Study Proposal: Studying Therapeutic Efficacy of Psilocybin on Major Depressive Disorder in Females During Late Follicular Phase, Mid-Luteal Phase, and Early Follicular Phase, and Across Estradiol-Progesterone Ratios.

Author’s Note

This paper was inspired by and written in the context of Dr. Stolzenberg’s seminar about “Research in Biological Psychology” during the Fall ‘24 quarter. The specific topic of the seminar was “Psychedelics as Therapeutics” and “Women's Brain Health.” After doing a thorough literature review, I discovered a huge lack of research concerning the effects and interactions of hormonal changes during the menstrual cycle and the therapeutic efficacy of drugs in general. I learned that sex as a biological variable was often disregarded or actively excluded from research findings. Moreover, recently, psychedelics have received more attention as highly effective treatments for depressive disorders. I argue that female sex steroid hormones must be considered and examined as biological variables that influence the therapeutic efficacy of psychedelics due to their modulatory and interactive effect on the serotonin system. Thus, I designed a study proposal that aims to examine the therapeutic efficacy of psilocybin on females with major depressive disorder, depending on their menstrual cycle stage. On a larger scale, my goal is to contribute to overcoming health inequity and the exclusion of females and other minorities from scientific research. 

Study Proposal: 

Studying Therapeutic Efficacy of Psilocybin on Major Depressive Disorder in Females During Late Follicular Phase, Mid-Luteal Phase, and Early Follicular Phase, and Across Estradiol-Progesterone Ratios.

Major Depressive Disorder (MDD) is the largest cause of disease-related disability worldwide and accounts for 50% of suicides occurring per year (Chesney, 2014). Neurobiologically, MDD involves dysregulation of the serotonin system on different levels including serotonin synthesis, genetic regulation of serotonin pathways, or receptor dynamics at a circuit level (Frontier Paper). Serotonin (5-HT) is a neurotransmitter that binds to different receptor subtypes, including 5-HT2ARs, which are highly expressed in cortical areas and modulate excitability and plasticity, thus by playing a critical role for  affective and cognitive processes (Harmon et al., 2025).

Despite this critical role, standard pharmaceutical treatments such as Selective Serotonin Reuptake Inhibitors (SSRIs) often show small effect sizes, delayed onset, numerous side effects, and require chronic dosing (Li, 2024). On the contrary, current findings suggest therapeutic psychedelics  produce rapid and sustained antidepressant effects following a single dose (Vargas, 2023). Specifically, psilocybin has demonstrated therapeutic efficacy in clinical trials compared to placebo (Raison et al., 2023; von Rotz et al., 2023) and SSRIs (Carhart-Harris et al., 2021). Psilocybin is a compound that naturally occurs in mushrooms of the psilocybe species. It is classified as serotonergic psychedelic, because psilocybin and its metabolites are hypothesized to achieve hallucinogenic and therapeutic effects through serotonin receptor agonism. More specifically, research suggests that psilocybin exerts antidepressant and anxiety-relieving  effects through 5-HT2AR agonism, triggering enhanced neurogenesis and neuroplasticity while reducing reactivity and activity in the amygdala and default mode network (Wang et al., 2023).

Notably, females are twice as likely as males to experience the distress and impairment of MDD symptoms (Weissman & Olfson, 1995). The difference may indicate a link to sex hormones. Estradiol (E2) and progesterone (P4) are the primary female sex hormones produced by the ovaries and are crucial for normal reproductive processes. Sex hormones fluctuate throughout the lifespan and during a monthly menstrual cycle. During the menstrual cycle, luteinizing hormones (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, together with E2 and P4 from the ovarian follicles, drive ovarian and uterine processes. E2 and P4 achieve different levels and ratios depending on the menstrual cycle stage (MCS). During the follicular phase, P4 remains low while E2 increases, peaking just before ovulation following an LH surge.  Following ovulation, P4 peaks midway through the luteal phase and exceeds the levels of estradiol’s secondary peak. Both P4 and E2 are relatively low during menses. 

Substantial evidence links other mood disorders, such as premenstrual dysphoric disorder, which is characterized by severe mood and physical symptoms during the late luteal phase postpartum depression, and perimenopausal depression, to fluctuations in sex hormones that dysregulate  serotonergic signaling, resulting in disorder-specific symptomatology. Specifically, E2 is associated with 5-HT and depressive disorders. E2 acts via estrogen receptors (ERs), specifically ERβ. ERβ functions as an intracellular transcription factor and leads to the upregulation of 5HT2AR expression, elevated 5-HT levels, and improved depressive symptoms (Shadani et al., 2024). In contrast, the absence of ERβ increases susceptibility to depression (2024), suggesting a relationship between female sex hormones, the serotonin system, and depressive disorders. 

Consequently, considering the modulatory effects of E2 on 5-HT2AR expression and 5-HT levels, as well as the hormonal dynamics of the menstrual cycle, it is plausible to suggest a relationship between menstrual cycle stage (MCS) and the efficacy of therapeutics targeting 5-HT2AR (Shadani et al., 2024). Psilocybin represents one such therapeutic and exerts its antidepressant effects through 5-HT2AR agonism (Wang et al., 2023). Researchers have not conducted studies to test the interaction between sex hormones and 5-HT in the context of psychedelics (Shadani et al., 2024). Remarkably,  there aren’t any studies addressing the relationship between MCS, and any drug’s therapeutic efficacy. My study proposal aims to address both gaps by conducting a double-blind randomized controlled trial assessing the reduction in depression scores. The study aims to compare psilocybin to an active placebo administered to females with MDD across different MCSs and in association with estradiol-progesterone ratios (E2:P4). Greater therapeutic effects of psilocybin during the late follicular phase (LFP), compared to the midluteal (MLP) and early follicular phase (EFP), as well as in association with higher E2:P4 ratios, are hypothesized.

To test these hypotheses, we suggest the following experimental design. Eligible participants for this study are naturally and regularly cycling females diagnosed with MDD without any other psychiatric, endocrinological, or cardiovascular comorbidities or past exposure to psychedelics. For a full overview of the exclusion criteria, see Appendix A. The first independent variable (IV) is the treatment condition. Researchers randomly assign participants to either the experimental group, receiving a dose of 25 mg/70 kg of psilocybin, or the control group, receiving 100 mg of niacin as an active placebo. Researchers and participants are unaware of which dose they receive. Both groups receive non-directive therapy as well as integrative therapy following the visit. In this approach,  the therapist avoids interpreting the psychedelic experience or directing the patient towards a specific psychotherapeutic meaning or goal. Instead, therapists provide a supportive space for patients to reflect and integrate their experiences into their life. This approach reduces additional confounding factors. Researchers will monitor patients for 6 hours following administration or until all subjective side effects have worn off. 

The second “IV” is MCS, differentiated into LFP, EFP, and MLP. By tracking menses before and after the lab visit, in addition to self-administered urine LH testing, researchers can determine cycle stage retrospectively. A surge in LH indicates ovulation will occur shortly thereafter, and thereby divides the cycle into follicular and luteal phase. Specifically, according to Schmalenberg et al. 's phasing procedures (2021), LFP comprises day two (-2) and one before (-1), and MLP day six (+6) to ten (+10) after a positive test (0). EFP will be defined as the time of menses. Participants record menses and LH test results in an online database. Additionally, a doctor draws blood samples at a fixed time on the day of the lab visit, frozen, and collectively sent to the lab for radioimmunoassay to determine E2:P4. 

The dependent variable (DV) in this study is therapeutic efficacy, measured by reduction in depression scores on the HAM-D, one week prior to the day of administration and one week after. HAM-D, the Hamilton Depression Rating Scale, is one of the most prevalent depression assessment scales. The study runs over multiple years and recruits participants in parallel with the experiment process to ensure a powerful sample size. Appendix B provides a chronological overview of the study.

For statistical analysis we decided on a 2x3 ANOVA as an appropriate test for the purpose of this study because it examines the effects of two independent variables (treatment condition and menstrual cycle stage) across their levels (psilocybin vs. control; LPF vs. MLP vs. EFP). This test allows us to detect the main effects of treatment and MCS, as well as their interaction. We expect the highest mean reduction score for psilocybin during the LFP and the lowest during the MLP and EFP. To test this, we will run a 2x3 ANOVA, expecting a main effect of MCS and treatment condition as well as an interaction between them (see Appendix C). An interaction effect indicates an antidepressant response to psilocybin which differs from the control, depending on MCS. 

To specifically test the original hypothesis that psilocybin produces greater therapeutic effects during the LFP compared with the MLP and EFP, we will conduct a post-hoc analysis for  pairwise comparison: psilocybin & LFP versus psilocybin & menses, psilocybin & LFP versus psilocybin & MLP. We expect a significant difference between both pairs, which would support the hypothesis. To test the second part of the hypothesis, whether higher E2:P4 ratios are associated with increased therapeutic efficacy of psilocybin, we will run a Pearson correlation between HAM-D reduction scores and E2:P4 for both treatment conditions. We expect a significant positive correlation in the psilocybin condition, providing further support for the hypothesis (see Appendix C). 

While the study design offers important insights, certain limitations must be acknowledged. These include the study’s duration, complexity, and reliance on participants' active collaboration in tracking menses, conducting LH testing, and providing documentation. These factors may reduce adherence and high attrition rates, complicating data collection. Furthermore, the selected MCS and phasing procedures do not capture data across all days of a complete cycle, limiting precise comparisons across cycle phases. These limitations demonstrate a broader issue of menstrual cycle research. High variability both between and within individuals’ cycles create difficulties for designing reliable procedures to determine an individual's MCS. Furthermore, no standardized method currently exists to determine MCS accurately. 

Future research should aim to address these gaps so that the effect of the menstrual cycle on any condition or therapeutic can be studied with more accuracy. Future studies should also include self-assessment depression scales such as the BDI in addition to clinician-administered scales, as recent reviews suggest higher efficacy measures (Metaxa & Clarke, 2024). Researchers could also use multiple assessment scales as secondary outcomes. To determine long-term antidepressant effects, assessments could extend up to six months post-treatment. Another consideration for future studies is the dose-response relationship, as the maximum efficacy of psilocybin appears to differ in dose between primary and secondary depression. Thus, it may be beneficial  to specify subject selection or adjust dosage based on differentiated diagnoses (Perez et al., 2023). Ultimately, future experiments should assess adverse side effects related to dose and MCS to determine the most effective and safest way of treating MDD in females.

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Appendix A

Exclusion criteria

Appendix B

Timeline 

Appendix C

Statistical Analysis of Hypothetical Data 

Figure C1. This table depicts hypothetical data, specifically the mean HAM-D reduction scores for each treatment condition. Assuming there is statistical significance, the difference between means averaged across the levels of the other factor for both MCS and treatment condition suggests main effects for both.

Figure C2. This depicts the hypothetical data in the form of a bar graph. The effect of psilocybin compared to placebo in the LFP differs from the effect of psilocybin compared to placebo in the MLP and during menses, suggesting an interaction between MCS and treatment condition.

Figure C3. This graph depicts the hypothetical correlation between E:P and HAM-D reduction scores for both treatment conditions. The steep slope of the psilocybin condition suggests a positive correlation between E P and depression reduction scores for psilocybin.