Jaroslava Durdiaková

The effects of saliva collection, handling and storage on salivary testosterone measurement

Several endocrine parameters commonly measured in plasma, such as steroid hormones, can be measured in the oral fluid. However, there are several technical aspects of saliva sampling and processing that can potentially bias the validity of salivary testosterone measurement. The aim of this study was to evaluate the effects caused by repeated sampling; 5 min centrifugation (at 2000, 6000 or 10,000g); the stimulation of saliva flow by a cotton swab soaked in 2% citric acid touching the tongue; different storage times and conditions as well as the impact of blood contamination on salivary testosterone concentration measured using a commercially available ELISA kit. Fresh, unprocessed, unstimulated saliva samples served as a control. Salivary testosterone concentrations were influenced neither by repeated sampling nor by stimulation of salivary flow. Testosterone levels determined in samples stored in various laboratory conditions for time periods up to 1 month did not differ in comparison with controls. For both genders, salivary testosterone levels were substantially reduced after centrifugation (men F=29.1; women F=56.17, p<0.0001). Blood contamination decreased salivary testosterone levels in a dose-dependent manner (men F=6.54, p<0.01, F=5.01, p<0.05). Salivary testosterone can be considered A robust and stable marker. However, saliva processing and blood leakage can introduce bias into measurements of salivary testosterone using ELISA. Our observations should be considered in studies focusing on salivary testosterone.

Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing

Identification of foreign biological material by genetic profiling is widely used in forensic DNA testing in different cases of sexual violence, sexual abuse or sexual harassment. In all these kinds of sexual assaults, the perpetrator could constrain the victim to kissing. The value of the victims saliva taken after such an assault has not been investigated in the past with currently widely used molecular methods of extremely high sensitivity (e.g. qPCR) and specificity (e.g. multiplex Y-STR PCR). In our study, 12 voluntary pairs were tested at various intervals after intense kissing and saliva samples were taken from the women to assess the presence of male DNA. Sensitivity-focused assays based on the SRY (single-copy gene) and DYS (multi-copy gene) sequence motifs confirmed the presence of male DNA in female saliva after 10 and even 60min after kissing, respectively. For specificity, standard multiplex Y-STR PCR profiling was performed and male DNA was found in female saliva samples, as the entire Y-STR profile, even after 30min in one sample. Our study confirms that foreign DNA tends to persist for a restricted period of time in the victims mouth, can be isolated from saliva after prompt collection and can be used as a valuable source of evidence.

Comparison of different collection procedures and two methods for DNA isolation from saliva

Abstract Background: The non-invasive, flexible and easy sample collection makes saliva an interesting source of DNA for research and diagnostic purposes. The aim of our study was to find the most suitable collection method for biological material from the oral cavity and the most effective DNA isolation technique for further analytic applications. Methods: DNA was isolated from swabs, Salivette saliva, whole saliva and samples collected with a commercial set for scraping of buccal cells. Phenol-chloroform extraction and isolation using a silica membrane based commercial kit were compared. Quantity of bacterial and human genomic DNA was estimated using real time PCR. The effects of storage conditions on DNA recovery were assessed. Results: Sample collection techniques significantly affected the quantity of DNA for both, silica membrane based and phenol-chloroform isolations. Whole saliva provided the largest number of bacterial and human genome copies after both extraction methods. Storage for 36 months at –20°C reduced recovery of human genomic DNA five times after silica membrane based extraction and 10 times after phenol-chloroform isolation. Conclusions: Whole saliva was found to be the most suitable material for human and bacterial DNA isolation. Both compared methods are useful considering the quantity of extracted DNA.

Mental rotation in intellectually gifted boys is affected by the androgen receptor CAG repeat polymorphism

Testosterone was shown to organize brain and modulate cognitive functions. It is currently unknown whether mental rotation is also associated with prenatal testosterone exposure and testosterone-related genetic polymorphisms. The aim of our study was to analyze associations between mental rotation performance, the actual testosterone levels, the prenatal testosterone level (expressed as 2D:4D ratio) and the androgen receptor CAG repeat polymorphism in intellectually gifted boys. One hundred forty-seven boys aged 10-18 years with IQ>130 were enrolled. Saliva samples were collected and used for ELISA of actual levels of salivary testosterone. The 2D:4D finger length ratio as an indicator of prenatal testosterone was measured on both hands and averaged. Amthauer mental rotation test was used for the assessment of this spatial ability. The CAG repeat polymorphism in the androgen receptor gene was analyzed using PCR and capillary electrophoresis. Linear regression revealed that 2D:4D finger length ratio and the number of CAG repeats in the androgen receptor gene were associated with mental rotation. Actual levels of testosterone did not correlate significantly with mental rotation. Multivariate analysis of covariance revealed that after adjustment of age as a confounding variable, only the effect of the genetic polymorphism was significant. The results are in line with our previous genetic analysis of intellectually gifted boys showing the importance of CAG repeat polymorphism in the androgen receptor gene. Details of the interactions between androgen signaling, testosterone levels and its metabolism especially during the prenatal development of brain function remain to be elucidated.

Testosterone and Androgen Receptor Sensitivity in Relation to Hyperactivity Symptoms in Boys with Autism Spectrum Disorders.

Introduction Autism spectrum disorders (ASD) and hyperactivity symptoms exhibit an incidence that is male-biased. Thus androgen activity can be considered a plausible biological risk factor for these disorders. However, there is insufficient information about the association between increased androgen activity and hyperactivity symptoms in children with ASD. Methods In the present study, the relationship between parameters of androgenicity (plasmatic testosterone levels and androgen receptor sensitivity) and hyperactivity in 60 boys (age 3–15) with ASD is investigated. Given well documented differences in parent and trained examiners ratings of symptom severity, we employed a standardized parent`s questionnaire (Nisonger Child Behavior Rating Form) as well as a direct examiner`s rating (Autism diagnostic observation schedule) for assessment of hyperactivity symptoms. Results Although it was found there was no significant association between actual plasmatic testosterone levels and hyperactivity symptoms, the number of CAG triplets was significantly negatively correlated with hyperactivity symptoms (R2 = 0.118, p = 0.007) in the sample, indicating increased androgen receptor sensitivity in association with hyperactivity symptoms. Direct trained examiner´s assessment appeared to be a relevant method for evaluating of behavioral problems in the investigation of biological underpinnings of these problems in our study. Conclusions A potential ASD subtype characterized by increased rates of hyperactivity symptoms might have distinct etiopathogenesis and require a specific behavioral and pharmacological approach. We propose an increase of androgen receptor sensitivity as a biomarker for a specific ASD subtype accompanied with hyperactivity symptoms. Findings are discussed in terms of their implications for practice and future research.

Topoisomerases interlink genetic network underlying autism.

DNA topoisomerases belong to the group of proteins that play an important role in the organizational dynamics of the human genome. Their enzymatic activity solves topological strain rising from DNA supercoiling occurring during transcription. DNA topoisomerases are especially important for transcription of genes involved in neurodevelopment. Disruption of topoisomerase activity in animal models resulted in impaired neurodevelopment and changed brain architecture. Recent research revealed that topoisomerases induced expression of the same group of genes as those associated with autism. Transcriptional inhibition of neuronal genes during critical stages of brain development may be responsible for pathology of neurodevelopmental disorders such as autism. In this review we aim to outline the role of topoisomerase in neurodevelopment and its possible linkage to neuropathology of autism.

Spatial abilities are not related to testosterone levels and variation in the androgen receptor in healthy young men.

Androgens modulate brain functions such as cognition, emotions and ability. Several studies have shown a correlation between testosterone levels and mental rotation. The aim of the present study was to confirm the influence of salivary testosterone levels, 2D/4D ratio (such as a putative marker of prenatal testosterone), and sensitivity of androgen receptor on the mental rotation in healthy young men. Seventy-five healthy young men (age, 21.86 year) volunteered in this study. Mental rotation scores of our subjects were assessed using the Vandenberg and Kuse Mental Rotation Test. The 2D/4D finger length ratio as an indicator of prenatal testosterone was used as an average measurement of both hands. Correlation analysis revealed no correlation between salivary testosterone levels and mental rotation. However, we have observed a trend towards a negative correlation. There were no statistically significant results between 2D/4D ratio and mental rotation or between polymorphic three-nucleotide (CAG) repeats and mental rotation tests. Future studies should focus on other genetic determinants of spatial abilities, potentially genes involved in testosterone metabolism.

The effect of mental rotation on changes in plasma testosterone and cortisol levels

Testosterone level has an influence on cognitive functions, especially spatial abilities. The relationship is, however, bidirectional and brain activity also affects testosterone levels. The aim of this study was to analyze the effects of an intensive 3D mental rotation task on testosterone levels in young healthy men and women. In the mental rotation task, men reached a higher top score (P=0.027) and total score (P=0.004) compared to women. In 8 out of 9 women (P=0.008) but not in men (P=0.129) testosterone levels decreased after one hour of mental rotation testing. A significant gender difference was shown (P<0.0001). In all women, plasma cortisol levels was significantly lower after testing (P=0.004). In men cortisol levels decreased in 7 out of 9 subjects (P=0.039). A significant gender difference was not found (P=0.19). No association was found in women between baseline testosterone levels and mental rotation total score (P=0.810). In men there was a positive correlation between baseline testosterone and mental rotation total score (P=0.015). A significant difference gender difference was seen in the association between testosterone and mental rotation score (P<0.05). Mental rotation stimuli caused significant changes in hormonal levels of testosterone and cortisol. A gender-specific response was detected in testosterone fluctuation.

Problem Behavior and Testosterone in Pre-pubertal Boys with Autism Spectrum Disorder

Aim The aim of this study was to compare testosterone level with clinical features of problem behavior in children with ASD. Methods The study sample consisted of 35 pre-pubertal boys with clinical diagnosis of ASD. In all children (ages 3-10) diagnosed as ASD, parents completed Nisonger Child Behavior Rating Form (NCBRF) (version for intellectual disabilities)-parent version. Rating scale assessed child´s behavior as observed in previous 1-2 months. Specific problem behavior was rated on subscales for conduct problems, anxiety, hyperactivity, self-injury/stereotypic behavior, self-isolated/ritualistic and overly sensitive. On this rating form parents also assessed social behavior: compliant behavior and adaptive social behavior. Total serum testosterone levels were determined after the parents completed rating forms, according to standardized procedure. Results It was found positive correlation between total serum testosterone levels and conduct problem subscale (p=0.007, r=0.445) and as well as total serum testosterone levels and overly sensitive subscale (p=0.03, r=0.348) of NCBRF. In other subscalesof NCBRF(problem behavior or social behavior) were no significant correlations. Conclusions The results suggest that there might be some relationship between problem behavior (specifically conduct problems and overal sensitivity) and testosterone levels in boys with ASD. Testosterone levels had no significant correlation with other subscales of NCBRF. However further research is needed for investigation of complex androgen activity (free TST levels, SHBD levels) and potential roles of other hormones in complex etiology of conduct problems and overall sensitivity in children with ASD. This findings rise interest in further investigation of relationship between testosterone and conduct problems and overall sensitivity in children with ASD and indicate that therapeutic strategies targeting testosteronecould be useful in the treatment of problem behaviors in children with ASD. This study was part of broader projects focused on ASD conducted in Autism Research Center for Autism (ARCA), Comenius University and was supported by grants APVV 02544-11 and VEGA1/0086/14.

The Applicability of Autosomal Short Tandem Repeats Genotyping for Minor Contributor DNA Profiling from Mixed Saliva Samples

Objective: In our previous study focused on Y-chromosome sequence identification and genotyping we revealed the possibility of male minor fraction identification and genotyping in mixed salivary samples obtained from females 60 and 30 minutes after intense kissing. The aim of this study was to test the applicability of an autosomal STR (aSTR) profiling kit for male fraction detection and genotyping on salivary samples obtained from females 1 – 60 minutes after intense kissing. Methods: The aSTR typing was performed on DNA samples originated from buccal swab and saliva samples collected from 12 heterosexual pairs before and after 2 minutes of intense kissing, respectively. The success of minor contributor allele identification was quantified as the ratio between Counts of Identified Obligatory Alleles and Counts of Potentially Identifiable Obligatory Alleles. For the estimation of proportion of minor contributor DNA the Y/X Amelogenin peak height ratio was used. Results: In samples collected immediately after kissing has stopped the Amelogenin Y/X signal ratio varied between 0 and 63%. The ratio was associated with aSTR profiling success as in samples with higher than 7% Y/X ratio more than 80% of minor contributor male alleles were identified. In one sample collected 5 minutes after kissing expected male signal was detected with Y/X ratio reaching 15% and 77% of obligatory male alleles were identified. In comparison of previously and currently utilized methods for minor contributor male DNA detection and identification the concordance in their performance was recorded. Conclusion: We confirmed that in salivary mixtures, female saliva DNA analysis with use of aSTR genotyping kit is possible, but with limited effectiveness. The male admixture was detected with aSTR genotyping kit in salivary samples collected up to 5 minutes after intense kissing.