In the last two decades, a wave of studies have sought to investigate the role of oxytocin in human normal and impaired socio-affective behavior and cognition (Donaldson and Young, 2008; Dyball and Paterson, 1983). Several methodological approaches have been adopted to this effect. Predominantly these include: (i) the measurement of psychological or neurobiological outcomes after the intranasal administration of exogenous oxytocin compared to placebo (Leng and Ludwig, 2016); (ii) genetic association studies between variations in candidate genes of the oxytocin pathway and behavioral and brain phenotypes (Onodera et al., 2015; Verhagen et al., 2020); (iii) the assessment of associations between single measurements of the concentration of endogenous oxytocin in peripheral fluids (mainly blood and saliva) at rest (Valstad et al., 2017) and individual differences in neurobehavioral phenotypes (Crockford et al., 2014), psychiatric disorder status and/or symptom severity (Rutigliano et al., 2016).

The latter approach is based on two assumptions. The first is that single measures of baseline levels of endogenous oxytocin in the biological fluids of peripheral compartments can accurately index oxytocin release in the brain (Valstad et al., 2017). The second is that single measures provide reliable estimates of baseline levels of endogenous oxytocin in plasma or saliva. Definitive answers regarding the first assumption, whether it regards baseline or evoked release following some intervention, remain to be obtained (MacLean et al., 2019).

Here, we investigate the second assumption, which is a prerequisite if single measurements of baseline levels of endogenous oxytocin are to be used as a valid trait markers of the physiology of the human oxytocin system (Wang et al., 2006). Currently, we lack robust evidence that single measures of endogenous oxytocin in saliva and plasma at rest are stable enough to provide a valid trait marker of the activity of the oxytocin system in healthy individuals. Indeed, previous studies have claimed within-individual stability of baseline plasmatic and salivary concentrations of oxytocin in both adults and children based on moderate-to-strong correlations between salivary and plasmatic oxytocin concentrations measured repeatedly within the same individual over time using ELISA in unextracted samples (Feldman et al., 2013; Schneiderman et al., 2012; Gordon et al., 2017). However, these studies have a number of methodological limitations that raise questions about the validity of their main conclusion that baseline plasmatic and salivary concentrations are stable within individuals. First, measuring oxytocin in unextracted samples has been postulated as potentially erroneous, given the high risk of contamination with immunoreactive products other than oxytocin (Szeto et al., 2011). It is conceivable that these non-oxytocin immunoreactive products might constitute highly stable plasma housekeeping proteins (Zhu et al., 2019), which masked the true variability in oxytocin concentrations. Second, a simple correlation analysis cannot provide information about the absolute agreement of two sets of measurements – which would be a more appropriate approach to study within-subject reliability/stability. Third, it is not clear whether these findings generalize beyond the early parenting (Feldman et al., 2013) or early romantic (Schneiderman et al., 2012) periods participants were in when the studies were conducted, since these periods engage the activity of the oxytocin system in particular ways (Gordon et al., 2010). Hence, establishing the validity of salivary and plasmatic oxytocin as trait markers of the activity of the oxytocin system in humans remains as an unmet need. Such evidence is urgently required, given reports that plasma and saliva levels of oxytocin are frequently altered during neuropsychiatric illness and that they co-vary with clinical aspects of disease (Rutigliano et al., 2016).

The measurement of salivary oxytocin has been proposed as a surrogate for blood plasma levels (Valstad et al., 2017). Compared to blood sampling, saliva collection presents several logistical and measurement advantages (i.e. relatively clean matrix) (Gröschl, 2008). However, the validity of this approach remains unclear. While elevations in plasmatic oxytocin after intranasal administration are likely to result from capillary absorption in the nasal cavity (Gossen et al., 2012), elevations in saliva oxytocin could result from mucociliary clearance of intranasally delivered oxytocin from the nasal cavity to the oropharynx (‘drip-down’ oxytocin) (Huffmeijer et al., 2012; van Ijzendoorn et al., 2012). This question can be illuminated by ascertaining changes in salivary oxytocin following the intravenous administration of oxytocin, eliminating the confound of drip-down oxytocin. In a previous study using enzyme-linked immunosorbent assay, the administration of low and medium doses of intranasal oxytocin (8 and 24 IU) elevated oxytocin concentrations in saliva, but the administration of a low dose (1 IU) intravenously had no effect. Moreover, concentrations of oxytocin in plasma and saliva did not correlate at baseline or after the administration of exogenous oxytocin for either route (Quintana et al., 2018). These data suggest that salivary oxytocin is a weak surrogate measure for peripheral blood levels. However, questions remain about whether the same results would have been achieved by using a more sensitive method of quantification, such as radioimmunoassay (currently the gold-standard for oxytocin quantification [McCullough et al., 2013]), or higher doses of oxytocin administered intravenously.

Here, we aimed to characterize the reliability of both salivary and plasmatic single measures of basal oxytocin in two independent datasets, to gain insight about their stability in typical laboratory conditions and their validity as trait markers for the physiology of the oxytocin system in humans. Additionally, we investigated whether salivary oxytocin concentration reflects plasmatic oxytocin by examining (i) if the intravenous administration of a large dose of oxytocin which produces sustained increases in plasmatic oxytocin over the course of 2 hr also increases the concentration of salivary oxytocin; (ii) how potential changes in salivary oxytocin compare between different routes of administration (intranasal versus intravenous) and methods of intranasal administration (spray versus a nebuliser); and (iii) the correlation between plasmatic and salivary oxytocin levels at baseline and after the administration of exogenous oxytocin using two different methods of intranasal administration (spray versus nebuliser) and the intravenous route. For all analyses, we followed current gold-standard practices in the field and assayed oxytocin concentrations using radioimmunoassay in extracted samples, which has shown superior sensitivity and specificity when compared to other quantification methods (McCullough et al., 2013).

Using radioimmunoassay, a gold-standard method for oxytocin quantification, we showed that i) single measures of baseline oxytocin concentrations in saliva and plasma are not stable within the same individual across different days; (ii) for plasma, we replicated this finding in an independent cohort; (iii) intranasal administration of exogenous oxytocin, despite method of administration, increases salivary oxytocin, but intravenous administration of a considerable dose does not produce any changes; iv) salivary and plasmatic oxytocin do not correlate with each other either at baseline or after the intranasal or intravenous administration of exogenous oxytocin. We discuss our main findings below.

We reported poor reliability indexes for single measurements of baseline oxytocin in both plasma and saliva. This suggests that single measures of baseline oxytocin in either fluid cannot be consistently measured for the same individual across different days. The reliability estimates found in the current study are in the range of those already described for other hypothalamic hormones such as vasopressin (Quintana et al., 2017) or prolactin (Muti et al., 1996). These poor reliabilities are unlikely to be explained by variability in the time-interval between visits of the same individual. Three lines of converging evidence support this conclusion. First, we also found poor reliability indexes for both saliva and plasma when we restricted our analysis to a subset of our sample controlling for the exact number of days spacing visits. Second, we did not find any significant effect of time-interval on our estimated ICCs. If time-interval was driving the poor reliabilities then we would have expected that in our pairwise analyses reliability would be consistently higher for samples closer in time and drop as the time-interval between sessions increases. This was not what we found. Third, variability in the within-subject intervals between samples did not correlate with within-participant variance in oxytocin concentrations across participants.

We note that the mean CV for baseline concentrations of oxytocin in saliva and plasma is higher than four times the intra-assay variability of the radioimmunoassay we used (<10%). Furthermore, in a further analysis assessing the within-session stability of plasmatic oxytocin using two measurements collected 15 min apart from each other in the placebo visit (one sample collected at baseline and the other after the intravenous administration of saline), we found excellent within-session reliability (ICC = 0.92, CV = 20%). Together, this suggests that the low reliability of endogenous oxytocin measurements across visits in the current study results from true intrinsic individual biological variability and not technical variability/error in the method used for oxytocin quantification.

Although we made efforts to minimize variability in the conditions in which biological samples were collected across sessions in both datasets (i.e. asking participants to abstain from exercise the day before study; controlling the time of sample collection; processing samples immediately after collection to minimize differences in peptide degradation between samples), several additional factors might underlie the intra-individual biological variability in plasmatic and salivary oxytocin that we report here. These include differences across participants in sexual activity in the days preceding each visit, perceived stress, the amount of social interactions, or other non-acknowledged biological rhythms conditioning variations in oxytocin secretion throughout time (Jong et al., 2015; Engel et al., 2019). For instance, one study showed that oxytocin is secreted in a pulsatile manner in healthy males even at rest (Baskaran et al., 2017). Therefore, sampling during different phases of the pulsatile release of oxytocin for plasma across sessions could explain the discrepancies in oxytocin measurements observed in the current study across visits. The time-interval between measurements do not seem to significantly impact on the reliability of baseline oxytocin, as suggested by the overlap of the 95% confidence intervals of the ICCs estimated for each pair of sessions. Although higher reliabilities could be identified between some consecutive pairs of sessions (separated by an average of about 6 days from each other: sessions 1–2 and sessions 3–4 for plasma measurements), other consecutive pairs of sessions (for instance, sessions 2–3) did not produce significant reliability estimates. If there are specific reasons explaining the higher reliability indices observed for the specific pairs of sessions, these reasons remain to be elucidated. However, it is not implausible that we might have found higher reliabilities for these specific two pairs by chance, since the 95% confidence intervals for the ICCs for all pairs of samples overlapped.

Our observation of poor reliability questions the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans. Instead, we suggest that, at best, these measurements can provide reliable state markers within short time-intervals (5 min in our study). Our data does not support previous claims of high stability of plasmatic and salivary oxytocin within individuals over time. For instance, in one study, Feldman et al., 2013 assessed plasmatic oxytocin in recent mothers and fathers at two time-points spaced 6 months apart during the postpartum period. The authors found strong correlations between the two assessments for both mothers and fathers (Feldman et al., 2013). In another study, Schneiderman et al., 2012 found strong correlations between plasmatic oxytocin concentrations measured at two different instances spaced 6 months apart in both single and individuals recently involved in a new romantic relationship (Schneiderman et al., 2012). Two important differences between these studies and ours are i) the method used for oxytocin quantification and (ii) the particular states participants were in when the studies were conducted. Regarding the first difference, these previous studies used ELISA without extraction, reporting concentrations of plasmatic oxytocin well above the typical physiological range of 1–10 pg/ml detected in extracted samples (in their studies, the authors report concentrations above 200 pg/ml). The inclusion of extraction has been postulated as a critical step for obtaining valid measures of oxytocin in biological fluids (Szeto et al., 2011). Unextracted samples were shown to contain immunoreactive products other than oxytocin (Szeto et al., 2011), which contribute largely to the concentrations of oxytocin estimated by this method. It is possible that these non-oxytocin products might represent highly stable plasma housekeeping molecules (Zhu et al., 2019) that masked the true biological variability in oxytocin concentrations between assessments in these previous studies that we could detect in extracted samples in our study. Regarding the second difference, these previous studies on within-individual stability were conducted during the early parenting (Feldman et al., 2013) or early romantic (Schneiderman et al., 2012) periods, which engage the activity of the oxytocin system in particular ways (Gordon et al., 2010). Instead, we used a normative sample that did not specify these inclusion criteria. Hence, we cannot exclude that during these specific periods the reliability of salivary and plasmatic oxytocin concentrations might be higher. We note though that our sample more closely resembles the samples used the vast majority of studies in the field (which sometimes even exclude participants during early parenthood [Bui et al., 2019). Hence, our estimates of reliability are a better starter point for all studies where specific circumstances potentially affecting the activity of the oxytocin system have not been specified a priori.

Our data poses questions about the interpretation of previous evidence seeking to associate single measurements of baseline oxytocin in saliva and plasma with individual differences in a range of neuro-behavioral or clinical traits. Almost all these previous studies have relied on the collection of single samples from each individual. Our findings suggest measures of oxytocin could be inconsistent and thus it is unlikely that a single sample may accurately represent oxytocin physiology and thus capture relevant inter-individual differences. Reliability of measurements also impacts power to detect correlations against these measurements (Kanyongo et al., 2007). To illustrate this point, in Figure 5 we provide the results of a set of simulations investigating how different reliabilities of oxytocin measurements impact the sample sizes necessary to detect significant correlations between oxytocin concentrations in peripheral fluids and neurobehavioral outcomes of different effect sizes. Given the less-than-perfect reliability of oxytocin measurements (ICC around 0.30) we show here, to detect the small-to-medium (r = 0.20–0.50) correlations typically reported in studies using these measurements (Torres et al., 2018), researchers would need sample sizes between 102 and 651 participants to reach minimally acceptable power (80%). Sample sizes in association studies of endogenous oxytocin measurements are typically below 100 participants (Torres et al., 2018).

Figure 5

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Turning to our second main finding, salivary and plasmatic oxytocin did not correlate at baseline or after administration of exogenous oxytocin (irrespective of route), replicating previous observations of a null association between measurements in these two compartments (Javor et al., 2014). Furthermore, we could not find any significant correlation between changes in salivary or plasmatic oxytocin from baseline to 115 min after the end of our last treatment administration in any of our four treatment conditions. The lack of significant associations between salivary and plasmatic oxytocin (and respective changes from baseline) was further supported through our Bayesian analyses which demonstrated that given our data the null hypotheses were at least three times more likely than the alternative hypothesis. Two hypotheses could account for the lack of correlation between plasmatic and salivary oxytocin. First, as in all other studies in the field, we did not control or manipulate the rate of saliva flow in the current study. Lipid insoluble molecules, such as oxytocin, enter into saliva mainly via the tight junctions between acinar cells through ultrafiltration (Vining et al., 1983). If oxytocin does reach the saliva compartment through an ultrafiltration mechanism (which depends on saliva flow rate [Gröschl, 2008]) then it is possible that when saliva flow is stimulated, oxytocin measurements may better index its plasmatic concentrations. Second, we cannot discard differences in oxytocin degradation rates between saliva and plasma. Future studies should investigate these hypotheses further.

Studies have been using increases in salivary oxytocin after the intranasal administration of exogenous oxytocin to index systemic absorption and establish putative time-windows during which neurobehavioral effects of oxytocin administration may be expected. If oxytocin increases after intranasal administration reflected systemic absorption and transport from the blood then we would have expected that intravenous oxytocin would have also increased salivary oxytocin. Moreover, we would also have expected that increases in plasmatic and salivary oxytocin after intranasal administration would reflect the typical ratio of their concentration as observed during baseline (with lower concentrations in saliva than in plasma). Our findings were not consistent with these expectations. We could replicate previous evidence that intravenous oxytocin does not increase salivary oxytocin (Quintana et al., 2018) and extended it by showing that the lack of increase in salivary oxytocin is not limited to the specific low dose of intravenous OT that was previously used (1IU) and that it is not driven by the insufficient sensitivity of the OT measurement method which had resulted in more than 50% of the saliva samples being discarded in the previous study (Quintana et al., 2018). Therefore, our data supports the notion that increases in salivary oxytocin after its intranasal administration most likely reflect drip-down oxytocin from the nasal cavity (Quintana et al., 2018). If this is the case then oxytocin elevations in saliva are mainly driven by non-absorbed exogenous oxytocin and therefore their use to estimate levels of systemic absorption or predict treatment effects after intranasal oxytocin administration is not valid. We expect this phenomenon to be particularly pronounced for higher administered volumes. Further studies should examine the impact of different administered volumes on increases in salivary oxytocin.

The lack of increase in salivary oxytocin after the intravenous administration of exogenous oxytocin that was consistently found in our study and in a previous study (Quintana et al., 2018) also raises the question of how oxytocin reaches the saliva if not from the blood. Currently, there is no evidence of direct acinar secretion or direct nerve terminals release of oxytocin to the saliva; therefore, transport from the blood remains as the most plausible mechanism of appearance of oxytocin in the saliva. Clarifying these mechanisms of transport is paramount, given the current hypothesis that salivary oxytocin might be superior to plasma in indexing central levels of oxytocin in the CSF (Martin et al., 2018).

One may argue the absence of significant elevations of oxytocin in saliva after its intravenous administration may be explained by significant differences in the kinetics of oxytocin concentration variation between these two compartments. This may include a significant delay in the elevation of oxytocin in saliva after the beginning of its increase in the plasma (explaining why saliva and plasma concentrations of oxytocin are not correlated after its exogenous administration). While this may be possible, in a previous companion paper we showed that the peak in plasmatic oxytocin occurs immediately after the end of its intravenous administration – 115 min before our post-administration saliva sample collection (Martins et al., 2020). It is therefore unlikely that this large interval of time would not have captured a significant delay in saliva elevation of oxytocin if it really existed, especially when after this time-interval plasma oxytocin still remained elevated, compared to baseline levels.

A strength of our study is the replication of poor reliability for baseline plasmatic oxytocin in a second independent dataset, which strengths our confidence in the robustness of our reliability findings. However, we acknowledge the following limitations. First, we only considered baseline measures in our reliability analyses. As for other hypothalamic-pituitary-adrenocortical markers where evoked measures present higher reliability than baseline measures (Coste et al., 1994), stimuli-evoked release of endogenous oxytocin (i.e. after social interaction, stress, pain) might also present higher reliability. Our conclusions are also restricted to male participants and to the radioimmunoassay method of oxytocin measurement, precluding extrapolations to female populations or to other quantification methods. Also, due to time and logistical constrains during our MRI setup, we could only sample saliva before administration and at the end of the scanning period, leaving our analyses on the effects of the administration of exogenous oxytocin on its concentration in saliva restricted to one single-time point post-administration. It is possible that we may have missed peak increases in saliva oxytocin after the intravenous administration of exogenous oxytocin if they occurred between treatment administration and post-administration sampling. This is unlikely given that the dose we administered intravenously resulted in sustained increases in plasmatic oxytocin over the course of 2 hr. Unless the half-life of oxytocin in saliva is much shorter than in the plasma, it would be surprising to not find any increases in salivary oxytocin after intravenous oxytocin given that concentrations of oxytocin in the plasma were still elevated at the specific time-point of our second saliva sample. Currently, we have no estimate for the half-life of oxytocin in saliva; however, given that previous studies have found evidence of increased salivary oxytocin after single intranasal administrations of 16IU and 24IU oxytocin up to seven hours post-administration (van Ijzendoorn et al., 2012), it is unlikely that the half-life of oxytocin is shorter in the saliva than in the plasma.

In summary, single measurements of baseline levels of endogenous oxytocin in saliva and plasma are not stable in typical laboratory conditions and therefore their validity as trait markers of the physiology of the oxytocin system is questionable. Salivary oxytocin is a weak surrogate for plasmatic oxytocin; hence, salivary and plasmatic oxytocin should not be used interchangeably. Finally, increases in salivary oxytocin after the intranasal administration of exogenous oxytocin most likely represent drip-down transport from the nasal to the oral cavity and not systemic absorption. Therefore, increases in salivary oxytocin after intranasal oxytocin administration should not be used to predict treatment effects.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Daniel Martins

   Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0239-8206

2. Anthony S Gabay

   1. Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
   2. Centre for Human Brain Health, University of Birmingham, Birmingham, United Kingdom

   Contribution

   Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6946-1046

3. Mitul Mehta

   1. Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
   2. Centre for Human Brain Health, University of Birmingham, Birmingham, United Kingdom

   Contribution

   Supervision, Funding acquisition, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

4. Yannis Paloyelis

   Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   yannis.paloyelis@kcl.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4029-3720

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