Fathers care for their offspring in only 5–10 % of mammalian species (Clutton-Brock, 1991; Moller, 2003). Among species exhibiting paternal care, the transition to fatherhood can involve changes in levels of hormones such as testosterone (T), oxytocin (OT), vasopressin (AVP), prolactin, estradiol and cortisol (Cort) (Bales and Saltzman, 2016; Storey and Ziegler, 2016). In some cases, these changes are correlated with paternal behaviors. For example, among marmoset monkey fathers, infant carrying is associated with both increased prolactin levels and decreased T levels (Dixson and George, 1982; Nunes et al., 2001).

Many men are involved in raising their offspring, so they too may experience hormonal changes that support their involvement in paternal caregiving. Cross-sectional comparisons of human fathers and nonfathers are consistent with this possibility, as new fathers tend to have lower T levels (Grebe et al., 2019; Meijer et al., 2019), and higher OT levels (Grumi et al., 2021). However, these cross-sectional studies cannot discern whether fatherhood causes hormonal changes from the alternative that men with particular hormonal profiles are more likely to become engaged or invested fathers. Within-subject longitudinal studies that follow men across their transition to fatherhood are more informative in this regard. One such study of men living in the Philippines conclusively demonstrated within-subject decreases in T in men who became partnered fathers across a four year interval (Gettler et al., 2011a). However, that study did not address the specific timing of the hormonal changes. Several subsequent longitudinal studies have not identified significant decreases in T across the perinatal period (Berg and Wynne-Edwards, 2001; Cárdenas et al., 2023; Diaz-Rojas et al., 2023; Perini et al., 2012). Others have documented modest within-subject decreases in T (5–6 %) during the perinatal period (Bakermans-Kranenburg et al., 2022; Corpuz and Bugental, 2020; Edelstein et al., 2017), but the observed changes have not matched the magnitude of change in the study of Filipino men (26 %) or the magnitude of differences between fathers and non-fathers in cross-sectional comparisons (Berg and Wynne-Edwards, 2001; Gray et al., 2006; Mascaro et al., 2014). These longitudinal studies have included a limited number of pre and/or postnatal measurements, suggesting that additional changes may occur outside of the measured time points.

Discrepancies between cross-sectional and longitudinal studies also exist for OT. One study reported a modest 8 % increase across the first 6 months of fatherhood (Gordon et al., 2010), but this magnitude does not match reported cross-sectional differences between fathers and nonfathers on the order of 33 % (Mascaro et al., 2014). The Gordon et al. study did not include any prenatal measurements, and may have therefore missed changes that occur earlier in the transition to fatherhood. Another study found no significant change in OT across the perinatal period, but measurements were restricted to late gestation and the early postnatal period (Bakermans-Kranenburg et al., 2022). Although AVP has been implicated in both rodent and non-human primate paternal care (Kozorovitskiy et al., 2006; Wang et al., 1994), one recent study found that AVP levels decreased from the late prenatal to the early postpartum period in new fathers (Bakermans-Kranenburg et al., 2022). Finally, one longitudinal study found that Cort levels are highest just prior to birth in new fathers (Storey et al., 2000), whereas another showed that both T and Cort were more likely to be low in partnered fathers compared with single men (Gettler et al., 2011b).

Variation in hormone levels among new fathers have also been linked with paternal behavior (Grebe et al., 2019). For example, fathers with lower T levels tend to be more involved in caregiving (Gettler et al., 2011a; Mascaro et al., 2013), to report more sympathy for newborn infant crying (Fleming et al., 2002), and to be more responsive to their infants compared with fathers with higher T levels (Weisman et al., 2014). On the other hand, fathers with higher plasma OT and AVP levels tend to engage in more stimulatory behaviors with their infants such as playful touch and moving their infant's body through space (Apter-Levi et al., 2014; Gordon et al., 2010; Morris et al., 2021). However, these studies cannot establish the causal direction of these associations. That is, they cannot determine whether hormone levels cause paternal behavior or vice-versa. Better insight into causality comes from studies that measure fathers' change in T from baseline in response to interacting with their infant. Although some studies have shown decreases in paternal T following interactions with their infant, a recent meta-analysis concluded that the overall effect of exposure to child stimuli on paternal T levels is not significant (Meijer et al., 2019).

In this study, we aimed to elucidate the trajectory of hormonal changes in new fathers with greater temporal resolution than previous studies, and to provide further insight into the direction of causality between hormone levels and paternal behavior by asking whether hormone levels at one time point predict behavior at subsequent time points, or vice-versa. Specifically, we recruited a sample of first-time expecting fathers and measured levels of plasma T, OT, AVP and Cort at four time points spanning 4–5 months gestation through 4 months postnatally, and we compared these changes to those found in a control sample of adult male nonfathers. We also examined the concurrent and lagged relationships between hormone levels and fathers' self-reported paternal behaviors. Our pre-registered hypothesis was that OT and AVP levels will increase, and T levels will decrease, across the transition to fatherhood but not across the equivalent time period in the control sample of non-fathers, and that these changes will be correlated with paternal involvement and attachment. Note that the directionality of the AVP hypothesis was specified before publication of the recent study finding decreases in AVP across the perinatal period in new fathers (Bakermans-Kranenburg et al., 2022). Although Cort was not included in our hypothesis, the above evidence suggests it is expected to increase in fathers just before birth and to decrease postnatally.

Peripheral OT levels have been measured using a variety of different techniques, and concern has been raised that results using different techniques are often uncorrelated. Multiple different assays methods have been employed, including radioimmunoassay (RIA), enzyme immunoassay (EIA), and liquid chromatography-mass spectrometry (LC-MS/MS), and some labs perform an extraction step prior to assay, while others do not. A recent review article argued for the importance of sample extraction to eliminate interfering molecules, and noted the superior sensitivity of LC-MS/MS for measuring lower levels of OT that often fall below the limit of detection of RIA and EIA (Tabak et al., 2023). In this study, we use a newly developed LC-MS/MS method on extracted samples to measure all four hormones of interest.