Salivary oxytocin in children with and without OCD: A statistical analysis plan
Creators: name: Anna-Rosa Cecilie Mora-Jensen
name: Clemmensen, Line
name: Pagsberg, Anne Katrine
name: Lebowitz, Eli
name: Jørgensen, Niklas R
name: Quintana, Daniel
name: Lønfeldt, Nicole Nadine
Publisher: name: Open Science Framework
Year: 2023
Subjects: subject: Psychiatry and Psychology
subject: Medicine and Health Sciences
subject: pediatric
subject: menstrual cycle
subject: diurnal variation
subject: psychiatry
subject: age
subject: obsessive-compulsive disorder
subject: child
subject: mental health
subject: youth
subject: pubertal stage
subject: oxytocin

description: Salivary oxytocin in children with and without OCD: A statistical analysis plan of a cross- sectional study Anna-Rosa Cecilie Mora-Jensen1,2, Line Katrine Harder Clemmensen3, Eli R. Lebowitz4, Daniel S. Quintana5,6,7,8, Niklas Rye Jørgensen9, Anne Katrine Pagsberg1,2, Nicole Nadine Lønfeldt1,2 1Child and Adolescent Mental Health Center, Copenhagen University Hospital – Mental Health Services CPH, Copenhagen, Denmark 2 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 3Technical University of Denmark, DTU Compute, Kgs. Lyngby, Denmark 4Child Study Center, Yale University, New Haven, CT, USA 5Department of Psychology, University of Oslo, Oslo, Norway 6Norwegian Centre for Mental Disorders Research (NORMENT), University of Oslo, Oslo, Norway 7K.G. Jebsen Centre for Neurodevelopmental Disorders, University of Oslo, Oslo, Norway 8NevSom, Department of Rare Disorders, Oslo University Hospital, Oslo, Norway 9Department of Clinical Biochemistry, Rigshospitalet, Glostrup Background The association between endogenous oxytocin activity and obsessive-compulsive disorder (OCD) in youth is a sparsely researched area. Based on animal studies and a few smaller studies in adults, oxytocin concentrations have been hypothesized to be increased in patients with OCD[1]. However, oxytocin has also been associated with anxiolytic effects and negative correlations between oxytocin and ratings of anxiety in children have been described in separation anxiety[2]. Endogenous oxytocin in humans shows large variability, making it difficult to understand findings between groups and over time[3]. Therefore, understanding and including relevant covariates in the populations being studied is important. Objective The primary objective with this study is to investigate if salivary oxytocin concentration levels are associated with OCD status in children. The secondary objective is to investigate if salivary oxytocin is associated with age, pubertal stage, and sex. For the primary objective we hypothesize that salivary oxytocin levels are different in children with OCD compared to healthy controls. We have no preconceived hypothesis on the direction of a difference given the limited number of previous studies and possible theoretical explanations in both directions. Methods The study will include data on 130 youth with OCD and 90 sex- and aged matched healthy controls and their parents from the TECTO trial (Treatment Effects of Family Based Cognitive Therapy in Children and Adolescents with Obsessive Compulsive Disorder)[4]. We will examine salivary oxytocin collecting saliva with Salivettes (Sarstedt, Rommelsdorf, Germany) and analyzing with immunoassay using Enzo® (NY, USA) oxytocin kits. Statistical analysis Power The sample size of this study is based on the sample size calculation of the primary outcome of the TECTO trial. In this sub study of oxytocin assuming n=220, α=0.05 (2-sided) and power (80%) we will be able to reliably detect effect sizes of at least a Cohen's d of 0.4. There is a large difference in reported standard deviations between endogenous oxytocin studies. In addition, differences in means of oxytocin levels between patients with OCD and healthy controls have only been described in cerebrospinal fluid and plasma. We have therefore chosen to describe the effect size (described with Cohen's-D) we can detect, but not describe the expected difference in mean and SD. Outcome The primary outcome is child salivary oxytocin concentration. This will be included as a continuous variable. In an exploratory model we will also include parent salivary oxytocin as an outcome. Covariates In the primary statistical model, we will investigate the following covariates: OCD status, age, pubertal stage and sex. OCD status and sex will be included as dichotomous variables. Age will be included as continuous variables. Pubertal stage measured with the Tanner Staging[5,6] will be included as an ordinal variable. In an exploratory analysis we will also include a family variable (family variable implying participants related as a family). Family will be included as a categorical variable. In the supplementary analyses we will investigate the variables “time of sample across the day”, self-reported menarche, functional level measured with the Children's Global Assessment Scale (CGAS)[7],IQ total score measured with the WISC[8] or WAIS[9] test, social responsiveness scale total score [10] and OCD severity measured as total score from a CY-BOCS interviews [11]. The use of these variables will be specified further below. General analysis principles Frequentist statistical analyses will be performed using R (R Core Team, Vienna, Austria). Bayesian hypothesis testing will be performed using JASP. Data Exclusion Outliers will be defined as values in salivary oxytocin above mean + 3SD. Analysis will be performed both with and without outliers and difference between the two will be reported. Results without outliers will be presented in the main results section. Result without outliers will be provided in supplementary section. Data transformation We will systematically assess the statistical assumptions underlying each statistical model. If the distributional assumptions do not hold, methods like logarithmic transformation will be used, which has also been used in previous studies of salivary oxytocin. In the section below we have described which parametric test will be used. If the assumptions are not met, we will use the corresponding non-parametric tests. Correction for multiplicity We will not adjust for multiple comparisons, but report results of all comparisons made and be aware of the choice of no mathematical correction in our interpretation. Principle for statistical analysis Analyses will be performed with both frequentist and Bayesian statistics. Bayesian statistics will be performed using a Cauchy distribution prior centered around zero with a scale parameter of r=0.5. We will conduct three primary and one exploratory analysis. In addition, we will conduct exploratory analyses on variables that are not part of our primary focus e.g., time of sample across the day and menarche. These results will be presented in a supplement. If we find associations between variables in primary analysis, we will include them in the analyses of the exploratory analyses in the supplement. If we find associations between oxytocin and variables in the supplementary exploratory analyses, we will show results of analyses with and without the variables included in the primary models. These will be described in the supplement. Primary statistical models Model 1: Difference in child salivary oxytocin between children with OCD and healthy controls (OCD status) will be tested using Welch’s t-test. Model 2: Association between salivary oxytocin in all children and the variables OCD status, age and sex will be tested with an analysis of covariance. Analysis of a possible interaction between age and sex will be included in the model. Model 3: To investigate if pubertal stage is associated with salivary oxytocin, we will repeat Model 1 with Tanner stage instead of age. We expect age and Tanner stage to be highly correlated and thus we will not include the two predictors in the same model. Exploratory analyses Model 4: To further investigate the importance of age and sex we will include all participants (children, mothers, and fathers) in an ANCOVA with age and sex and a possible interaction between age and sex. Since previous studies have shown a correlation between mothers' and fathers' salivary oxytocin[12] and children's and parents' oxytocin[13], a family variable will be included as a covariate. This analysis will allow us to increase our sample size from Model 1 in the analysis of age and sex but without OCD-status as we do not have information on parent diagnostic status. Supplementary analysis We will analyze the variables: - Time of sample. This will show possible associations between oxytocin and diurnal variation. We will include all participants and the family variable if this shows significant association in Model 4. Time of sample will be included as a categorical variable with four categories (8am-10am, 10am-12am, 2pm-4pm). - Menarche. This will be analyzed in all female children and included as a dichotomous variable. - Menstrual cycle. We will investigate a difference in oxytocin levels between the menstrual phase and ovulation. Phase of menstrual cycle will be calculated from self-reported first date of last menstruation. We will include participant in menstrual phase or ovulation only and analyze this as a dichotomous variable. We will show results from all female participants together but also show results after stratification between children (&lt;18 years) and adults (≥18 years). - Functional level. This will be investigated using the Children's Global Assessment Scale (CGAS)[7] with a range from 0-100. We will include this as a continuous variable and investigate for an interaction with OCD-status. - Social responsiveness scale (SRS)[10]. The SRS assessed in patients only and we will thus only include the patients with OCD in the analysis. We will analyze the total score of the social responsiveness scale and include it as a continuous variable. - Intelligence quotient. We will analyze the total HIK score from the WISC[8] or WAIS[9] test assessed in all children and included it as a continuous variable. - CY-BOCS[11]. CY-BOCS is assessed with patients only and we therefore only include patients in this analysis. We will analyze the total score of the CY-BOCS interview[11] from the children with OCD as a continuous variable. Handling of missing data In the primary statistical models, data sets with missing data are excluded. In our exploratory model 4 we will include data with salivary oxytocin of at least two family members, which enables us to analyze the family variable. Results Participants were included between July 2018 and July 2022. Data analysis will start after pre-registration of the statistical analysis plan. We plan to submit results for publication in August 2023. 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