in females, and decreases in DHEAS and 20α DHP levels in males and 5α-DHADIONE in females were significant (Fig. 4B). The slight decrease in T in males was not significant (*p* = 0.06 before multiple testing correction). A significant sex difference in response to MS was observed for DHEAS, which appears to be increased in remyelinated lesions of females, but decreased in males, and 3β5α-THP (*p* = 0.0005 and *p* = 0.002 respectively after multiple testing correction). 20α-DHPREG and ALLO, which were altered in mixed and active lesions no longer showed changes in remyelinated lesions (Fig. 4B). The remaining detected steroids did not show any significant differences (Supp. Fig. S7). GC/MS steroid measurements are also summarized as means and standard deviations in Table 5, and the full dataset is provided in Supplemental Data.

In the remyelinated lesion cohorts only two female control donors received glucocorticoids and one female MS donor received aromatase inhibitors. Steroid measurements for these donors were well within the ranges of the other donors (not shown).

### 4. Discussion

We previously found that in female MS patients, steroid synthetic enzyme expression changes occurred within mixed and inactive MS lesions that indicate a shift towards increased progesterone signalling. This was accompanied by an increase in PR expression (Luchetti et al., 2014). In order to establish a more complete view of steroidogenesis in MS lesions, we here performed steroid profiling by a well-validated,
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Effects of steroid treatment on gene expression changes, measured by qPCR, in human fetal astrocytes (3 donors with 2-4 replications per donor). Steroid or vehicle was given first, and after 3 h pro-inflammatory cytokines (IL1B or TNFA) or no cytokine (vehicle) were added for a further 3-h incubation. Estradiol (E2) induced increases in neuroprotective genes and both pro- and anti-inflammatory cytokines, but only under inflammatory conditions. In particular APOE, BDNF, GLT1 and IGF1, were increased by E2 treatment. IL1B and IL6 were up-regulated in the presence of TNFA, while LIF expression was induced by E2 in both inflammatory conditions. # *p* < 0.05, F-test of linear model over all concentrations; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 post-hoc test of specific concentrations (Dunnett's test, vs 0 nM as control).</figcaption></figure>

precise and sensitive GC/MS methodology (Gonzalez Deniselle et al., 2016; Hertig et al., 2010; Liere et al., 2004). Especially marked was the decrease in 20α-DHPREG (pregnenediol) in MS females, which could reflect an increased upstream steroid metabolism (Koyama et al., 2016). Steroid profiling revealed very specific changes in metabolic pathways. In lesions of female donors we found an overall decrease in levels of androgens and increases in the concentrations of metabolites of PROG, ALLO and corticosterone (Fig. 5). This is consistent with the changes in enzyme gene expression we previously reported and suggests a shift towards progesterone signalling in females.

Notably, we found a marked increase in ALLO levels in MS lesions in females but not in males. This neuroactive derivative of PROG, which acts via the GABAA receptor as a positive allosteric modulator, has neuroprotective, anti-inflammatory and pro-myelinating properties (Faroni and Magnaghi, 2011; Ghoumari et al., 2003; Guennoun et al., 2015; Noorbakhsh et al., 2014). The observed increase in ALLO could be an endogenous protective response to promote lesion resolution and remyelination in females. It was accompanied by a reduction in 3β5α-THP, which antagonises the effects of ALLO on GABA signalling (Bäckström et al., 2005). This indicates a coordinated shift in steroid production to promote GABA signalling and to promote anti-inflammatory effects, since macrophages, microglia and T-cells all express GABA receptors. Such effects of ALLO have been shown directly on macrophages and microglia (Ghezzi et al., 2000; Jolivel et al., 2021; Müller and Kerschbaum, 2006; Noorbakhsh et al., 2011). An anti-proliferative effect on T-cells was also reported by (Sparrow et al., 2021), although not by (Noorbakhsh et al., 2011).

The increase in ALLO and other changes in steroid levels may be

partly due to local synthesis and partly due to changes in plasma steroid levels. The majority of steroids freely cross the blood-brain barrier although the relationship between brain and plasma levels differs between steroids (Caruso et al., 2013; Zhu et al., 2017). ALLO was previously found to be reduced in male MS patient plasma and CSF (Caruso et al., 2014), but we do not see a decrease in ALLO in male lesional white matter. Female MS patients were not included in that study. Plasma ALLO levels were unchanged between MS patients and healthy controls in another study of both sexes (Cheng et al., 2021). However ALLO plasma levels have not been measured separately in female MS patients so it is unclear what influence circulating ALLO might have on brain levels in these patients. The changes that we observe are, however, consistent with changes in local synthesis due to the changes in enzyme expression we previously observed, in particular the shift towards progesterone and its metabolites in females indicated by increased 3β-HSD and 5α-reductase type 1 (Luchetti et al., 2014).

Demyelinated lesions have a reduced fat content which might lead to reduced steroid content due to the lipophilic nature of these molecules. However, this would result in consistent reductions in many steroids in both sexes, whereas we see sexually dimorphic changes and also increases in some steroids. Therefore our data do not support this as being an explanation of the results we obtained.

The hypothalamic-pituitary-adrenal stress axis is activated in MS and increased levels of corticosteroids in MS patients are associated with less severe disease. HPA activation is also associated with increased expression of genes involved in regulation of inflammation and with myelination (Melief et al., 2019; Melief et al., 2013). The increase of corticosterone in female MS lesions may be beneficial via a local anti-
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Steroid-related gene expression and steroid content in remyelinated lesions, NAWM and control white matter. (A) Changes in gene expression, measured by qPCR, in remyelinated lesions and adjacent normal-appearing white matter (NAWM) nearby. The enzyme AKR1C3, which synthesises E2 from E1 or T from androstenedione (ADIONE), is increased in remyelinated lesions, more markedly in females, where it is also increased in nearby NAWM compared to control white matter (CTR). In males the increase is milder and significant only with respect to NAWM, which is no different to controls. All three main nuclear steroid receptors are also upregulated in the remyelinated lesions and NAWM in females. However in males only the estrogen receptor ERα is upregulated in remyelinated lesions as compared to NAWM and control white matter. (B) Changes in steroid content, measured by GC/MS, in remyelinated lesions, compared to white matter from control donors. No changes in steroid levels between MS and control white matter were significant after multiple testing correction, although sex differences in the responses of DHEAS and 3β5α-THP levels to MS was found (##: p < 0.01, ###: p < 0.001, robust linear model, interaction of sex and steroid content). Before multiple testing correction, the increase in E1 level in females, and the decreases in DHEAS and 20α DHP levels in males and 5α-DHADIONE in females were significant. No changes were apparent in other steroids measured. Steroid concentrations are shown on a logarithmic scale. Limits of quantification are indicated by horizontal dashed lines.
* p < 0.05, ** p < 0.01, *** p < 0.001, post-hoc test (Tukey's) following a significant F-test in a linear model.</figcaption></figure>

inflammatory effect. As this response is also lacking in males, the corticosterone response may be an additional mechanism that contributes to better lesion resolution in females.

In the light of recent experimental results, the female lesion-specific decrease in androgens is of particular interest. The proportion of microglia expressing AR was significantly greater in MS lesions of females when compared to males (Zahaf et al., 2023). Furthermore, in female mouse models of demyelination, androgen signalling via AR was shown to play an important role in polarizing microglia towards a regenerative phenotype. This unexpected importance of androgen signalling in demyelinating lesions of females points to the possibility that decreased androgens within MS lesions may favour a chronic demyelinated state in both mixed and inactive lesions.

In males, we previously found increased aromatase and AKR1C3 expression, consistent with an upregulation of E2 production (Luchetti et al., 2014). This was accompanied by increased ERβ expression. However, we did not find here any increase in estrogen production. We found instead an increase in 20α-DHPREG and 5α-DHDOC and a reduction in the testosterone metabolite 3β5α-THT. It is plausible that the increased AKR1C3 expression may be responsible for the stimulation of 20α-DHPREG synthesis, since the AKR1C enzymes also have 20-keto-steroid reductase activity (Penning et al., 2000). Notably, 5α-DHDOC is an intermediate for the production of 3α5α-THDOC. Both molecules, like ALLO, act as positive allosteric modulators of the GABA receptor (Reddy and Rogawski, 2002), but 5α-DHDOC only weakly so. This intermediate was undetectable in females and so this may represent a male-specific
<figure><img src="image_9.png" /><figcaption>Fig. 5. Overview of steroid synthesis pathways and changes in tissue steroid content that occur in male and female MS in mixed and inactive lesions compared to control donor white matter. Red triangles indicate significant changes in steroid content in female MS lesions, while blue triangles indicate significant changes in males (Wilcoxon tests, MS lesion vs control). Hash symbols indicate significant sex differences in the response to MS lesion formation (interaction of sex with disease status, robust linear model). When a significant sex difference was found, but no significant differences within sexes, empty triangles indicate the direction of change in each sex. In females only, an increase in the neuroactive steroid ALLO, and a decrease in the oppositely acting 3β5α-THP occurs, indicating an increase in anti-inflammatory neurosteroid activity, via these neurosteroids' actions on the GABA<sub>A</sub> receptor. Females also increase corticosterone, which may add to the overall anti-inflammatory response. At the same time, androgens, including DHEA and a number of testosterone metabolites are reduced. This suggests a general shift away from androgen production towards progesterone and its derivatives, as indicated by the large red arrows and grey dotted line. In males, responses in terms of steroid content were much more limited, with increases only in the corticosteroid 5α-DHDOC and the pregnenolone metabolite 20α-DHPREG. The former has only weak activity as a positive modulator of the GABAA receptor, and conversion to the more potent THDOC appears not to occur. The 20α-DHPREG increase in male lesions may be an inadvertent effect of the increase in AKR1C activity in lesions as previously reported (Luchetti et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

GABA<sub>A</sub>-R activating pathway, induced instead of the ALLO upregulation that takes place in females. However the final step in this pathway, i.e. the conversion of 5α-DHDOC to the more potent 3α5α-THDOC, appears to be very low in males. Increased levels of ALLO and corticosterone in female lesions, contrasting with lower levels of ALLO and 3α5α-THDOC in male lesions, might contribute to the sex differences in lesion development and the absence of MRI 'black hole' lesions in female patients.

A role for ALLO in MS was already highlighted by Noorbakhsh et al. (2011, 2014), who found that ALLO production was deficient in MS white matter, although sex differences were not investigated. The discrepancy with our results may be due to the fact that they looked at peri-lesional white matter, while we looked within the lesions. Thus, it is possible that ALLO deficiency contributes to susceptibility to lesion formation in MS white matter, but once a lesion has formed, there is an endogenous response with the upregulation of ALLO synthesis in females. Overall both studies support a beneficial role of ALLO in MS, in prevention of lesion formation, damping lesion development or improved lesion resolution.

In contrast to mixed and inactive lesions, we found no significant differences in steroid content in remyelinated lesions (shadow plaques) when compared to control white matter, although a possible increase in estrone (E1) may occur in females (in our data significant only before multiple testing correction). Furthermore, the gene expression changes

we found in remyelinated lesions are a subset of those previously found in mixed and inactive lesions (Luchetti et al., 2014). These findings are consistent with the idea that in remyelinated lesions, steroid levels and steroidogenic enzyme expression are likely to have partially returned to normal (i.e. NAWM) levels. However, our data also show that gene expression changes in MS lesions of female patients persist more into the remyelinated phase than in males. The AKR1C3 enzyme catalyses the formation of the more potent androgens and estrogens with respect to activity at their respective receptors (Penning et al., 2015). Its expression was found to be elevated in mixed and inactive lesions of females and even more pronounced in males (Luchetti et al., 2014). In the remyelinated lesions and NAWM examined in this study, a marked upregulation of AKR1C3 persisted only in females. Consistent with the notion of stronger androgen signalling in female remyelinated lesions, AR expression remained elevated. The same was true for PR expression, and only ERα remained elevated in remyelinated lesions and adjacent NAWM in both sexes. In contrast, in inactive and mixed lesions, ERα, AR and PR were increased in both sexes (Luchetti et al., 2014). Therefore it appears that in remyelinated lesions, gene expression mostly returns to baseline (NAWM) levels in males, but in females a continuing response is still detectable. Females thus have not only a stronger response in terms of steroids produced in mixed and inactive lesions, but more durable changes in gene expression in lesions that have been repaired.
We tested steroid effects on microglia and astrocytes in vitro using steroid concentrations of 1-100 nM. In our GC/MCS results the higher mean levels of E2, T and P correspond to about 1-2 nM, similar to the lower active dose of E2 added to the microglial cells and astrocytes. Plasma levels of E2 reach concentrations of 1-2 nM during the menstrual cycle and 20-40 nM during pregnancy (Frederiksen et al., 2024). Progesterone levels during the cycle are in the range 20-40 nM and during pregnancy typically 150-250 nM, while circulating reference levels of testosterone in men are about 20 nM (Berkane et al., 2018; Bhasin et al., 2011). Furthermore, in the brain local concentrations of steroids may greatly exceed those measured in whole tissue. For example, aromatase, the estrogen biosynthetic enzyme, is localized within specific cellular compartments, such as synapses, and is strongly upregulated in response to injury within astrocytes (Arevalo et al., 2015; Balthazart et al., 2006). Therefore the concentrations that we used reflect the likely possible range of concentrations that may occur in vivo. Furthermore we note that in most cases effects were seen at the lower doses (1-10 nM) which are closer to the measured values that we report here.

Steroid effects on glial cells were somewhat mixed. Microglia have an important role in MS, being widespread in active lesions, although it is not clear whether they are harmful or beneficial overall. For example, microglia are thought to promote remyelination, by clearance of myelin debris and production of growth factors (Lloyd and Miron, 2019; Pons and Rivest, 2020). In fact this may depend on their phenotype, in particular on their cytokine and trophic factor profiles, where anti-inflammatory cytokines may promote remyelination and neural repair.

Here we find that primary microglia from MS lesions appear to have reduced levels of anti-inflammatory cytokines which are restored by E2, PROG and partially by T. Paradoxically, E2 also reduced the M2 phenotype marker CD206 in our experiments. Nonetheless, E2 appeared to be the most potent steroid in the promotion of an anti-inflammatory phenotype, followed by PROG. Notably steroids had no effects on control donor microglia, consistent with a previous report (Saijo et al., 2011) where E2 failed to induce an anti-inflammatory profile in primary human microglia, although synthetic estrogen receptor ligands did.

In normal brain it appears that microglia express ERα and ERβ but not PR (Sierra et al., 2008) and a subset express AR (Zahaf et al., 2023). However, in MS lesions, expression of all three receptors was increased and was found in both microglia and astrocytes in chronic active lesion rims (Luchetti et al., 2014). In addition, microglia express the non-classical progesterone receptors mPRα,β and γ and PGMRC1 (Bali et al., 2013; Roche et al., 2016) in animal models. Female microglia may be more responsive to steroids since expression of PR was higher in female MS lesions than in males (Luchetti et al., 2014), while the proportion of microglia expressing of AR shows a female-specific increase in MS lesions (Zahaf et al., 2023). Numerous reports in rodents support an anti-inflammatory effect of E2 on microglia in animal models (Barreto et al., 2007; Bruce-Keller et al., 2000; Liu et al., 2005; Smith et al., 2011; Vegeto et al., 2001; Wu et al., 2016; reviewed by Yilmaz et al., 2019).

Astrocytes play an important role in the context of steroid actions in MS. They are a target for neuroprotective actions of neurosteroids (Asbelaoui et al., 2024; Spence et al., 2013; Spence et al., 2011; Yilmaz et al., 2019) and also a site of synthesis of multiple steroids (Zwain and Yen, 1999). The enzyme gene expression changes we previously identified took place primarily in astrocytes in MS lesions (Luchetti et al., 2014). Furthermore, astrocytes may contribute to MS pathology, since they become reactive in MS lesions. Healthy CNS astrocytes promote an anti-inflammatory environment but in active lesions they become hypertrophic and are a source of cytokines that promote recruitment of macrophages/microglia and lymphocytes (Ponath et al., 2018). An inflammatory subtype of astrocyte was identified specifically at mixed lesion rims (Absinta et al., 2021). Astrocytes also have an important role in neuroprotection in MS since they are a source of the neurotrophin BDNF and take up glutamate, and so have an anti-excitotoxicity function (Liberto et al., 2004). E2 is known to regulate BDNF (Sohrabji and Lewis, 2006). E2 has been shown to induce NGF and BDNF in cultured rat

cortical astrocytes (Langhnoja et al., 2018), while estrogen activation of astrocytes was required for expression of BDNF, IGF1 and GLT1 in an ischemia model (Lu et al., 2020). Our results in human fetal astrocytes are consistent with these findings. Here E2, but not DHT or PROG, was effective in promoting neuroprotective gene expression under inflammatory conditions. In the case of IGF1, GLT1 and BDNF, expression levels appear to be lowered by the inflammatory stimulus and E2 treatment counteracts this effect. However, under these conditions, E2 promoted expression of both pro and anti-inflammatory cytokines. It is worth noting that IL6 is now known to have a dual role and can be anti-inflammatory in the CNS (Willis et al., 2020), and pro-inflammatory cytokines such as IL1β and TNFα have been shown to be beneficial for CNS remyelination (Arnett et al., 2001; Mason et al., 2001). Thus, the overall balance of estrogen-induced astrocytic gene expression may yet be beneficial. Estrogens, acting on astrocytes, are known to be beneficial in EAE (Spence et al., 2013; Spence et al., 2011).

The steroidogenic enzyme regulation we previously observed took place mainly in astrocytes (Luchetti et al., 2014) and microglia are thus not likely to contribute to changes in steroid synthesis. However, while we found broadly beneficial effects of the steroids tested on microglia and astrocytes, other cell types in MS lesions may also be important for mediating effects of steroids on MS lesion development, including oligodendrocytes, oligodendrocyte precursor cells and infiltrating T-cells. For example, P promotes OPC proliferation and differentiation into oligodendrocytes (Ghoumari et al., 2005; Labombarda et al., 2009) and ALLO has an anti-proliferative effect on T-cells (Sparrow et al., 2021).

In conclusion, our study reveals sex differences in neurosteroid levels in MS lesions, suggesting a female-specific neuroprotective ALLO response to lesion formation. These findings align with our previous observations of altered steroidogenic gene expression in female patients. This response contrasts with a failure in males to produce neuroprotective steroids, despite increases in estrogen-producing enzymes. Steroids had mixed effects on cell types that participate in MS pathology, with estrogen being most clearly beneficial on primary microglia. These results build on our previous findings of sex differences in the steroid response to MS and provide a biological basis for sex differences in lesion development and progression in this disease.

## Ethics approval and consent

All donors gave permission to the Netherlands Brain Bank for brain autopsy and the use of tissue and clinical information for research purposes. Human fetal brain tissue was collected from donors from whom a written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic, The Netherlands. Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code provided by the Medical Ethics Committee of the Amsterdam UMC.

## CRediT authorship contribution statement

**Sabina Luchetti:** Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization. **Matthew R.J. Mason:** Writing – original draft, Visualization, Formal analysis. **Philippe Liere:** Writing – review & editing, Methodology, Investigation. **Antoine Pianos:** Investigation. **Sara Cossu:** Investigation. **Marloes Hofstee:** Investigation. **Arja Sluiter:** Investigation. **Adelia:** Investigation. **Jackelien van Scheppingen:** Investigation. **Eleonora Aronica:** Resources. **Michael Schumacher:** Writing – review & editing, Resources. **Inge Huitinga:** Writing – review & editing, Project administration.

## Funding

This work was funded by the Italian Multiple Sclerosis Foundation (Fondazione Italiana Sclerosi Multipla) grant no. FISM 2012/R/10 and the MS Research Foundation of the Netherlands (Stichting MS
Research), grant no. 12-793.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

In memory of the first author, Sabina Luchetti, who sadly passed away before the manuscript could be submitted.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2025.107040.

Data availability

Data will be made available on request.

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