Hormonal contraceptives prevent stress enhancement of fear memory via blunted stress responsivity

Daniella Ludmir, the University of Michigan

Abstract:

Hormonal contraceptives (HCs) are an essential part of healthcare. Many users experience improved mood, however up to 10% experience adverse mood effects. Given the suppression of the acute stress response by HCs, we aimed to determine how HCs modulate stress-related learning. Previous studies demonstrated that stress enhances fear conditioning, therefore we hypothesize HCs will prevent stress-enhancement of fear. Young adult female BALB/c mice were given daily doses of sucrose (control) or ethinyl estradiol (EE, 0.02μg) and levonorgestrel (LVNG, 0.75μg) in sucrose. Half of each treatment group was exposed to acute restraint stress 3 hours prior to contextual fear conditioning (CFC) training (AS group), and half received no stress prior to CFC (NS group). Control + AS mice exhibited significantly higher freezing compared to control + NS mice. In contrast, HC-treated mice showed similar freezing levels regardless of acute stress exposure. This suggests that HC exposure alters the relationship between stress and CFC. Corticosterone (CORT) levels demonstrated that, after multiple stressors, control mice showed a decrease in CORT, whereas HC mice showed an exacerbated CORT response. These findings add to our understanding of how HCs affect stress modulation and fear responses, with implications for mental health outcomes in HC users. 

Project summary

1) Introduction/Background

            Hormonal contraceptives (HCs) are a vital part of healthcare for users all around the world. They have contributed to unprecedented economic and reproductive control, and due to the benefits in alleviating dysmenorrhea, hormonal acne, and reducing the risk of certain cancers, have helped users regain control over their health (Michels et al., 2018, Schindler, 2013). In the United States, oral contraceptives are the most common method of contraception, accounting for

25% of contraceptive users (Cooper & Patel, 2024). Despite the prevalence of oral contraceptives, research surrounding the side effects remains unclear and inconsistent. Up to 10%, translating to 30 million women, report adverse changes in mood as a side effect of HCs (United Nations, 2019). Women taking oral contraceptives have shown suppression in the stress response (Hertel et al., 2017; Kirschbaum et al., 1995). Using a mouse model of oral contraceptive exposure developed in our lab, we replicated the findings from the human data and found that HC exposure blunted stress-induced corticosterone (CORT) response, with no significant changes in basal CORT levels (Schuh et al. 2023). CORT is the main stress-responsive hormone in rodents, a glucocorticoid which is produced by the adrenal glands. 

Fear is a normal part of the body’s response to danger for survival, however, consistent or exacerbated fear symptoms may be symptoms of trauma. Although stress-mediated disorders like Post-Traumatic Stress Disorder (PTSD) are inherently unpredictable due to the stressors that can lead to PTSD not being able to be foreseen, other factors may influence a person’s ability to respond appropriately to fear. Although females do not experience more traumatic events than males, they are still two times as likely to develop PTSD than men (Kilpatrick et al, 2013, Kessler et al., 1995). A known disruptor to one’s ability to adequately respond to fear is stress, making prior exposure to stress a risk factor for trauma, PTSD, and anxiety disorders.

In order to truly understand PTSD and anxiety disorders, a good animal model is crucial. Pavlovian fear conditioning does this by creating a dramatic fear reaction in a situation where this response is disproportionate (Fanselow & Sterlace, 2014). In PTSD, a fear response to reminders of the trauma is present long after the traumatic event occurred. Pavlovian fear conditioning simulates this response by creating an association between an otherwise neutral environmental stimulus and a painful or dangerous stimulus. These stimuli, which were not threatening in the first place, evoke a response used to protect against threats (Fanselow, 1984). The effect of stress on trauma has been demonstrated in animal models, as prior exposure to acute stress enhances contextual fear in mice (Tronson et al., 2010). 

Due to the effect of HCs in modulating the stress response by blunting CORT levels, we hypothesize that they will interact with stress, modifying an adequate response to fear. 

2) Methods

Animals: 48 nine-week-old female BALB/c mice were individually housed in an enclosed animal cubicle, at a 12/12 dark light cycle (7:00 AM–7:00 PM), 40% to 50% humidity, and 20°C (± 2°C). All studies were approved by the Animal Care and Use Committee at the University of Michigan. 

All animals received daily oral contraceptives (HC) or sucrose (control) for four weeks prior to the start of behavioral testing. The experiment consisted of 3 days of testing. On the first day, half of the animals were exposed to an acute stressor (immobilization stress). Three hours later the training element of the CFC test was run: animals were placed in the context box for 3 minutes followed by a 30 second tone and 2 second footshock. Then, the context test was run 24 hours after the training was completed. Finally, 10 days later, blood was drawn before a restraint stressor and trunk blood was collected 30 minutes after stress exposure.

HC Exposure: Animals were given 0.075μg/mL ethinyl estradiol (EE) and 3 μg/mL levonorgestrel (LVNG) orally in 0.25mL of 10% sucrose (HC group) or 0.25mL of 10% sucrose water (control group) daily just prior to lights out for 4 weeks before and throughout testing (Schuh et al. 2024). This model was designed to mimic oral contraceptive usage daily, at the same time, as in humans.

Acute restraint Stress: Mice assigned to the acute stress group were immobilized for one hour. Mice were lightly anesthetized with isoflurane and placed on their backs on a flat surface. Their forelimbs and hindlimbs were taped to the surface for one hour. Control mice were left in their home cages. On the final day, all mice were exposed to tube restraint to measure differences in stress-induced CORT levels. Animals were restrained in 50 mL conical tubes taped to a flat surface to prevent rolling. 

Fear Conditioning: Fear conditioning was performed three hours after restraint stress was completed to assess fear learning (Tronson et al., 2010). Mice were exposed to the conditioning box (context) for three minutes, followed by a 30-second tone, terminating in a 2-second footshock (.7 mA). Mice were returned to their homecage immediately after training. Twenty-four hours later, the contextual memory tests were performed, re-exposing the mice to the conditioning context for 3 minutes, without a tone or shock. Percent freezing and total locomotion activity were measured using VideoFreeze (MedAssociates, VT). Boxes were cleaned using 70% ethanol between each trial. 

Blood draws: Saphenous vein blood draws were performed for all mice 30 minutes after CFC training and on the final day before exposing mice to the acute stressor, tube restraint. Finally, trunk blood was collected 30 minutes after tube restraint was completed. An ELISA assay was run to analyze circulating CORT levels.

3) Results Behavior:

            To assess the effects of HC treatment and stress exposure on fear learning and memory, we analyzed freezing behavior during CFC. We observed a main effect of stress in the change in percent freezing (context test – training) driven by the non-HC treated animals (F (1, 38) = 3.814, p = .01) and an interaction that approaches significance (Figure 1a; F (1, 38) = 3.814, p = 0.06). This trend is consistent in the percent freezing data from the context test, with the main effect of stress approaching significance (Figure 1b; F (1, 40) = 3.634, p = 0.06). In the percent freezing data from CFC training, we see an overall effect of HC (F (1, 43) = 9.039, p < 0.01) and an overall effect of stress (Figure 1c; F (1, 43) = 3.905, p  = 0.05). Here, HC + NS mice showed significantly higher levels of freezing compared to Control + AS mice (p < 0.01). 

CORTICOSTERONE:

            To determine how HC and previous stress exposure influence CORT levels, we measured CORT from blood serum both at baseline and following different stress exposures. There is no significant difference in baseline levels of CORT amongst groups before being exposed to a stressor, after treatment exposure (Figure 2a). Although there was no significant difference in post-context test CORT levels, the trend across groups was consistent with HC-treated animals not exposed to acute stress showing blunted CORT responses, indicating an interruption in stress response activation (Figure 2b). This observation was supported by an interaction effect between stress and HC exposure in CORT changes from baseline (F (1, 29) = 4.281, p < 0.05; Figure 2c), consistent with prior findings (Schuh et al., 2024). Based on the trend in Figure 2b, the blunting of CORT concentration appears to be driven by the NS + HC group, as this effect was not observed in HC-treated animals previously exposed to acute stress or in either control group. The context test, acting as a stressor, shows blunted CORT levels in HC animals not previously exposed to AS, however, doesn’t show this blunting of CORT levels in HC animals previously exposed to AS and in the control NS or control AS groups (Figure 2b and Figure 2c). Similarly, there is also an interaction in CORT levels (F (1, 29) = 5.611, p < 0.05) 30 minutes after tube restraint stress further highlighting the interaction in CORT modulation based on stress and HC exposure (Figure 2d).

4) Discussion

The findings of this study reveal the modulation of HCs on the stress response using a mouse model. Consistent with previous human and animal research, HC usage blunts CORT responses to acute stress in animals, suggesting a fundamental modification of stress regulation (Schuh et al., 2024). This effect is particularly important given the critical role of CORT in the body’s response to stress, and the role of stress in fear memory.

One of the key behavioral observations in this study was that animals exposed to HCs showed similar freezing behavior under AS and NS conditions during CFC. This contrasts with control animals, where prior AS exposure significantly enhanced freezing responses during CFC, consistent with the established role of prior stress in fear learning. The lack of distinction in freezing behavior among HC-treated mice suggests that HCs disrupt the adaptive sensitization of the fear response, commonly observed following stress exposure. This is further supported by the CORT data as interactions between stress and HC exposure were observed.

These findings further the understanding of how HCs may influence susceptibility to anxiety disorders and PTSD. By blunting CORT responses and altering fear learning, HCs may increase vulnerability to maladaptive fear responses in some users, potentially explaining the increased incidence of adverse mood effects and anxiety-related symptoms reported by a subset of HC users. The lack of significant differences in baseline CORT levels across groups indicates that the observed effects are driven by acute stress responses rather than chronic dysregulation of the HPA axis.

In addition, CORT is crucial for more than just stress, as it also plays an important role in the sleep/wake cycle, immune function, and feelings of excitement. This is also consistent with research from our lab, as animals exposed to HC show anhedonia-like symptoms in response to the sucrose preference test (Schuh et al., 2024). A possible explanation for this is that HC-treated animals’ lower CORT levels may reduce their sensitivity to stimuli that would typically elicit higher levels of pleasure or excitement.

Further research will identify the underlying mechanisms by which HCs modulate stress and fear processing, including the roles of estrogen and progesterone receptors in regions required for fear memory, including the amygdala and hippocampus. Identifying genetic factors that may interact with HC usage to influence stress responses could provide valuable insights into individual risks for HC-related mood effects, allowing for a priori predictions of who may be more susceptible to adverse mood effects.

This study explores the importance of evaluating the impact of HCs on stress regulation and fear processing. Understanding these effects is crucial for developing personalized interventions to minimize adverse outcomes and optimize the beneficial effects of HCs. This ensures they remain both effective and safe for all users, particularly those at risk for anxiety and stress-related disorders.

5) Impact Statement

            Ensuring that HCs remain safe and accessible is vital, not only for current users but also to increase access for those who currently may not benefit from HCs due to side effects. By investigating the effects of HC usage on stress modulation and the development of anxiety disorders and PTSD, this research aims to identify specific biological mechanisms, such as the role of CORT regulation and its interaction with stress. These insights can guide the development of targeted interventions, such as modified contraceptive recommendations for different patients or additional monitoring that minimizes adverse mental health outcomes while maintaining the effectiveness and benefits of contraceptives. By understanding the biological mechanisms underlying these effects, and communicating these insights beyond academic settings, this work has the potential to enhance global access to these life-changing medications, driving future research and clinical advancements toward more comprehensive, equitable care for all users.

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