Pregnancy initiates a cascade of physiological changes that span the brain and body, many of which are thought to coordinate behavioral changes essential for offspring survival. Some of these behavioral changes, such as nest building, prepare for the arrival of offspring, while others adjust to new status-specific threats. One such threat is the risk of infanticide, a well-described phenomenon in mammals, typically performed by competing males to increase reproductive access to females (Elwood and Stolzenberg, 2020; Hrdy, 1979; Agrell et al., 1998; Lukas and Huchard, 2014; Vom Saal, 1985). In response to these risks, mothers exhibit heightened aggression toward unfamiliar conspecifics, particularly toward novel males (Lonstein and Gammie, 2002; Mann and Svare, 1982; St. John and Corning, 1973). Therefore, an unfamiliar male that once represented a potential mate transforms into a threat (Martín-Sánchez et al., 2015a). These behavioral shifts suggest that reproductive status reshapes the meaning and salience of social signals, such that a female's response to the same social situation changes across reproductive states. Yet, despite strong evidence that maternal behavior depends on social context, little is known about how pregnancy modulates female responses to social stimuli. It remains unclear whether the valence of male signals shifts during pregnancy, potentially reflecting a preemptive strategy to avoid future threats and enhance offspring survival.

The social stimuli we focus on are urine-based male odors in house mice. House mice, like many mammals, scent mark their environment (Hurst and Beynon, 2004; Desjardins et al., 1973; Johnson, 1973; Hurst, 1987; Arakawa et al., 2008; Crowcroft and Rowe, 1963; Ferkin, 2015). These urine marks are rich with information about the sex, status, health, and identity of individuals (Desjardins et al., 1973; Demir et al., 2020; Coombes et al., 2018; Cheetham et al., 2007; Roberts et al., 2012a; Roberts et al., 2014; Sheehan et al., 2016; Sheehan et al., 2019; Lee et al., 2017; Kaur et al., 2014; Ferkin, 2019; Ferkin and Johnston, 1995). Consequently, these social signals are a powerful system for studying social decision-making across reproductive stages. Moreover, the presence of males and their urine marks have been well-described to shape female behavior (Martín-Sánchez et al., 2015a; Roberts et al., 2012a; Ramm et al., 2008; Moncho-Bogani et al., 2002; Hurst, 1990; Garratt et al., 2011; Rich and Hurst, 1998; Rich and Hurst, 1999; Drickamer, 1989a; Johnston, 1979). Sexually receptive female mice in estrus find male urine attractive and rewarding (Roberts et al., 2014; Mucignat-Caretta et al., 1998; Drickamer, 1989b). In early pregnancy, novel male odors can trigger pregnancy block (i.e., the Bruce effect) (Bruce, 1959; Brennan, 2009). During lactation, dams exhibit maternal aggression toward male intruders, specifically toward gonadally intact adults (Martín-Sánchez et al., 2015b; Svare et al., 1981). This maternal aggression can be recapitulated by swabbing adult male urine odors onto castrated males (Coombes et al., 2018). Critically, the expression of maternal aggression, as well as other forms of parental care, requires an intact olfactory system (Lonstein and Gammie, 2002; Cowley and Cooper, 1977; Gandelman et al., 1972; Chamero et al., 2011; Hasen and Gammie, 2009; Kimchi et al., 2007), highlighting the central role of social odors in regulating maternal behavior.

While prior work has demonstrated that female responses to male odors vary with reproductive status, most studies have focused on the endpoints of this trajectory—estrus and lactation—leaving the transitional period of late pregnancy largely unexplored. Yet this period may be especially critical for behavioral reorganization. In terms of reproductive availability, estrus marks peak sexual receptivity, while late pregnancy represents complete reproductive unavailability. Hormonally, estrous is characterized by a dip in estrogen and sustained progesterone levels (Miller and Takahashi, 2014; McLean et al., 2012; Smith et al., 1975; Wood et al., 2007), whereas late gestation features massive hormonal peaks after which progesterone declines as parturition (birth) approaches (Barkley et al., 1979; Duarte-Guterman et al., 2019; Finn and Martin, 1969) (Fig. 1A). The hormonal fluctuations during pregnancy are known to drive preparatory behaviors such as nest building (Topilko et al., 2022) and maternal aggression (Mann and Svare, 1982; Martín-Sánchez et al., 2015b; Svare et al., 1981; Sandnabba, 1993; Svare, 1988; Palanza et al., 1996).These behavioral adaptations are anticipatory, often emerging before birth in the absence of direct pup contact. Importantly, maternal aggression—a hallmark of offspring protection—begins to ramp up during the final days of pregnancy (Mann and Svare, 1982; Svare, 1988), suggesting that the maternal brain begins transitioning toward defensive behaviors prior to parturition. Furthermore, late gestation is a uniquely vulnerable period. Females are less mobile, have high energetic demands, and cannot terminate the pregnancy or relocate easily. This likely creates strong selective pressure to avoid risky or unstable social environments. Strategic avoidance of unfamiliar males during this time could help secure a safe environment for birth and the survival of young pups.

Despite this, it remains unclear whether the behavioral shift toward defense includes a change in how female mice perceive or evaluate male odors. Specifically, does pregnancy re-tune social odor preferences in a way that discourages risky interactions with unfamiliar males? And if so, what components of male odor drive this shift? Addressing these questions is essential for understanding how reproductive status shapes social perception and for identifying early predictors of parental behavior.

In this study, we test whether reproductive status alters female social odor preferences in house mice by comparing responses to male urine and individual major urinary proteins during two distinct stages: estrus (ovulation) and late pregnancy. These time points differ along multiple biological axes—sexual receptivity, hormonal profile, ecological vulnerability, and preparatory parental behaviors—making them ideal for examining how the brain recalibrates social behavior in anticipation of offspring. We hypothesized that female mice would shift from attraction to aversion toward unfamiliar male odors during late pregnancy, reflecting an adaptive prepartum form of threat avoidance. We further sought to identify the components of male odor driving these responses. We focused on major urinary proteins (MUPs)—key signals in mouse urine—and used a novel two-day trial design to test female preference or avoidance of social stimuli. This approach confirmed findings from previous one-day or multi-day assays, while revealing unexpected complexity in female responses across days. In doing so, this work fills a key gap in the maternal behavior literature and offers new insights into how the pregnant brain anticipates and prepares for the challenges of motherhood.