Why the estrous cycle matters for neuroscience

BACKGROUND: Ovarian hormone fluctuations over the rodent estrous cycle and the human menstrual cycle are known to significantly impact brain physiology and disease risk, yet this variable is largely ignored in preclinical neuroscience research, clinical studies, and psychiatric practice. METHODS: To assess the importance of the estrous cycle information for the analysis of sex differences in neuroscience research, we re-analyzed our previously published data with or without the estrous cycle information, giving a side-by-side comparison of the analyses of behavior, brain structure, gene expression, and 3D genome organization in female and male mice. We also examined and compared the variance of female and male groups across all neurobehavioral measures. RESULTS: We show that accounting for the estrous cycle significantly increases the resolution of the neuroscience studies and allows for: (a) identification of masked sex differences; (b) mechanistic insight(s) into the identified sex differences, across different neurobehavioral outcomes, from behavior to molecular phenotypes. We confirm previous findings that female data from either mixed- or staged-female groups are, on average, not more variable than that of males. However, we show that female variability is not, at all, predictive of whether the estrous cycle plays an important role in regulating the outcome of interest. CONCLUSIONS: We argue that "bringing back" the estrous cycle variable to the main stage is important in order to enhance the resolution and quality of the data, to advance the health of women and other menstruators, and to make research more gender-inclusive. We strongly encourage the neuroscience community to incorporate the estrous cycle information in their study design and data analysis, whenever possible, and we debunk some myths that tend to de-emphasize the importance and discourage the inclusion of this critically important biological variable. Highlights Ovarian hormone fluctuation impacts brain physiology and is a major psychiatric risk factor, yet this variable has been overlooked in neuroscience research and psychiatric practice. From rodent behavior to gene regulation, accounting for the estrous cycle increases the resolution of the neuroscience data, allowing identification and mechanistic insight(s) into sex differences. Female variability does not equal (and is not predictive of) the estrous cycle effect and should not be used as a proxy for the effects of ovarian hormones on the outcome of interest. Neuroscience researchers are advised to incorporate the estrous cycle information in their studies to foster more equitable, female- and gender-inclusive research. Studies of the ovarian cycle are especially important for improving women's mental health.