Prenatal Stress Differentially Shapes Adult Behavior in Male and Female Offspring

Background

The prenatal period is a critical window of neurodevelopment highly sensitive to environmental perturbations. Prenatal stress (PNS) has been identified as a major risk factor for neuropsychiatric vulnerability and is associated with an increased incidence of anxiety, depressive and substance use disorders in adulthood. In animal models, PNS has also been linked to a range of behavioral alterations in offspring, including heightened anxiety-like behaviors, impaired cognitive flexibility and disrupted social functioning.

Importantly, the effects of PNS are sexually dimorphic. Males and females follow distinct neurodevelopmental trajectories, shaping their differential sensitivity to prenatal stress. Clinical evidence suggests that males are more affected by early gestational stress, increasing risk of schizophrenia and autism-related disorders, while females are more sensitive to late gestational stress, linked to higher rates of depression and post-traumatic stress disorder.

PNS has also been implicated in sex-specific susceptibility to alcohol-related outcomes. Males exposed to PNS tend to exhibit more externalizing phenotypes, whereas females are more prone to internalizing, stress-related responses, thereby increasing their vulnerability to alcohol use disorders.

Despite these findings, relatively few preclinical studies have directly compared male and female offspring within the same experimental framework, limiting the ability to isolate true sex-dependent effects. Consequently, the neurobiological mechanisms underlying these sex differences remain poorly understood.

To address this gap, the present study employs a murine model of gestational stress induced by repeated restraint stress on a daily basis from embryonic day 7 through birth to systematically investigate the impact of the PNS on male and female offspring. Behavioral outcomes assessed include locomotor activity, anxiety-like behavior, sociability, learning, fear extinction, memory and alcohol-related responses. This approach aims to elucidate the mechanisms underlying sex-specific vulnerabilities to prenatal stress.

Key Findings

Animal model of prenatal stress:
The authors used a gestational restraint stress model in mice, a well-established and widely used experimental paradigm to induce prenatal stress. Pregnant Swiss albino ND4 dams were individually housed under a 12-hour light-dark cycle with ad libitum access to food and water. From embryonic day 7 until delivery, stressed dams were subjected to repeated restraint stress. Specifically, they were placed in a narrow, transparent Plexiglas tube under bright light three times daily for 45 minutes per session.

Prenatal stress increases anxiety-like behaviors in females:
PNS significantly increased locomotor activity in both sexes during the open field test. In the elevated plus maze, anxiety-like behaviors were also heightened, as reflected by reduced exploration of the open arms. In the light dark box test, females exposed to PNS showed greater avoidance of the illuminated compartment, suggesting that females may be more sensitive to anxiogenic environments.

Prenatal stress alters sociability in males:
In the three-chamber social test, males exposed to PNS exhibited a significant reduction in social interactions. In contrast, sociability in females was not affected by stress, although they showed lower baseline social engagement.

Prenatal stress disrupts cognitive functions:
PNS impaired fear extinction and recognition memory in both sexes. In fear conditioning, PNS-exposed animals maintained higher freezing levels. In the novel recognition task, stressed animals showed a lower ability to distinguish between familiar and novel objects. Although deficits appeared more pronounced in females, sex differences did not reach statistical significance. These findings indicate that prenatal stress induces broad cognitive disruptions, with a potential trend toward heightened female vulnerability.

Prenatal stress increases alcohol vulnerability in females:
Female offspring exposed to PNS consumed significantly more ethanol than males and control groups, suggesting greater susceptibility to alcohol intake. Furthermore, PNS modulated sensitivity to the sedative effects of ethanol in both sexes.

Implications

These findings support the notion that early-life stress programs neurobehavioral outcomes in a sex-dependent manner. Hyperlocomotion and cognitive impairments are observed in both sexes, whereas sex-specific effects include increased anxiety and alcohol vulnerability in females, and altered sociability in males. Using identical paradigms to directly compare males and females, this study provides one of the most sex-balanced characterizations across internalizing, externalizing, cognitive and substance-related phenotypes. Future studies integrating neuroendrocrine, epigenetic and cross-species approaches will allow a better understanding of the biological mechanisms underlying these sex-specific effects and enhance translational relevance.

Why we highlight this preprint

What led us to choose to highlight this preprint is that it addresses a central question relevant to our respective work in prenatal research. We are both working on the prenatal period, which explains our particular interest in this article. What stood out to us was the clear emphasis on sex differences in the effects of prenatal stress, an aspect that remains largely overlooked in the field. This resonates with our own research, highlighting the importance of stress in our current mouse models and reinforcing the need to consider this paradigm.

More broadly, stress is an environmental factor that affects a large portion of the population, making this article highly pertinent to broader public health questions. We found that it provides valuable insights that will help guide and enrich our future work.

Questions for the authors

1. Given that males and females may adopt different behavioral strategies and exhibit distinct biological trajectories, do you think using the same behavioral paradigms truly allows for a fair comparison between sexes or would it be more relevant to adapt certain paradigms to better capture these differences?
2. Since some studies suggest males are more vulnerable to certain early-life stressors, while females are sometimes more affected by stressors occurring later in gestation, do you think the timing of gestational stress exposure could differentially influence developmental trajectories in males and females? Have you considered whether different gestational windows might modulate male and female vulnerability to PNS differently?
3. Did you assess whether prenatal stress altered postnatal maternal behaviors and whether this could contribute to the behavioral phenotypes observed in the offspring?

Tags: behaviour and cognition, mouse model, neurodevelopment, prenatal stress, sex differences

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