Assessment of the levels of vasopressin and androgen in the sample of Iraqi children with Autism spectrum disorder

level in Saliva was decreased in the whole autistic group compared to the control, but there was a significant increase in the mean vasopressin level in the older age group 10-< years than in the younger age group. Mean salivary apelin level was a highly significant increase in autistic children compared to control and didn’t find statistically significant differences between the age groups of autistic children according to the apelin level in saliva . The levels of these hormones are altered in autistic boys than the normal behavior boys, there is a significant correlation (p<0.05) between vasopressin and androgen with simple linear regression.


Introduction
Recent epidemiological studies, conducted in different regions of the world, indicated that at least one in every 100 people has some form of autism.Although there is no known unique cause of autism, there is growing evidence that autism can be caused by a variety of disorders, however, the exact pathophysiology is unknown [1][2][3].
No disease markers for the diagnosis of autism have been validated, the discovery of such biomarkers something like a blood test, or saliva, for autism, may eventually lead to, or would allow earlier and potentially more reliable diagnoses of autism, and would also help researchers achieve an understanding of the biological basis of this disorder [4,5].
It is four times more common among boys than girls.Its pathogenesis remains unclear, the biological hypotheses concentrating on neuroanatomy, physiology and genetics of CNS.Several endocrine hormones are directly or indirectly linked with the encoding of social behavior, recent studies have focused on whether and how these peptides are involved in the pathogenesis of ASD [6].
Neuroactive steroids(NASs) have the capability of rapidly modifying neural activities and producing a rapid non-genomic effect in the brain, the term neurosteroids used for NASs that are synthesized from cholesterol de novo in the brain or formed by metabolism in the brain from precursor compounds coming from endocrine sources [7].
The majority of studies conducted worldwide have found that a number of factors, including gender differences and biological and genetic susceptibilities as well as the effects of certain hormones, contribute to the development of autistic traits.These factors must be investigated in order to better understand the behavioral disorder autism [8].
Many studies in the world showed that some steroid hormones are correlated with autism, but others showed different results, one of them is the androgen hormone deficits for its role in brain functioning [9].The neuropeptides arginine-vasopressin (AVP), which are synthesized in the paraventricular and supraoptic nuclei of the hypothalamus and transported to the posterior pituitary and released into the blood, are closely connected and have attracted significant interest in research on the genesis of mental illnesses [10].
Numerous projections into other regions of the brain, primarily the limbic system, are made by the vsopressinergic neurons that are generated in the hypothalamus.Act in a neuromodulatory way, for example, while processing emotions or exhibiting social behavior [11,12].Despite having a strong affinity, this neuropeptide has an opposing impact in most brain regions.Vasopressin is shown to be associated with the emotions of fear and anger [13].
Similar to vasopressin, apelin is a relatively recent neuropeptide that regulates the vasopressinergic system and is produced by magnocellular neurons in the paraventricular and supraoptic nuclei of the hypothalamoneurohypophysial system.A tiny peptide found in the brain in 1998 by Fujinos' team, along with indications that apelin and APJ are also abundantly distributed there [14,15].
Although less documented, the effects of apelin on neurobehavior have been addressed and its beneficial effects on emotionality have been proven.secreted by the adipose tissue, has been recently characterized as human apelin and its receptor are found in several brain regions including the hypothalamus, thalamus, cerebral nuclei, and pons, the potential effect of apelin, especially its potent formapelin-13, on mood and anxiety had been studied because of high expression in hypothalamus and indicated that APJ includes some effect in the regulation of the HPA axis as a result to stress [16].Due to the lack of similar study in Iraq, aimed to assessment the salivary androgen and vasopressin and apelin levels in children with Autism to investigate whether those levels differ from those of sexand age-matched healthy controls for casecontrol study

Subjects
Children include 45 boys with a confirmed professional diagnosis of autism spectrum disorder (early childhood autism) from Al-Safa Center in Zayoonah, Baghdad, Iraq and from Baghdad Teaching Hospital -Medical City-Ministry of Health, with the ages ranging from 6-13 years were enrolled in this study.
The control group included 45 boys -Iraqi pupils from Zamzam primary schools in Baghdad city, who corresponded to the first group in their age, in order to compare the salivary hormones to allow for case-control design.All pupils involved in this study were interviewed.This study was performed from November 2022 to the end of April 2023.
All the subjects recruited to this study were of similar ethnic backgrounds to Iraqi Arabs people depending on the history taken from them.

Inclusion criteria
Autistic children (mild and moderate) with early diagnosis by the specialist child psychiatric will be subject in this study and the normal children were free of neurological and psychiatric disorders

Exclusion criteria
Include no present or history of other psychiatric disorders, comorbid disorders were excluded, no significant illness and all children were free medication affect saliva hormonal levels.

Samples collection
Salivary sample from each subject was collected under standardized conditions, after being asked to sit down and relax as much as possible, then by tilting the head forward, allowing to saliva gather on the floor of the mouth, the entire mixture of unstimulated saliva was collected, and placed in sterile plastic collection tube, centrifuged at 3000 rpm for 10 minutes, clear supernatant frozen and stored at -20 o C [17].Salivary samples were collected from the study subjects between 8 to 10 a.m., before breakfast.
Commercially available human androgen, vasopressin and apelin kits, all purchased from the company (human Gesellschaft for biochemical and diagnostic mbHmax-Wiesbaden-Germany, were used for determining the level of these hormones. The samples were measured by using an indirect enzyme-linked immunosorbent assay (IELISA).Hormones analysis was carried out in the National Center of Teaching Laboratory-Ministry of Health.

Statistical Analysis
The Statistical Analysis System-SAS (2018) program was used to detect the effect of different groups (patients and control) on study parameters.T-test was used to significantly compare between means in this study.

Results
The mean salivary androgen level for all the children included in this study are presented in Table 1, the mean salivary androgen level was highly significantly increased (P≤0.01) in the autistic group 2.24± 0.16, than that of the control 1.67± 0.126 ng/ml.Also, it was found that as appears in the same table highly significant increase of salivary androgen level in the older age group between 10 to 13 years old than in the age group from 6 to 10 years, 1.70 ±0.23 ng/ml.The mean vasopressin level in Saliva was significantly lowered in the whole autistic group compared to the control, but there was a significant increase in the mean vasopressin level in the older age group 10-< years than in the younger age group( Table 2 ).In the current study, as appears in Table 3  and 4, mean salivary apelin level was highly significantly decreased in autistic children compared to control and didn't find statistically significant differences between the first group and second group in the group of autistic boys according to the apelin level in saliva.

Discussion
Autism is four times more common in boys than girls, these sex differences suggest that there may be a male vulnerability to developing the major subgroups of autism conditions, this findings caused the extreme male brain theory, which has led the researchers to look for the possible role of testosterone and or androgen in the cause of ASD [18].To our best knowledge, there is no such study about androgen, vasopressin and apelin levels in autistic primary school children in Iraq.
The results of the current study showed that the androgen level in saliva was significantly increased in the autistic group than in normal boys in the control group, which also suggests the role of androgen hormone in the behavior of those children.These results came in agreement with that of previous research [19], who found significantly higher salivary testosterone levels in boys at prepubertal and concluded that salivary testosterone levels remained stable in this period [19].Also, it has been found a relationship between serum testosterone levels and psychological behavior in adolescent girls [20].
It has showed alteration of adrenal gland metabolites in relation to some behavioral disorders like Kanner's and Asperger syndromes and stated that the steroid hormone profiles, especially those of androgens may be useful clinical markers that support the diagnosis of autism and further imply that the mild more severe forms of autism can be distinguished and might be a promising target for pharmaceuticals for the severe form [7].
it has been disclosed that the concentrations of some steroid hormones like 20-alpha dihydropregnenolone also DHEA were high statistically when studied and compared in the saliva of children diagnosed with autism and children without autism (epiandrosterone-C , allopregnanolone) than control children and were more prominent in older autistic children and boys when analyzed using gas-chromatography-mass spectrometry and radioimmunoassay.In this study, the age of children was allocated into two groups, group one between 6 -10 years old and group two from 10 to less than 13 years [9].It has been stated that the testosterone level was found to be associated with autistic traits in children aged 2 years or younger [21].In contrast to the results of the present study about higher androgen levels in the older age group, it has stated an association between elevated androgen levels in early development and autistic behaviors [22].
Many previous studies in different parts of the world using different methods have indicated that testosterone or androgen may play a central role because of its association with the modulation of psychomotor activity and executive function which are the main clinical features of individuals with autism [23][24][25][26].The results of the present study about reduced vasopressin levels in autistic children agree with the data published by previous research in the Riyadh area when measured vasopressin levels in autistic and control children and found statistically significantly lower plasma levels of vasopressin in autistic children when compared to the controls.Which explained that, might be related to the abnormal social behavior in autistic children [27].
In contrast, it has found elevated concentrations of vasopressin in adults with ASD [28].It has not find any change in plasma vasopressin concentration and statistical differences in the concentration of vasopressin when they compared children with autism to normal children from China, even with a relatively large sample size (>70 in each group), well established AVP measurement method, and related that too implies the normal hypothalamic AVP synthesis or release in ASD children [29].In fact, it has suggested that there is promise for the development of precise and sensitive techniques for laboratory diagnosis of ASD early in infancy since measuring the AVP level in conjunction with the assessment of gene expression levels for the vasopressin receptor properly discriminated neurotypical and ASD people in 84% of the instances.In this study, the salivary apelin level was higher in an autistic group with no significant differences when compared to the control group [30].Similarly, it has showed No significant difference was found between the groups for girls or across the entire subject cohort, indicating that apelin-13 may play a role in the etiopathogenesis of ADHD either directly affecting the apelin receptor or via its antagonistic effect on the vasopressinergic system.Plasma male children with ADHD had significantly higher mean apelin-13 levels than plasma male control subjects [31].
It has detected higher serum apelin-12 levels in patients who scored higher in the Beck depression inventory [32].It has found that the serum apelin level was significantly lowered in patients with major depressive disorder than in healthy controls [14].It has reported that intracerebroventricular apelin-13 infusion reversed memory impairment and depressive-like behaviors in chronically stressed rats [33].This study also showed that the concentration in saliva hasn't any statistical difference in apelin levels between the two age groups of autistic boys.
It has showed [34] that With observation, it becomes clear that the role of this hormone in the regulation of emotional states is more nuanced.The severity of depression symptoms in elderly individuals is positively connected to plasma apelin levels.These findings highlight apelin's multifaceted role in the control of emotional state, which may vary depending on the participants' ages and metabolic conditions.To emphasize the therapeutic potential of influencing the apelinergic system in mood-related illnesses, more research is necessary.
Vasopressin and androgen showed the highest correlation in the studied groups in this study (r = 0.28, P = 0.048) in correlation matrices (Table 4).Whereas the opposite was observed for apelin and androgen (r = -0.079,P = 0.62).Recently, several patient groups have favoured using saliva as their specimen of choice for steroid detection due to its convenience, non-invasive nature, and stressfree collection [9,35].In particular for autistic kids who frequently exhibit excessive stress reactions?Steroid concentrations in saliva mirror those in blood and are significantly lower than those in serum [36,37], and steroid polarity affects the ratios of various steroids in these bodily fluids.Androgens, which are unconjugated lipid-soluble steroids, May easily cross capillary walls, and salivary concentrations of these substances have been estimated to account for 10% of serum levels [34,36,37].
As the adrenal cortex is the primary source of DHEA and DHEAS in people, whose levels were raised, it appears to be the major source of amplified steroid production in autistic children.Despite this, every research subject was a prepuberta.The adrenals also release very little androgen throughout the early years of life.Prior to gonadal maturation, the reticular zone forms during adrenarche changes the adrenal cortex and produces DHEA, DHEAS, and other androgens [38].

Conclusion
Finally, steroid hormone profiles may provide as helpful clinical indicators for the diagnosis of autism.It is important to look into how biochemical and genetic factors contribute to increased steroid production in autism.International research should be done to examine any potential links between variations in steroid production and autism and environmental or iatrogenic variables.

Table 1 .
Comparison between autistic patients and control groups in salivary Androgen levels

Table 2 .
Comparison between autistic patients and control groups in salivary Vasopressin levels

Table 3 .
Comparison between autistic patients and control groups in salivary apelin levels

Table 4 .
Correlation between parameters study in Patients Group