In a pilot sub-study of a Phase 3 trial (MDMA n=16, placebo n=7), changes in DNA methylation across HPA-axis genes predicted PTSD symptom reduction, with two CpG sites surviving FDR correction and the MDMA group showing greater methylation change at one NR3C1 (glucocorticoid receptor) site. These preliminary findings suggest NR3C1 methylation may be associated with response to MDMA‑assisted therapy and warrant replication in larger cohorts.
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Background
Previous research has demonstrated that epigenetic changes in specific hypothalamic-pituitary-adrenal (HPA) genes may predict successful psychotherapy in post-traumatic stress disorder (PTSD). A recent Phase 3 clinical trial reported high efficacy of 3,4-methylenedioxymethamphetamine (MDMA)-assisted therapy for treating patients with severe PTSD compared to a therapy with placebo group (NCT03537014). This raises important questions regarding potential mechanisms of MDMA-assisted therapy. In the present study, we examined epigenetic changes in three key HPA axis genes before and after MDMA and placebo with therapy. As a pilot sub-study to the parent clinical trial, we assessed potential HPA epigenetic predictors for treatment response with genomic DNA derived from saliva (MDMA, n = 16; placebo, n = 7). Methylation levels at all 259 CpG sites annotated to three HPA genes (CRHR1, FKBP5, and NR3C1) were assessed in relation to treatment response as measured by the Clinician-Administered PTSD Scale (CAPS-5; Total Severity Score). Second, group (MDMA vs. placebo) differences in methylation change were assessed for sites that predicted treatment response.
Results
Methylation change across groups significantly predicted symptom reduction on 37 of 259 CpG sites tested, with two sites surviving false discovery rate (FDR) correction. Further, the MDMA-treatment group showed more methylation change compared to placebo on one site of the NR3C1 gene.
Conclusion
The findings of this study suggest that therapy-related PTSD symptom improvements may be related to DNA methylation changes in HPA genes and such changes may be greater in those receiving MDMA-assisted therapy. These findings can be used to generate hypothesis driven analyses for future studies with larger cohorts.
Earlier research has implicated epigenetic alterations in hypothalamic-pituitary-adrenal (HPA) axis genes as markers or predictors of psychotherapy response in post-traumatic stress disorder (PTSD). Recent clinical trials have shown robust clinical benefit of MDMA-assisted therapy versus placebo with therapy for severe PTSD, but molecular mechanisms that might underlie treatment response remain unexplored. Epigenetic marks such as DNA methylation of HPA-related genes (notably NR3C1, FKBP5, and CRHR1) have previously been associated with trauma exposure, PTSD diagnosis, and psychotherapy outcomes, making them candidates for investigation in the context of MDMA-assisted therapy. This pilot sub-study, embedded within a recent Phase III randomized trial, aimed to test two hypotheses: (1) changes in DNA methylation of NR3C1, FKBP5, and CRHR1 would predict symptomatic reduction in PTSD (measured by CAPS-5) across both MDMA- and placebo-with-therapy groups; and (2) the MDMA-assisted therapy group would show greater methylation changes than the placebo group at sites that predicted treatment response. The study was exploratory and intended to generate loci and hypotheses for future, larger investigations.
This sub-study sampled participants from a multicentre Phase III trial of MDMA‑assisted therapy for severe PTSD. Of 90 participants treated in the parent trial, 33 consented to the epigenetics sub-study; due to COVID-19 interruptions 10 of these lacked post-treatment saliva, leaving 23 participants with paired pre- and post-treatment samples (MDMA n = 16; placebo n = 7). Participants had completed medication taper and preparatory therapy before randomization, which was stratified by site and allocated 1:1 to MDMA-assisted therapy or placebo with therapy. The intervention mirrored the parent trial: three 8‑hour experimental sessions about 4 weeks apart, each with a single divided dose of 80–180 mg MDMA or inactive placebo, followed by three 90‑minute integration sessions. Primary clinical outcome was CAPS-5 assessed at baseline and ~8 weeks after the third experimental session; saliva for methylation analysis was collected at the same baseline and outcome visits. Salivary DNA was extracted and bisulfite-converted, and genome-wide methylation was assayed using the Illumina Infinium MethylationEPIC BeadChip. Standard quality-control and preprocessing in R (minfi) were performed, probes on sex chromosomes were removed, and estimated epithelial cell proportion (derived with EpiDISH RPC) was used as a covariate. M-values were used for statistical modelling. All CpG probes annotated to NR3C1, FKBP5, and CRHR1 on the EPIC array were examined; sites differing between MDMA and placebo at baseline were excluded from longitudinal analyses. Statistical analyses compared the epigenetics subsample to the parent trial on demographics and CAPS-5 scores, and compared treatment groups within the sub-sample. Multivariate linear regression tested whether methylation change (independent variable) predicted CAPS-5 change (dependent variable), controlling for sex, age, and epithelial cell proportion. Because prior studies report heterogeneous directions of methylation change, the investigators tested both raw methylation difference scores and absolute value change scores (abs) to allow detection of predictive changes irrespective of direction. Given the pilot size, results are reported both uncorrected and after false discovery rate (FDR) correction (FDR threshold noted as < 0.1). For sites that predicted treatment response after FDR correction, group differences in methylation change were assessed using ANCOVA controlling for the same covariates.
10.3389/fpsyt.2023.959590 such changes may be greater in those receiving MDMA-assisted therapy. These findings can be used to generate hypothesis driven analyses for future studies with larger cohorts. KEYWORDS PTSD, MDMA, MDMA-assisted therapy, HPA, NR3C1, glucocorticoid receptor, epigenetics, DNA methylation
Recent investigations demonstrate significant benefits of MDMA (3,4-methylenedioxymethamphetamine)-vs. placebo with therapy in the treatment of PTSD. A pooled analysis of six Phase 2 trials demonstrated that 54.2% of persons treated with MDMAassisted therapy no longer met criteria for PTSD at study end compared to 22.6% in the control group, and gains continued to be sustained over time. These results were recapitulated in a more recent randomized, double-blind, placebo-controlled Phase 3 study. The placebo with therapy also resulted in a rather high rate of PTSD loss of diagnosis, though far less substantial than that observed in the MDMA-assisted therapy arm; 67% of MDMA participants no longer met criteria for PTSD at study end compared to 32% in the placebo group. These findings raise important questions about the biological changes that occur in association with treatment response after MDMA-assisted therapy. To date, however, there have not been molecular studies examining predictors of successful MDMA-assisted therapy for PTSD. Epigenetic mechanisms, such as DNA methylation, particularly of certain hypothalamic-pituitary-adrenal (HPA) axis-related genes, have been implicated in mediating adaptations to life conditions and may potentially serve as molecular markers of brain-body health. Importantly, alterations of the HPA axis were one of the earliest findings in PTSDand have been repeatedly replicated. There is now ample support for the idea that epigenetic alterations underlie neuroendocrine abnormalities in PTSD and may be implicated in conferring risk for PTSD following trauma exposure. Epigenetic marks on HPA axis genes have been associated with the prediction and successful outcome of psychotherapy in PTSD. These and other genes were recently confirmed in a second study examining epigenome-wide correlates of successful psychotherapy in PTSD. As such, epigenetic alterations on HPA axis genes may be markers, or even predictors, of successful psychotherapy in PTSD. Taken together, HPA axis gene methylation appears to be a promising epigenetic mechanism for treatment response in PTSD, motivating the current study focus. Using a sub-sample from the recent Phase 3 clinical trial, we conducted a pilot study investigating the methylation of three key PTSD-relevant HPA genes in association with MDMA-assisted therapy treatment response (3); NR3C1, FKBP5, and CRHR1. The gene NR3C1 encodes for a glucocorticoid receptor (GR) which Abbreviations: HPA, hypothalamic-pituitary-adrenal; PTSD, post-traumatic stress disorder; MDMA, 3,4-methylenedioxymethamphetamine. plays a role in the HPA negative feedback loop. The FKBP5 gene encodes a molecular co-chaperone that interacts with cortisol-GR complexes to regulate its downstream transcription-factor activity. The CRHR1 gene encodes corticotropin-releasing hormone (CRH) receptor 1, which is one of two receptors in this gene family. These genes were chosen based on prior studies demonstrating a change in DNA methylation associating with treatment response in PTSD. We hypothesized that (1) symptomatic reduction of PTSD after placebo and MDMA-assisted therapy would be predicted by changes in DNA methylation on NR3C1, FKBP5, and CRHR1; and (2) MDMA-assisted therapy group would exhibit more methylation change compared to placebo, related to the additional efficacy conferred by MDMA relative to placebo conditions.
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Kachmarik, J. E., Loftis, J. M., Stauffer, C. S. · Frontiers in Neuroscience (2026)
Sarmanlu, M., Kuypers, K. P. C., Vizeli, P. et al. · Progress in Neuro-Psychopharmacology and Biological Psychiatry (2023)
The analysed sample (n = 23; MDMA n = 16; placebo n = 7) did not differ from the full trial sample on age, sex, baseline CAPS-5, or outcome CAPS-5. Within the sub-study, MDMA and placebo groups were similar on age and baseline CAPS-5 but differed in sex composition; sex was therefore included as a covariate. Clinically, the MDMA group showed greater reduction in CAPS-5 scores than placebo, consistent with the parent trial. Across the three candidate genes, 259 CpG sites on the EPIC array were evaluated. Baseline comparisons identified five CRHR1, two FKBP5, and three NR3C1 sites that differed between treatment groups; these were removed from subsequent predictive analyses. Correlations between salivary beta values and publicly available brain methylation beta values were strong for all three genes (NR3C1 r = 0.73; CRHR1 r = 0.88; FKBP5 r = 0.83; all p < 0.00001), indicating saliva measurements related to brain methylation patterns at these loci. When testing whether methylation change predicted PTSD symptom change across groups, 37 of 259 CpG sites showed uncorrected significant associations. Broken down by gene, before FDR correction 17 CRHR1 sites, nine FKBP5 sites (15.8% of FKBP5 sites examined, 9 of 57), and 11 NR3C1 sites (10% of NR3C1 sites examined, 11 of 111) predicted CAPS-5 change at p ≤ 0.05. After FDR correction, two CpG sites remained significant: CRHR1 cg08276280 (open sea; pre-FDR B = 42.12, p = 0.005) and NR3C1 cg01391283 (absolute change score; open sea). For FKBP5 no sites survived FDR correction; the largest FKBP5 effect cited was cg16012111 (island site; B = 32.52, p = 0.012, pre-FDR). For NR3C1, the largest raw-effect site reported was cg6222722 (open sea; B = 19.32, p = 0.003). Of the two FDR-significant predictive sites, NR3C1 cg01391283 also exhibited a significant group difference in methylation change: the MDMA group showed greater absolute methylation change than placebo at this site (ANCOVA controlling for sex, age, epithelial proportion; F(1,18) = 4.78, p = 0.042). The extracted text does not report effect-size estimates for this group comparison beyond the F statistic and p-value.
Hendricks and colleagues report the first pilot evidence linking DNA methylation changes at HPA‑axis genes with treatment response to MDMA‑assisted therapy for severe PTSD. In this small sample, methylation change at multiple CpG sites across CRHR1, FKBP5, and NR3C1 was associated with PTSD symptom reduction; after FDR correction one CRHR1 site (cg08276280) and one NR3C1 site (cg01391283, assessed as an absolute change) remained significant. Notably, the NR3C1 cg01391283 site showed a larger magnitude of methylation change in the MDMA group than in the placebo group. The authors situate these findings within the extensive literature on NR3C1 and stress biology: NR3C1 encodes the glucocorticoid receptor that mediates HPA negative feedback, and prior studies have linked trauma and PTSD to differential methylation of this gene. The investigators note that most prior work has focused on CpG islands in the NR3C1 promoter, whereas the significant sites in this pilot were in open sea regions where functional consequences are less well understood. Because methylation at different positions can have different effects (for example, promoter methylation often represses transcription while gene-body methylation may not), the authors caution against simple directional interpretations and justify their use of absolute change scores to capture predictive changes regardless of sign. Several limitations are acknowledged. The pilot sample was small (n = 23 with paired samples), limiting statistical power and the ability to control for potential confounders such as race, smoking, genetic ancestry, or detailed trauma history. Use of the Infinium EPIC array meant some previously studied CpG loci (for example, certain FKBP5 promoter/intron sites) were not assayed. Peripheral saliva samples were used, which cannot definitively represent brain tissue methylation, although saliva–brain correlations for these genes were reported as strong. Finally, the study was candidate‑gene focused because of limited power; larger, better-powered studies are needed to validate the suggested loci and to determine whether greater clinical efficacy of MDMA‑assisted therapy is mechanistically linked to larger epigenetic changes. Overall, the authors interpret the data cautiously: results support the idea that psychotherapy-related symptom improvements may be accompanied by methylation changes in HPA genes and suggest specific CpG sites for hypothesis-driven follow-up in larger cohorts. They propose a plausible model in which MDMA produces a physiological state (including transient increases in cortisol and monoamines) that could render the epigenome more labile while eliciting a psychological state conducive to therapeutic processing, but they emphasise that mechanistic links remain speculative and require further investigation.
Participants represent a subsample of patients with severe PTSD from a Phase 3 clinical trial (NCT03537014; N = 90 treated) who consented to be in the epigenetic sub-study (N = 33; MDMA n = 16; placebo n = 7) (3). For the parent clinical trial, participants were recruited at 15 study sites through print and internet advertisements, referrals from treatment providers, and by word of mouth. Of the 11 sites located in the USA, seven participated in recruiting for the epigenetic study. Following an initial phone screening for the clinical trial, participants provided written informed consent and underwent further screening assessments for eligibility in the clinical trial. Eligible participants were enrolled in the trial and began psychiatric medication taper if needed, lasting from 0 days (no taper needed) to 103 days. Only participants who enrolled in the study and met criteria for severe PTSD after the taper period and preparatory therapy were offered the opportunity to proceed with the clinical trial and enroll in the present epigenetics sub-study. A total of N = 33 participants in the parent clinical trial consented to participate in this epigenetics sub-study; however 10 of the 33 did not provide posttreatment salivary samples due to COVID-19 related interruptions to the study, leaving a total of N = 23 (MDMA, n = 16; Placebo, n = 7) participants with both pre-and post-salivary samples for our analysis. This study was conducted in accordance with the principles of the Declaration of Helsinki-for full information on the larger study, see here. Ethics approval for this epigenetics sub-study was obtained from University of Southern California Institutional Review Board.
For full information on the intervention in the parent clinical trial, see. Participants were randomized and allocated 1:1 to either the MDMA-assisted therapy group or the placebo with therapy group. Randomization was stratified by site and occurred following enrollment confirmation. The treatment period consisted of three 8-h experimental sessions of either MDMA-assisted therapy or therapy with inactive placebo control, spaced ∼4 weeks apart. In each experimental session the participants received a single divided dose of 80-180 mg MDMA or placebo. Each experimental session was followed by three 90-min integration sessions that were spaced ∼1 week apart to allow the participant to understand and incorporate their experience. The first integration session always occurred on the morning after the experimental session, and the remaining two integration sessions occurred over the following 3-4 weeks. A blinded and centralized independent rater pool was used to conduct DSM-5 (The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition) diagnostic assessments at screening and to administer the primary outcome measure [i.e., Clinician-Administered PTSD Scale (CAPS-5) for DSM-5]. The independent rater measurements were conducted at baseline and ∼3 weeks after each of the first two experimental sessions via video interviews. The primary outcome assessment for the clinical trial was the final CAPS-5 conducted ∼8 weeks after the third experimental session (∼18 weeks after the baseline assessment). Saliva samples were collected from participants on days corresponding with the baseline and outcome CAPS-5 assessments to determine epigenetic changes associated with the clinical intervention and primary outcome of PTSD symptoms as measured by CAPS-5.
Saliva samples were collected with Oragene-DNA saliva kits (Ottawa, Ontario, Canada) from 33 participants during the baseline visit. Due to COVID-19 related interruptions to the study, 10 participants were unable to provide follow up samples at the final visit, providing a total of 23 participants with both baseline and final saliva samples (MDMA, n = 16; Placebo, n = 7). DNA was extracted with a DNA Genotek isolation kit (PT-L2P; Ottawa, Ontario, Canada). Sample yield and purity were assessed spectrophotometrically using a NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE, United States). Approximately 500 ng of DNA was treated with sodium bisulfite using the EZ-96 DNA Methylation Kit (Zymo Research, Irvine, CA, United States). DNA methylation was quantified using the Infinium MethylationEPIC BeadChip (IlluminaEPICarray) run on an Illumina iScanSystem (Illumina, San Diego, CA, United States). Raw Intensity Data (IDAT) files were exported for preprocessing in R with the minfi package. A filter was applied to remove probes located on the sex chromosomes. Data was subjected to quality control analyses, which included quantile normalization, checking for sex mismatches, and excluding low-intensity samples (p < 0.01). All samples passed quality control. Using the minfi package, data were normalized and annotated with Illumina CpG site probe names. Using the R package EpiDISH (Epigenetic Dissection of Intra-Sample Heterogeneity, 3.8) Robust Partial Correlation (RPC) method, the proportion of estimated epithelial cells was used as a covariate in our statistical models. M-values were used for methylation analysis as has been recommended, especially for the homoscedasticity.
To ensure generalizability of the results, the epigenetics subsample and the parent clinical trial sample were compared on demographic variables and CAPS-5 baseline and outcome scores. Variables were tested for normality using the Shapiro-Wilk's method. A Student's t-test was used for normally distributed variables, a non-parametric Kruskal-Wallis rank sum test for non-normally distributed variables, and a χ2 test for categorical variables. The same procedures were used to compare the MDMA and placebo groups in the sample. All sites annotated to the genes of interest on the Illumina Infinium MethylationEPIC BeadChip were analyzed. For all sites of interest, a Student's t-test was used to compare baseline DNA methylation levels between placebo and MDMA groups. All sites significantly different in DNA methylation between groups pretreatment were removed from further analyses. Multivariate linear regression modeling was conducted to assess if changes in DNA methylation predicted treatment response across groups (hypothesis 1). CpG change scores were used as an independent variable with CAPS-5 change score (final-baseline) as the dependent variable with sex, age, and proportion of estimated epithelial cells as covariates. Because prior associations between methylation of CpG sites on HPA genes with trauma have mixed directions (either hypermethylation, hypomethylation, mixed findings, or differential effects with no direction specified) [see Figurein Watkeys et al.], we allowed for either an increase or decrease in methylation to predict treatment response by calculating absolute value (abs ) methylation changes scores to test in the same regression models. Because this is a small pilot study, we report both non-corrected and false-discovery rate (FDR < 0.1) corrected results. Lastly, to determine if there was a group difference in methylation change, we tested if sites that significantly predicted treatment response across groups (FDR corrected) demonstrated significant group differences in methylation change (hypothesis 2). We compared MDMA and placebo groups on methylation change score with analysis of covariance (ANCOVA) models while controlling for sex, age, and proportion of estimated epithelial cells. Analyses were performed using various packages in R. A note on nomenclature relevant to the following description of CpG genomic locations: CpG sites are often assessed in the CpG-rich islands in and around promotor regions of genes, and the methylation of these CpG sites are generally associated with gene silencing. If a CpG site occurs at a distance situated within 2 kb from an island, the location is referred to "north shore" or upstream and "south shore" or downstream from the island; if it occurs at a location within 2-4 kb then it is referred to as "north shelf " or "south shelf " and any CpG site located further than 4 kb from an island are referred to as "open sea". This study assessed all sites annotated to the candidate genes on the Infinium MethylationEPIC BeadChip (IlluminaEPICarray). Beta-and M-values for all three analyzed genes can be found in Supplementary Data Sheets.
Epigenetics sub-study participants The sub-study sample did not differ from the clinical trial sample in age, sex, baseline CAPS-5, or outcome CAPS-5 scores (Table; all p's > 0.05). Within the sub-study sample, treatment groups (MDMA vs. placebo) did not significantly differ on age or baseline CAPS-5 score but did differ on sex (Table). Sex is used as a covariate in later analyses to account for any bias due to the different group sex compositions. As expected, treatment groups significantly differed on final CAPS-5 score in favor of MDMA conferring more benefit than placebo (Figure). A comparison of DNA methylation levels measured from salivary DNA in this study and publicly available methylation level measured from brain can be found in Supplementary Tables.
Beta values (which represent percent methylation at each CpG site) can be visualized for all CpG sites that significantly predicted treatment response (Supplementary Figures). Correlation between beta values generated from saliva in this study and publicly available beta values generated from brain were compared. NR3C1 (r = 0.73, p = < 0.00001), CRHR1 (r = 0.88, p = < 0.00001), and FKBP5 (r = 0.83, p = < 0.00001) sites all show strong and significant correlations between saliva and brain samples (Supplementary Tables). Brain DNA methylation values were obtained from the Allen Brain Atlas BrainSpan data. 1
Five CRHR1 sites, two FKBP5 sites, and three NR3C1 sites were significantly different between MDMA and placebo group at baseline thus were removed from further analyses (p's < 0.05).). Ten of these sites resided in open sea regions, two on south shore regions, two on north shore regions, two on an island, and one on a south shelf. The site with the largest effect size survived FDR correction (open sea: cg08276280; B = 42.12, p = 0.005-pre-FDR correction) and is depicted in Figures.
Methylation change on 15.8% (9 of 57 sites; 8 ; 1 abs ) of sites annotated to FKBP5 predicted change in CAPS-5 score [p's < 0.05; Table]. Seven of these sites resided in open sea regions and two on an island. No sites from this gene survived FDR correction and the site with the largest effect size for this gene (island site: cg16012111; B = 32.52, p = 0.012) is depicted in Figures.
Methylation change on 10% (11 of 111 sites; 4 ; 6 abs ; 1 and abs ) of sites annotated to NR3C1 predicted change in CAPS-5 score (p's ≤ 0.05; Table). Nine of these sites resided in open sea regions, one on north shore regions, one on an island, and one on a north shelf. One site (abs ; open sea; cg01391283; Figure) from this gene survived FDR correction. The site with the largest effect size (open sea: cg6222722; B = 19.32, p = 0.003) is depicted in Figures.
Of the two sites that predicted treatment response across groups after FDR correction (CRHR1 cg08276280 and NR3C1 abs cg01391283), one site also had a significant difference in methylation change between the MDMA and placebo groups [NR3C1; open sea; cg01391283 abs ; F(1, 18) = 4.78, p = 0.042]. A figure depicting the absolute change score plotted from pre-to post-treatment can be found in Figure.
This is the first study to examine DNA methylation relationships with treatment response to MDMA-assisted therapy in patients with severe PTSD. Due to being a small pilot study, we report both non-corrected significant results and FDR-corrected results. Before FDR correction, seventeen CRHR1, nine FKBP5, and eleven NR3C1 CpG sites predicted treatment response across groups. After FDR correction, one CRHR1 (cg08276280) CpG site and one NR3C1 (cg01391283) CpG remained significant. The results reinforce previous research that symptom reduction after PTSD treatment correlates with DNA methylation changes on the CRHR1 and NR3C1 genes. Of the two sites that predicted treatment response across groups after FDR correction, NR3C1 cg01391283 had a significantly larger change in methylation in the MDMA group compared to placebo. Genes of the HPA axis have been extensively studied in the context of epigenetic response to trauma. The most commonly-studied epigenetic candidate gene in the HPA axis is the glucocorticoid receptor gene (nuclear receptor subfamily 3 group C member 1; NR3C1). Glucocorticoid binding at this receptor regulates the stress response through a negative feedback loop. Much research to date demonstrates that childhood trauma and early stress alter the methylation status of this gene and its expression [for reviews see, Jiang et al., and Palma-Gudiel et al.]. While the majority of results find early life stress leads to an increase in methylation and a decrease of expression of NR3C1, opposing directional findings are reported. In addition to trauma-related methylation changes, two seminal papers found NR3C1 CpG methylation was associated with PTSD symptoms and many have replicated these findings. Taking into consideration the dynamic nature of epigenetic marks and their malleability in response to psycho-social experiences, recent research has focused on potential epigenetic changes in response to psychotherapy for stress-related disorders [for review see here]. Our results add to this growing field, suggesting epigenetic change on the NR3C1 gene may relate to treatment response to MDMA-assisted therapy. Further, this pilot study was intended to inform hypothesis development, these results suggest specific sites to examine on three HPA genes in future studies with a larger sample size. The NR3C1 gene consists of nine non-coding first exons and seven of these are located within a CpG island spanning 3 kb along the proximal promoter region of the NR3C1 gene. The majority of studies assessing NR3C1 methylation in relation to stress, psychopathology, and treatment response have focused on differential methylation primarily in this CpG island. This work has shaped general discussions about the function of experience-dependent DNA methylation changes. There has been much discussion over the notion of epigenetic modifications turning gene regulation "up or down" or "on or off " since methylation in the immediate vicinity of the transcriptional start site inhibits the initiation of transcription. CpG sites with differential methylation are located all along the NR3C1 gene sequence and the position of methylation across the gene influences its functionality. For example, methylation in the gene body does not block and might even stimulate transcription elongation or have an impact on splicing. While the functional implications of upstream methylation is less understood, some data suggests it may increase expression. Indeed, others have found methylation of the NR3C1 promoter region regulates transcript expression and levels. In this study, the two sites that predicted change in PTSD symptoms are both in open sea regions. To date, little is known about the functional consequences of DNA methylation in open sea positions. Therefore, it is difficult to speculate on the potential downstream effects of open sea DNA methylation changes on the HPA system. While the majority of research to date has found increased NR3C1 promotor methylation associated with trauma, the results are not always consistent. A 2018 systematic review summarized results across studies and effectively visualized how CpG sites in the 1F region of NR3C1 have been associated with hypermethylation, hypomethylation, non-significant findings, mixed findings, or differential effects with no direction specified, in association with both depression and childhood trauma. This suggests that trauma may dysregulate the HPA axis through differing routes, i.e., increase or decrease in methylation at various CpG sites, and that both hyper-and hypo-methylation can be maladaptive. Therefore, it stands to reason that psychotherapeutic effects mediating treatment response may occur in opposite directions depending on the individual, gene, and CpG site. For this reason, we chose to not only assess raw difference scores as treatment predictors, but also absolute difference scores which allow for a change in either direction. This novel analytic approach highlighted interesting findings such that the CpG methylation absolute difference score was more sensitive at predicting treatment response than the raw difference scores for NR3C1 and CRHR1. Taken together, these results suggest that both trauma-induced dysregulation and psychotherapyinduced changes of the epigenome may entail either hyper-or hypo-methylation dependent on the individual. MDMA induces a physiological response similar to an activated stress response, such as increased heart rate, blood pressure, oral temperature, and cortisol levels. MDMA and acute stress also increase levels of serotonin, dopamine, and norepinephrine. While the mechanisms behind acute stress induced changes in DNA methylation are not known, it is plausible that the increased monoamine and cortisol exposure induced by psychological stress may be involved in inducing a transient state of epigenetic-malleability. Especially taken into consideration that the cortisol-bound glucocorticoid receptor directly up-and downregulates thousands of genes as a transcription factor and other mechanisms. Further, the glucocorticoid receptor may directly influence DNA methylation through decreasing activity of a transcription factor, p53, known to regulate DNA methyltransferase (DNMT). However, MDMA-induced psychological effects are quite opposite to an acute stressor or traumatic experience. MDMA produces unique subjective properties such as reduced anxiety, acute positive affect, increased insight, accelerated thinking and euphoria, and increased sense of trust and bonding. Taken together, MDMA may induce a transient epigenetic-malleable state similarly to that of stress but a psychological state highly conducive to successful psychotherapy. Therefore, MDMA-assisted therapy may serve as an "inverse trauma experience, " such that acute stress and MDMA produce similar physiological states and a highly salient psychological experience. However, trauma has the potential to alter epigenetics underlying symptom formation, whereas MDMAassisted therapy has the potential to alter epigenetics underlying symptom reduction. While this study highlights potential biological mechanisms underlying MDMA-assisted therapy for PTSD, there are limitations to address. This initial pilot study was small, intended to assess preliminary evidence to support a larger future investigation. As such, additional studies with more power should be conducted to validate these results, however, these results provide specific loci to be studied in the future which may negate the need for p-value correction. The small sample size also necessitated the use of a candidate gene approach; while this study focuses on HPA axis genes, there are other potentially informative genes not assessed here [e.g., immune/inflammatory genes such as IL-12 and IFN-γ, for example see]. However, our results corroborate a large body of literature associating HPA gene methylation and stress-related disorders. While we accounted for participant age, sex, and cell count in our analyses, we were not powered to control for other factors such as race, smoking, ancestry, or trauma histories. Of note, the Infinium EPIC array probes we utilized do not fully incorporate all CpG sites that have been investigated previously. For example, previous studies have focused on the promoter region in intron seven of the FKBP5 gene, demonstrating epigenetic changes correlating with trauma exposure and PTSD symptomatology. However, these sites were not included in the EPIC array so we cannot ascertain if methylation changes in this region were related to MDMA-assisted therapy efficacy. Finally, it is important to note the limitations of using peripheral samples, which cannot elucidate associations in relevant brain tissue. More than ever, it has become clear that epigenetic changes in response to trauma or stress co-occur with behavioral and physiological symptoms associated with stress-related disorders. Here, we add to a growing number of studies demonstrating that psychotherapeutic experiences may also lead to alterations in the epigenetic landscape underlying symptom improvement. With the small pilot study sample size, it is difficult to determine if the greater efficacy of MDMA-assisted therapy compared to placebo therapy, is driven by greater changes in epigenetic regulation of HPA genes. However, these findings do suggest potential epigenetic mechanisms of MDMA-assisted therapy and their role in symptom reduction is worthy of continued investigation.