Decreased oxytocin levels related to social cognition impairment in borderline personality disorder

Dysfunctions in the oxytocin system have been reported in patients with borderline personality disorder (BPD). Deficits could be related to interpersonal hypersensitivity, which has been previously associated with failures in social cognition (SC) in this disorder, especially in Theory of Mind (ToM) skills. The aim of this work is to study the links between the oxytocin system and SC impairments in patients with BPD.

Borderline personality disorder (BPD) is a severe and heterogeneous mental disorder characterized by intense emotional dysregulation and impulsive behavior, in which hypersensitivity to interpersonal rejection, fear of abandonment, and unstable and unsatisfactory interpersonal relationships are nuclear features. 1he heterogeneity of clinical symptoms has previously been related to diverse biological alterations that may underlie the varying clinical presentation diagnosed as BPD. 2 Bearing that in mind, identifying potential biomarkers is the cornerstone of personalized therapeutic approaches to tailor diagnosis and therapies to patients. 3mong the pathophysiological features that have been identified in the disease, genetic abnormalities, 4 dysfunctional serotonergic regulation, 5 hypothalamic-pituitaryadrenal (HPA) axis overactivity 6 and changes in the regulation of the inflammatory response 2,7 are the most consistent findings nowadays.
Another biomarker of interest due to its potential relationship with particular BPD symptoms, such as emotional instability, hypersensitivity to rejection and fear of abandonment, could be oxytocin (OXT). 8Oxytocin constitutes a neuropeptide of nine amino acids produced in the supraoptic and paraventricular nuclei of the hypothalamus, stored within the pituitary gland, and subsequently released into the bloodstream. 9OXT receptors (OXTR) are distributed across various brain regions, including the amygdala, ventromedial hypothalamus, nucleus accumbens, basal forebrain, and the cingulate cortex. 10,11Additionally, OXTR can be found in peripheral blood mononuclear cells (PBMC), modulating the inflammatory response by promoting the proliferation of PBMC. 12,13eripheral OXT may be involved in elemental processes, such as parturition and lactation. 14In contrast, central OXT can reduce the response of the amygdala, modulating the influence of stress hormones through the HPA axis and monitoring brain activity in regions associated with social psychosocial stress, social cognition, and aggressive-conflict behaviors. 15,16egarding patients with BPD, intranasal OXT administration has been proposed as a promising line of pharmacological treatment for this clinical group 17 and our group has recently published several studies showing decreasing plasma levels of OXT and PBMC OXTR. 16,18owever, results are still scarce, and more research is needed to study the role of OXT and OXTR in patients with BPD. 19ome studies support that emotional instability and impulsivity related to difficulties in interpersonal relationships in BPD may be derived from deficits in social cognition in these patients. 20,21Social cognition (SC) refers to the mental processes that enable individuals to perceive, interpret, manage and respond properly to social information from others. 22One of the main SC components is Theory of Mind (ToM). 23It is also known as mentalization or mental state attribution, and it involves the ability to infer other people's mental states, considering their intentions, beliefs and emotions. 24ollowing the theoretical approach derived from the Movie for the Assessment of Social Cognition (MASC), 25,26 two types of errors about inferring others' mental states have been described: overmentalization errors (excessive attribution of mental states) and inframentalization errors (deficient attribution of mental states).Specifically, inframentalization errors include undermentalization errors (insufficient attribution of mental states) and total absence of mental inference errors (e.g., lack of mental attributions to social situations, making wrong attributions of physical causality to social situations despite mentalizing explanations). 26ther studies on ToM ability in patients with BPD show how they present overmentalization errors.8][29][30] Recent studies have shown that ToM deficits in people with BPD are not caused by affective disturbances, but clear relationships with disorder chronicity and functional status have been reported. 21Specifically, incipient disorder stages were related to a tendency to misunderstand mental states by erroneous overattribution, whereas fewer mental state attributions may be shown as the disorder becomes chronic.

Significant Outcomes
• Oxytocin (OXT) levels are linked to deficits in social cognition in BPD patients • Lower OXT receptor levels were associated with more overmentalization errors in BPD • BPD patients showed more undermentalization errors

Limitations
• The reduced sample size due to the characteristics of the clinical sample and the difficulties in obtaining biological samples • The possible influence of medications for patients in the neurochemical results.• The sample was composed by patients with moderate-to-severe severity and high dysfunction, so this sample could not represent the average of BPD population.
In this way, this study aims to gain insight into the association of phenotypic and endophenotypic characteristics in BPD through the study of the relationship between the role of oxytocin and difficulties in ToM ability in these patients, with the objective of improving the biological and clinical understanding and examine more specifically therapeutic targets.

| Participants
Thirty-three adult patients (age: M = 28.85,DT = 8.83) diagnosed with borderline personality disorder according to DSM-V criteria, 31 with a moderate-to-severe degree of severity (BPD-CGI [Clinical Global Impression for Borderline Personality Disorder Patients] > 4) and a moderate level of dysfunction (GAF [Global Assessment of Functioning Scale] < 65), were assessed at the Personality Disorders Unit of the Hospital Clínico San Carlos (Madrid, Spain).
Patients were excluded from the study if they met the following criteria: (1) had a neurological or medical illness that could affect brain functions; (2) had an intelligence quotient (IQ) bellow 85; (3) had a lifetime history of schizophrenia, schizophreniform disorder or bipolar disorder; (4) had a current major depressive episode or a substance use disorder that could affect neuropsychological performance at the time of the assessment.All patients received detailed information about the study, and they signed an informed consent prior to their participation in the research.The clinical research study was approved by the Clinical Research Ethics Committee of the Hospital Clínico San Carlos.

| Clinical measurements
Sociodemographic data was collected through an ad-hoc interview, and pharmacological treatment was collected by consulting clinical records.The collection of data from clinical variables was conducted by experienced psychiatrists and psychologists.Exclusion of other mental or neurological disorders was performed through a clinical interview conducted by a senior psychiatrist with large experience (chief of the Personality Disorders Unit).Severity of the disorder was measured using the Clinical Global Scale for Personality Disorders (CGI-BPD) 32 and dysfunctionality was assessed through the Global Assessment of Functioning Scale (GAF). 33Finally, ToM ability was evaluated with the Movie for the Assessment of Social Cognition (MASC). 25The MASC is an instrument with high ecological validity.Like a movie, it comprises visual information (facial, gaze and emotions recognition), hearing information (different prosodic aspects) and verbal information (language content).It response format is a multiple-choice one, which allows to differentiate between correct ToM responses and three different kinds of errors: overmentalization (attributing a mental state despite there being no explanation for it), undermentalization (a mental state wrongly attributed) and total absence of mentalization (giving social situations and mental states purely physical attributions). 26To clarify this multiple-choice format for the MASC Spanish validation, 26 the four possible responses for a specific item (What is Sandra feeling?) are described: option (a) "the haircut does not fit her so well" (absence of mentalization); option (b) "she is happy for the compliment" (undermentalization); option (c) "it is exasperated for her that he is hitting on her so heavy" (overmentalization); option (d) "she feels flattered but somehow it catches her by surprise" (correct mentalization). 25,26According to the Spanish validation of the instrument, 26 the mean scores on the MASC responses in healthy controls is 33.56 for correct mentalization responses, 6.3 for overmentalization errors, 3.4 for undermentalization errors, and 1.7 for absence of mentalization errors.

| Specimen collection and preparation
Venous blood samples (10 mL) were collected between 8:00 and 10:00 h after fasting overnight.Blood tubes were centrifuged (641 g, 10 min, 4 C).The resultant plasma samples were collected and stored at À80 C. The rest of the sample was 1:2 diluted in RPMI 1640 medium (Life Technologies, Paisley, UK), and a gradient with Ficoll-Paque (GE Healthcare, Uppsala, Sweden) was used to isolate mononuclear cells by centrifugation (800 g, 40 min, room temperature [RT]).Peripheral blood mononuclear cell (PBMC) layer was aspirated and resuspended in RPMI and centrifuged (1116 g, 10 min, RT).The supernatant was removed and the mononuclear cell-enriched pellet was stored at À80 C.
Biochemical determinations in plasma Oxytocin levels were determined through a commercially competitive ELISA-based kit (ref.500440, Cayman Chemical, Estonia) following the manufacturer's instructions.The absorbance was measured at 405 nm (Synergy 2, Biotek).The intra and interassay CV were 7.2% and 7.0%, respectively.
To obtain valid assay results, plasma samples were previously purified using C18 Sep-Pak columns (Waters, UK) to remove molecules that could interfere with the assay. 34

| Biochemical determinations in peripheral blood mononuclear cell (PBMC)
PBMC samples were first fractionated in cytosolic and nuclear extracts as in previously published articles. 35,36he protein levels from cytosolic extracts were measured using the Bradford method based on protein-dye binding.
For Western Blot analyses, 15 μg of cytosolic extracts were loaded into an electrophoresis gel.Once separated based on molecular weight, proteins from the gels were blotted onto a nitrocellulose membrane (Transfer Pack, Bio-Rad, Hercules, CA, USA) with a semi-dry transfer system (Bio-Rad) and were blocked in 5% BSA for 1.5 h, and then, the membranes were incubated overnight at 4 C with specific antibodies against OXTR (1:750 BSA 0.5%, sc515809, SCB) and β-actin (1:15,000, A5441, Sigma, St. Louis, MO, USA).After washing with a TBS-Tween solution, the membranes were incubated with the respective horseradish peroxidase-conjugated secondary antibodies for 90 min at RT and revealed by ECL™ kit following manufacturer's instructions (Amersham Iberica, Spain).Blots were imaged using an Odyssey Fc System (Li-COR Biosciences, Germany) and quantified by densitometry (NIH ImageJ software).β-Actin was used as a loading control.All blots were performed at least three times in separate assays, and the densitometries are expressed in % from the control group.

| Statistical analysis
Sociodemographic data, MASC and biochemical variables were explored using means and standard deviations for quantitative scores, and proportion of cases for qualitative variables.Generalized linear regression was performed to study the relationship between the different types of ToM responses of the MASC and OXT levels, using age, sex and prescription of antipsychotic treatment as covariates.In the same line, generalized linear regression was used to study the relationship between the OXTR levels and each type of ToM response scores, controlling for the same covariates.The Akaike information criterion (AIC) was used to support that a regression model with the covariates may explain a higher proportion of the outcome variance than a model without covariates.The adjusted R 2 was used as an effect size estimate and the odds ratio (OR) estimate was used to explore loading magnitude.All the analyses were conducted using the R software v. 4.1.2,with lmer4, psych, and ggplot2 packages.

| RESULTS
Table 1 shows descriptive sociodemographic, clinical and biochemical data.
The relationships between the type of responses in the ToM task with OXT plasma and OXTR levels and other relevant clinical variables were studied through generalized linear regression models.Models which included plasma OXT levels were not found to be significant in relation to the type of ToM response in the MASC.However, the generalized linear regression model for the OXTR levels with covariates (Table 2) were significant, showing a significant inverse relationship between the OXTR variable and overmentalization errors (OR = 0.90; t = À2.38;p < 0.05).According to the Akaike information criterion (AIC), the model with covariates is the one that best explains the probability of make overmentalization responses (AIC = 19.01),as it has a lower AIC index than the model without covariates (AIC = 21.02)(adjusted R 2 = 0.17).To put it in another way, lower OXTR levels were associated with more overmentalization errors in patients with BPD.For the rest of the ToM response types (correct mentalization and absence of mentalization errors), OXT or OXTR were not significant variables for the model.Finally, age was directly and significantly related to undermentalizing errors (OR = 1.37; t = 1.98; p = 0.05), so that older patients will show more undermentalizing errors.

| DISCUSSION
Results of our study support the relationship between deficits in oxytocin levels and social cognition impairments, through evaluated ToM errors, observed in borderline personality disorder, broadening current knowledge about endophenotypes related to specific BPD phenotypic characteristics, to improve clinical knowledge and to develop potential therapeutic targets.
In previous works of our group, patients with BPD show a deficit in both oxytocin plasma levels and oxytocin receptor (OXTR) levels. 18The finding of this deficit in OXTR levels in patients with BPD is particularly important, as the effect of oxytocin at the central level is known to be dependent of the dynamics and distribution of its receptor. 37ow levels of oxytocin have been related to an altered response to interpersonal stress, which has also been associated with the emotional instability and interpersonal conflict pattern characteristic of patients with BPD 8 : limbic system may produce an unusually high response in these patients, which would not be sufficiently modulated by the prefrontal cortex, resulting in interpersonal hypersensitivity and emotional dysregulation.Likewise, interpersonal hypersensitivity and associated emotional instability have also been related to deficits in social cognition in people with BPD. 20n the other hand, regarding social cognition, performance on mental state decoding tasks may not be significantly worse in people with BPD, despite Richman and Unoka 38 reported a tendency for patients with BPD to attribute mental states of negative valence to neutral facial expressions.It seems that people with BPD do not have greater problems to recognize isolated emotional stimulus, 39 and difficulties may appear in social situations where a more complex mentalizing ability is needed. 40,41Regarding the studied types of ToM error, most of the literature shows a tendency for people with BPD to make overmentalization errors, an erroneous overattribution of mental states.][29][30] Results of our study show how a lower expression of OXTR in patients with BPD is significantly associated with higher levels of overmentalization errors.Therefore, our results may show a direct and multivariate relationship between the demonstrated deficit of oxytocin levels plus a deficit in the expression of its receptors and the ToM errors observed in these patients, specifically with overmentalization errors, which are frequently observed in the literature.OXTR expression would provide a better representation of the general functioning of the oxytocin system than plasma OXT levels because it is a measure of the specific state, as, for example, how the OXT system is functioning at a specific moment, rather than reflecting a more general functioning.
A deficit in the oxytocin system has been related to early attachment experiences, suggesting the importance of OXTR in mediating the impact of parental care on the emergence of psychopathology. 42Moreover, Jobst et al. 43 found a relationship between reduced plasma OXT levels and the existence of a disorganized attachment style.However, it is currently unknown whether this deficit in the oxytocin system could promote a biological vulnerability that contributes to an emotional hypersensitivity to certain stressful childhood situations, leading to the development of a disorganized attachment style, or whether the continued experience of stressful and traumatic situations produces, in the long term, a dysfunction of the oxytocin system.
On the other hand, the disorganized attachment characteristic of patients with BPD may contribute to engage in impulsive behavior and, to a greater degree, different types of aggressive behaviors to get any emotional regulation.This relationship between this deficit in the oxytocin system, disorganized attachment, and aggressive behaviors would explain the efficacy seen of intranasal oxytocin treatment for impulsive-aggressive behavior in patients with BPD. 17 Therefore, as our results show a relationship between low OXTR levels and overmentalization errors in BPD patients.These results go in line with the Fonagy and Bateman's developmental hypothesis 44 through this possible relationship between low OXT levels and early attachment dysfunctions.According to Fonagy and Bateman, 44 difficulties in identifying and understanding mental states may have their origin in an impaired of understanding mental states due to neuropsychological alterations and attachment disturbances due to a nonwell attuned upbringing, a non-sensitive environment and experiences of early trauma (i.e., abandonment, neglect, physical, and sexual abuse).Some authors 21 have recently established ToM profiles characterized by undermentalization and absence of mentalization in more chronic and severe patients with BPD.In line with these results, undermentalization and absence of mentalization errors would be more severe than overmentalization errors.Our results are also in line with Galvez-Merlin et al., 21 since older patients, and therefore with greater chronicity in their majority, will show more ToM errors characterized by undermentalization.In future works it would be interesting to examine the relationship between OXT levels and ToM errors in BPD, to establish different groups of patients according to OXT levels and to test whether higher OXT levels in patients are related more severe ToM errors (undermentalization and absence of mentalization).Furthermore, future investigations must establish linear regressions models which include other clinical variables such as chronicity and experience of traumatic events, in order to define whether the deficit in the OXT system produces ToM errors, and thus emotional instability, or vice versa.
In general, patients with BPD with greater difficulties in social cognition show a worse evolution and adherence to treatment. 45In addition, poor recognition of integrated emotional stimuli in these patients has been associated with greater interpersonal hostility, producing higher distrust and impulsive-aggressive behavior that is more difficult to manage. 40Therefore, our results lay the groundwork for future studies on possible pharmacological treatments based on the oxytocin system that improve ToM abilities of these patients, with the purpose of producing an improvement in aggressive symptomatology, interpersonal hypersensitivity, improving clinical evolution and adherence to treatment.
Some limitations of this study should be mentioned.First, the reduced sample size.The characteristics of the clinical sample (patients with high levels of severity and dysfunctionality) and the difficulties in obtaining biological samples voluntarily in this group of patients are the reasons for the reduced sample size.It has unable us from developing a more complex statistical analysis of the relationship between plasma OXT levels, OXTR expression, and other clinical features.Future studies must consider testing the OXTR expression levels in more chronic patients, comparing them to less chronic and more acute patients, and also, to define their relationship with impulsive-aggressive behavior.
Second, comorbidity with other Axis I disorders has not been examined in the current sample.Because BPD patients are frequently diagnosed with comorbidity disorders such as depression, anxiety disorders, post-traumatic stress disorder or dissociative disorders, not having examined this comorbidity could have influenced results.Future studies must consider possible comorbidities in order to exclude potential influences on results.
Furthermore, it is also important to highlight the use of pharmacological treatment by the patients in the sample.The discontinuation of the pharmacological treatment would have serious clinical consequences and, to preserve patients' health and reduce the clinical risk, current pharmacological treatment has not been eliminated in this study.However, pharmacological treatment was limited to the lowest needed dose to obtain moderate behavioral stability that would allow the proper treatment, but without including patients who showed signs of sedation or slowing.In future studies, all the drugrelated data should be collected in detail in order to deduce their possible consequences through a more complete statistical analysis.
Finally, the sample was composed of patients with moderate-severe (BPD-CGI [Clinical Global Impression for Borderline Personality Disorder Patients]) and high dysfunctionality (GAF [global activity assessment scale] < 65).Therefore, the findings of our study cannot be generalized to all patients with impulsive-aggressive personality disorders as our patients were severely affected by the disorder, so they do not represent the average population of people with BPD.
To conclude, this work confirms the relationship between deficits in the oxytocin system and SC deficits, though ToM errors, observed in BPD patients, enhancing the search for endophenotypes related to the phenotypic features of this disorder, with the aim to improve clinical knowledge and developing specific therapeutic targets.In particular, our results show an inverse relationship of the OXT receptor expression with ToM errors characterized by erroneous overattribution of mental states, the most frequently observed mentalizing error in BPD patients according to the literature.In general, BPD patients with greater difficulties in social cognition show worse evolution and adherence to treatment, as well as greater impulsive-aggressive behavior.In light of these previous results, our study paves the way for future studies on possible pharmacological treatments based on the oxytocin system to improve patients' ToM abilities, aiming to improve aggressive symptomatology, interpersonal hypersensitivity and treatment adherence.
T A B L E 1 Descriptive data of sociodemographic, clinical, functional, and biochemical variables.
Abbreviations: BPD, borderline personality disorder; CGI-BPD, severity of the BPD assessed through the Clinical Global Impression for Borderline Personality Disorder Patients; GAF, dysfunctionality assessed through the Global Assessment of Functioning Scale; M, mean; n, sample size; OXT, plasma oxytocin levels; OXTR, oxytocin receptor levels; SD, standard deviation.
Results of the generalized linear regression models for ToM responses in BPD patients.