The idea that people with autism are insensitive to pain is now largely a myth. For example, in an experimental study by (Carissa Cascio et al. 2008), researchers subjected people with ASD to a mild electrical shock, pressure, heat, or cold. The results showed that individuals with autism have normal pain thresholds or may even be more sensitive to pain. Another myth surrounding ASD patients is that they lack empathy. Two imaging studies (Bird et al. 2010, Fan et al. 2014) revealed that the insula and anterior cingulate gyrus, brain regions responsible for empathetic feelings for other people’s suffering, are equally activated in autistic people and healthy controls. However, individuals with ASD have difficulty displaying their emotions, which was associated with lower intensity of signals in the same brain areas.

Autism is a clinically complex disease in which, in addition to classic neuropsychiatric symptoms and signs, many sensory and motor pathologies are described (Bhat et al. 2011). The variety of these clinical manifestations includes auditory, visual, olfactory, tactile, vestibular, and other sensory disorders (Crasta et al. 2020, Jorquera-Cabrera et al. 2017, McCormick et al. 2016, Suarez 2012). Furthermore, many children with autism have different types of motor impairments, including fine and gross motor problems, in the form of poorly coordinated gait, difficulty manipulating objects, and troubles with writing (Bhat et al. 2011). Some have challenges coordinating movements between the left and right body sides (Thompson et al. 2017), which points to abnormalities in the corpus callosum that will be discussed in the next section. Others may have reduced muscle tone and hence problems maintaining posture, balance, or hand-eye coordination. Such a variety of pathologies can indicate both disorders in CNS and PNS, including afferent and efferent pathways.

Since ASD is primarily a neurodevelopmental disorder, it is intuitive to assume that autistic patients have more brain, spinal cord, and PNS problems. Examples of such issues include various types of headaches, including migraines, as well as epilepsy (Kursun et al. 2021, Lukmanji et al. 2019, Pan et al. 2020, Vetri 2020). Interestingly, the review by (Vezzani et al. 2015) described a variety of infectious and inflammatory conditions that could result in the development of epilepsy. Since infections and inflammation are almost universally present in ASD, as will be discussed later in the paper, epileptic seizures should not be surprising in these patients.

Understanding the possible etiological factors and pathogenetic mechanisms may contribute to recognizing and treating various medical diseases currently associated with autism. For example, children with autism suffer from frequent pain syndrome and such GI symptoms as abdominal pain, bloating, belching, diarrhea or constipation, flatulence, nausea, and reflux (Berry et al. 2015, Zeratsky 2019). Proper medical treatment of these diseases can improve affected patients’ quality of life, well-being, and behavior.

Endocrine issues are often underdiagnosed in ASD patients due to a multitude of other problems. Additionally, persistent inflammation, abnormal microflora, and restrictive nutrition contribute to the disruption of endocrine health (Loyacono et al. 2020). In fact, thyroid diseases are among the most frequently found in autistic individuals (Fini and Demeneix 2019). Since thyroid hormones are essential for the normal metabolism of various organs and tissues, including the nervous system, it is crucial to recognize and treat these problems in ASD patients to prevent further exacerbation of their symptoms. Interestingly, the study by (Petrov AM et al. 2017) found an association between the disruption of cholesterol metabolism and such neurodegenerative diseases as Alzheimer’s and Parkinson’s as well as ASD. Such metabolic disorder as type 2 diabetes mellitus was found to exacerbate ASD symptoms, likely due to the tissue damage from high blood glucose levels (Alhowikan et al. 2019). Moreover, dietary and environmental factors like folic acid and vitamin D deficiency were found to play a significant role in developing autistic phenotype (Alhowikan et al. 2019). Overall, it is evident that a broad spectrum of health problems seen in ASD patients, behavioral and somatic, should have a complex but common etiology, which we suggest to be various infections and persistent systemic inflammation.

Abnormal myelination in ASD occurs because oligodendrocytes, the cell responsible for nerve myelination in the CNS, are particularly susceptible to oxidative damage due to insufficient amounts of glutathione and abundance of sphingolipids (Thorburne and Juurlink 1996). In fact, the report by (Galvez-Contreras et al. 2020) describes multiple regions of the brain that have abnormal myelination. These regions include the orbitofrontal cortex, lateral prefrontal cortex, anterior cingulate gyrus, arcuate fasciculus, amygdala, medial lemniscus column, thalamus, temporoparietal junctions, and some others (Galvez-Contreras et al. 2020). Notably, magnetic resonance imaging (MRI) revealed asymmetry in these regions of the brain in a cohort of 411 ASD children (Richards et al. 2020). The state of persistent inflammation caused by various factors in ASD children made it inevitable for them to develop neuronal and glial damage from the byproducts of inflammatory process.

Apart from gross changes in the brains of autistic patient, disruptions of molecular pathways were also examined. The study by (Silva et al. 2019) showed that microdeletions in the 15q11.2 chromosomal region resulted in an increase in the white matter volume but a reduction in the size of the corpus callosum. At the same time, the disturbance of epidermal growth factor (EGF) or its receptor that are involved in promoting the proliferation of oligodendrocytes was also associated with ASD symptoms. Specifically, low levels of EGF were found in adult patients with high-functioning autism (Suzuki et al. 2007). The review by (Chen et al. 2014) demonstrated that the disruption of IGF-1/PI3K/AKT/mTOR signaling pathways was found to play a role in various neurodevelopmental disorders (NDDs), including ASD, because this pathway impacts such vital cellular processes as metabolism, response to hypoxia, and, in case of neurons, neurotransmitter synthesis (Pagani et al. 2021). Moreover, research by (Aida et al. 2015) showed that the GLT1 receptor knockout mouse models demonstrated an elevated ratio of excitation-inhibition in astrocytes, resulting in repetitive patterns of behavior. It may suggest that glucose transport disruption may be present in the glial cells of autistic children. Overall, these studies indicate that ASD pathophysiology and abnormal wiring in the brain are not caused by one change or several similar disruptions but by various factors that affect balanced myelination, oligodendrocyte proliferation, apoptosis, cellular metabolism, response to inflammatory stress, and abnormal neurotransmitter production. All these disruptions in the normal functioning of the CNS are caused by genetic and epigenetic changes that result in altered transcriptional regulation, which in turn contributes to the gross changes described earlier. Moreover, infectious agents and inflammatory mediators play an important role in these changes as well, making them as important potential targets for a curative treatment of ASD patients.

As seen from the information presented above, inflammation of the CNS is uniformly present in children with autism. Although the exact mechanism of how inflammatory mediators result in ASD is yet to be found, one of the reasons why persistent inflammation induces the development of autistic phenotype is that it alters the morphology of astrocytes and microglia in the hippocampus and the frontal cortex, two essential regions for memory formation, higher-order cognition, and behavior (Matta et al. 2019). Microglia and astrocytes play a critical role in synaptic plasticity under a normal physiologic state; however, when they are constantly exposed to infectious agents and pro-inflammatory cytokines, their morphology changes, affecting their normal functioning (Matta et al. 2019). As discussed later in the paper, these children are exposed to maternal infections and inflammation in utero, which contributes to abnormal brain development. Since the inflammatory state persists, glial cells remain in their abnormal shapes, unable to perform synaptic modification and pruning.

Autism is a multifactorial disorder in which genetic and external risk factors interact to trigger its development. If the search is performed on the SFARI GENE database (https://gene.sfari.org), the results show approximately 700 genes associated with ASD, including copy number variations (CNVs), while some papers report about 1000 candidate autism-related genes (Ramaswami et al. 2020). And amongst them, hundreds of genes have been identified as contributors to deficits in social communication, cognition, and behavioral problems of ASD patients (Cheroni et al. 2020). Still, none of these mutations were found to explain more than a small subset of autism cases. The candidate genes, potentially involved in ASD development, are known to participate in the formation of fetal brain architecture and some other organs and tissues (Masini et al. 2020). Examples of the genes include MECP2, CHD2, CHD7, FMR1, NF1, PTEN, CDKL5, and many others (Masini et al. 2020). Notably, genes that were found to be associated with ASD had also been described to be involved in other pathologies. For instance, germline mutations in the PTEN gene have been identified in up to 20% of children with ASD, macrocephaly, and white matter abnormalities (Busch et al. 2019). At the same time, this gene mutation is associated with a high risk of various malignancies and multiple hamartomas (Yehia et al. 2020). As another example, CHD2 mutations relate to various NDDs characterized by onset epileptic encephalopathy with refractory seizures and progressive cognitive decline associated with it (Carvill and Mefford 2015). CHD2-related disorders are autosomal dominant, caused by a de novo pathogenic variant, which means these mutations are sporadic (Carvill and Mefford 2015). As important as they may seem, genetic studies play a minor role in understanding this disease. Instead, epigenetic and transcriptomics research was found to be crucial to identifying the abnormal molecular pathways and mechanisms as future therapeutic targets.

Gene expression studies have been instrumental in comparing groups of people with ASD and control samples to measure which genes are dysregulated in ASD. Transcriptomic studies are essential as a key link between measuring protein levels and analyzing genetic information. In this field, research concentrates on epigenetic and transcriptional alterations in ASD development. For instance, hypermethylation of specific regions of MECP2 were associated with the behavioral changes observed in ASD (Masini et al. 2020). Moreover, the study by (Hughes et al. 2018) showed that activation of TLR2 and TLR4 genes with LPS exposure in monocytes from ASD patients resulted in consistently elevated inflammatory pathways. In contrast, similar activation in healthy controls ended with a gradual decline of these pathways. It means that these children are born with aberrant epigenetic changes in their immune system, causing a persistent inflammatory state. In fact, gene expression studies have shown that there is a significant upregulation of genes associated with the KEGG inflammatory pathway in ASD patients (He et al. 2019, Hughes et al. 2022). Moreover, transcriptome analysis studies revealed substantial abnormalities in gene expression patterns in microglia, astroglia, and cortical neurons of autistic children (Gupta et al. 2014, Velmeshev et al. 2020). Transcriptional regulations were also found to play a significant role in ASD phenotype. Specifically, a number of microRNAs (miRNAs), small non-coding RNA involved in transcriptional regulation, were reported to be downregulated (miR-23a-3p, miR-628-5p, miR-32-5p, and miR-140-39) or upregulated (miR-2467-5p and miR-7-5p) in these patients (Sehovic et al. 2020). These miRNAs are essential for regulating various processes during cell differentiation and proliferation, targeting messenger RNAs of the genes that were earlier found to be involved in ASD.

To summarize the genetics, epigenetics, and transcriptomics data presented above, although above 100 ASD-susceptibility genes have been found, the utility of this information remains elusive. On the other hand, gene expression studies allow to understand thresholds for differential expression of genes, which in turn helps to identify pathologic pathways (Ansel et al. 2017). Approximately 12,000 genes were differentially expressed between ASD compared to controls in gene expression studies since 2011. Among the 12,000 differentially expressed genes in various studies over the past decade three main sources were recognized: brain (about 3500 dysregulated genes), GI (approximately 2600 dysregulated genes), and lymphoblastoid cell lines (5600 dysregulated genes) (Ansel et al. 2017). Notably, these ASD gene expression studies allowed to find various pathways that lead to abnormalities in cell cycle, immune function, neurogenesis, and GI diseases in autistic patients. It suggests that targeting the causes of these dysregulations may lead to improvement of behavioral and somatic symptoms in children with ASD.

Various maternal infections during pregnancy were found to have a positive correlation with the clinical manifestation of ASD. A 2019 publication in the journal JAMA Psychiatry showed the invovlement of maternal infection in the development of ASD. The article presented the results of a study in which about 1.8 million children born in Sweden were observed from birth for over 40 years (Al-Haddad et al. 2019). The results of this study suggest that maternal infection requiring hospitalization increased the risk of autism and depression over the lifetime of observed children (Al-Haddad et al. 2019). In a Danish study that lasted from 1980 to 2005, it was found that children born to mothers who were hospitalized due to a viral infection in the first trimester and a bacterial infection in the second trimester had a higher risk of being diagnosed with ASD (Atladóttir et al. 2010). Another Swedish study (Lee et al. 2015) examined the association between maternal infection during pregnancy and the risk of ASD in the nationwide register-based birth cohort from 1984 to 2007. The total sample consisted of over 2 million people with 24,414 cases of ASD (Lee et al. 2015). The results showed that the odds ratio of developing the disease increased by approximately 30% in children born to mothers who had an infection at any point during their pregnancies. Remarkably, in the subsample analysis, ASD with mental retardation had a higher association with the term infections.

Other clinical and epidemiological studies also confirmed maternal infection’s role. In the study by (Fang et al. 2015), the data about 4184 children diagnosed with ASD and 16,734 non-autistic controls between 2000 and 2007 were retrieved from the National Health Insurance Research Database. Specifically, information about the maternal infection, medical history, and sociodemographic data was obtained and analyzed. The study found that there was a slight increase in the risk of ASD development in a child if a mother had two or more hospital visits for vaginal infections during pregnancy (Fang et al. 2015). The meta-analysis by (Tioleco et al. 2021) found a correlation between maternal infection and autism risk across 36 studies, assessing children with the definitive diagnosis of ASD and their prenatal exposure to infections. In fact, even adjustment for publication bias only mildly reduced the statistical significance. The prospective multicenter study by (Joseph et al. 2017) assessed the past medical records of 889 children with ASD and intellectual disability for prenatal exposure during 23-27 weeks of pregnancy from 2002 to 2004. The authors report that evaluation of the interviews obtained from the mothers showed that vaginal infections during the early stages of gestation seemed to have an increased association with the diagnosis of ASD with intellectual disability. Another meta-analysis by (Jiang et al. 2016) found that among the 15 studies assessed, any maternal infection during pregnancy increased the odds of ASD progression in a child by 12%. The authors suggest two possible mechanisms of involvement of maternal infections in the formation of autistic phenotype in children: direct exposure of a fetus to a pathogen and fetal brain damage from the maternal immune activation in response to infection.

One of the most interesting discoveries about the role of a mother’s illnesses in children’s autistic phenotype was a wall-free pathogen, also known as L-form. This phenomenon was found both in the intestines of autistic patients and their mothers (Markova 2017, Markova 2019). The pathogenic microflora was presented by Enterococcus faecalis, Pseudomonas aeruginosa, Morganella morganii, Chryseobacterium indologenes, Proteus mirabilis, Candida parapsilosis, Cryptococcus albidus, Rhodotorula mucilaginosa, Cryptococcus albidus, Aspergillus fumigatus and several others (Markova 2019). Interestingly, almost all autistic children had wall-deficient forms of Aspergillus fumigatus in their guts as well as the circulation, but it was not detected in healthy controls (Markova 2019). Moreover, Candida parapsilosis and Rhodotorula mucilaginosa, as well as cultures of opportunistic bacteria, often found as causes of urogenital infections were isolated from the blood of children with ASD. The increase in the specific characteristics of IgG, IgM, and IgA, together with the typical growth of mold, was a decisive argument in favor of the proven presence of Aspergillus fumigatus in almost all autistic children. A fungal infection like Aspergillus fumigatus or Candida albicans may suppress the immune system and damage the normal development of the nervous system through the production of toxic metabolites (Markova 2019). Notably, L-forms are capable of transforming into a fully-functional reproducing cell (Markova 2019). The theory is that infection may occur through vertical transmission from a mother to a fetus during pregnancy since L-forms were demonstrated to be capable of crossing the placenta (Markova 2019). This finding explains why no therapy used for the treatment of ASD is effective when used alone since improving the state of microbiota or exclusively targeting neurotransmitters only improves some symptoms. However, the fragmented approach is not curative because the chronic systemic inflammation present in autistic children is multifactorial in nature. Hence, evaluating pregnant women for prenatal infections and treating them accordingly may help reduce the incidence of ASD in children.

In autism, along with many symptoms associated with the pathology of the nervous system, children with ASD tend to have medical issues related to the GI tract (Zeratsky 2019). The problems show a dysbiosis which clinically manifests itself by a wide range of symptoms of digestive disorders, including diarrhea, cramping, constipation, bloating, abdominal pain, nausea, and vomiting (Holingue et al. 2018). Dysbiosis in children with autism contributes to a range of other health conditions with varying degrees of severity. These include cavities, periodontal inflammation, frequent infectious lesions in the form of stomatitis, as well as fungal and bacterial oral infections, accompanied by halitosis. In addition, ASD children’s stool often contains undigested food, has an unusual color and smell, and, in some cases, apparent signs of predominant fungal microflora in the form of moldy growth on the surface of feces. The abnormal intestinal microbiota is believed to be the main contributor to the manifestation of these symptoms.

The establishment and development of the early gut microbiome in infancy are essential for immune maturation and metabolic programming (Lozupone et al. 2012). Many factors can disrupt the formation of a healthy microbiome. One of the examples of such a factor is the delivery mode that largely determines the early gut microbial composition in children. Infants delivered by Cesarean section (CS) miss contact with the maternal vaginal microbiome compared to infants born by vaginal delivery (VD), suggesting that CS delivery perturbs the early establishment and development of the infant gut microbiome, increasing the risk of having skin microflora (e.g., Staphylococcus aureus, Streptococcus mutans, Propionibacterium acnes, and other bacteria belonging to four phyla – Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria) to be predominant in the intestines of the infants born through CS (Davis 1996, Dominguez-Bello et al. 2010). The list of possible pathologies associated with the formation of intestinal dysbiosis is diverse. For most, a link between intestinal dysbiosis and other pathological processes has not been established yet, but for some, a causal relationship and the underlying mechanisms have been demonstrated. Examples of the latter include type 1 diabetes (Vatanen et al. 2016, Vatanen et al. 2018) and allergies (Chua et al. 2018, Lee et al. 2018). For example, according to the results of a longitudinal study (Rogers and Kim 2020), maternal infections during pregnancy with rubella virus and cytomegalovirus increased the odds of developing type 1 diabetes in children 12- and 4-folds, respectively. The proven involvement of the TORCH pathogens and etiologic factors damaging the normal formation of the immune system may indicate that dysbiosis may be developing due to immune dysregulation and inflammation.

Shaping of an infant’s microflora, normal or pathologic, begins when one is born either through vaginal delivery or CS. In children with ASD, this microbiome consists of higher than usual, Staphylococci, Streptococci, Actinomycetes, pathogenic Clostridia, Egertella lenta, Propionibacterium acnes, as well as pathogenic fungi such as Candida, Aspergillus, and other Micromycetes (K. Alibek, MD, unpublished data, 2022). These microbes further contribute to the pathological state of autism by enhancing the chronic inflammation, oxidative stress, and intoxication effects produced by virulence factors or chemical toxins they secrete.

The research also shows that autistic children having an overgrowth of the spore-forming Clostridium filum in their intestines developed a regressive form of ASD, in addition to having severe GI symptoms (Hughes et al. 2018, Sandler et al. 2000). Although the administration of vancomycin improved communication and social behavior, ASD symptoms usually recurred soon after the removal of this treatment (Hughes et al. 2018). Another study by (Saleem et al. 2020) assesses plasma and serum levels of various amino acids, oxidants, and byproducts of biochemical reactions like ammonia. The authors report that ASD patients had a significant disruption of amino acid metabolism, high ammonia concentrations, and elevated oxidative stress (Saleem et al. 2020).

Another GI problem often seen in ASD patients is small intestinal bacterial overgrowth (SIBO), which is presented by bloating, distention, diarrhea, and abdominal pain after eating carbohydrate-rich meals, legumes, grains, or excess fiber (Adike and DiBaise 2018, Rana and Bhardwaj 2008). The study by (Wang et al. 2018) evaluated 310 ASD children for the presence of SIBO, and the results showed a statistically significant correlation between SIBO and autistic symptoms of social domain disruption. Overall, it appears that the dysbiotic microbiome participates in the formation of the pathological state of autism by strengthening the pro-inflammatory state, chronic inflammation, oxidative stress, and intoxication produced by toxic metabolites secreted by pathogenic microflora.

Cytomegalovirus (CMV) belongs to the family of human herpesviruses and infects approximately 60-90% of the world population (Söderberg-Nauclér 2012). Furthermore, congenital CMV infection is a leading cause of various neurologic disabilities and hearing loss in children (Boppana et al. 2013). Although the risk of developing long-term sequelae from congenital CMV is high, most infants with this infection are asymptomatic. Still, approximately 10%-15% of these children develop long-term complications (Boppana et al. 2013). Primary CMV infection commonly causes a mild illness in immunocompetent individuals, presenting with lymphopenia, lymphadenopathy, fever, transaminitis, and splenomegaly. In contrast, CMV may cause substantial harm to immunocompromised children (Bateman et al. 2021) and ASD patients are not protected from harmful effects of this virus.

CMV encodes numerous proteins and miRNAs that allow the virus to replicate and disseminate in the body, evading the immune system and resulting in detrimental health outcomes (Jackson et al. 2017). Primary CMV infection triggers a series of cell-mediated immune reactions, first by innate NK cells, then adaptive CD4+ and CD8+ T cells, and plasma cells (Jackson et al. 2011). The CMV can reactivate sporadically in these immune cells, leading to vicious cycles of stimulation of inflammatory mediators, autoimmunity, and, in some cases, cancer. The adaptive immune response to CMV, as well as to many other viruses, is characterized as a large number of effector-memory T cells, causing “memory inflation,” which is a clonal expansion of CMV-specific CD8+ T cells (O'Hara et al. 2012, Pardieck et al. 2018) Overall, CMV infection was shown to be associated with higher morbidity and mortality in immunocompromised hosts (Rahbar et al. 2013, Savva et al. 2013, Wolmer-Solberg et al. 2013).

The Epstein-Barr virus (EBV) is a highly successful human herpes virus that infects more than 90% of people globally at some point in their lives (Ressing et al. 2015). Transmission of EBV through saliva leads to infection of the epithelial cells of the oropharynx. From epithelial cells, EBV can infect B cells, which are the main reservoir for the virus. EBV can move between different types of cells, mainly B cells and epithelial cells. Systemic autoimmune diseases often occur as overlapping syndromes with symptoms and characteristic autoantibodies. Moreover, because the virus can switch between the latent and lytic life cycle, EBV can cause chronic reactivating infections (Houen and Trier 2021). EBV causes latent infection in B lymphocytes, characterized by limited expression of viral genes. To produce new viral offspring, EBV is reactivated from its latency. During the active phase, EBV uses cellular machinery to express more than 80 of its genes. Even though EBV-specific T lymphocytes can react and destroy infected cells, EBV remains in its host for a lifetime due to the multiple immune evading and suppressing mechansims (Ressing et al. 2015).

Although symptomatic EBV infection is rare in infants, it can sometimes can cause such neurologic disorders as meningitis, encephalitis, transverse myelitis, Guillain-Barré syndrome, cerebellar ataxia, sleep disorders, and psychoses (CDC 2020). Apart from these pathologies, EBV infection can cause hematologic and immune problems. The virus can induce severe lymphocytosis, neutropenia, hemophagocytic syndrome, hypogammaglobulinemia, and lymphoproliferative disease. These immunosuppressive conditions make infected individuals prone to secondary infections (CDC 2020).

Congenital rubella syndrome (CRS) is an infant disease that stems from maternal infection with rubella virus (Lanzieri et al. 2020). Infection with this virus in the first trimester may lead to stillbirths, miscarriages, and severe birth defects (Lanzieri et al. 2020). Fortunately, defects are rare after the twentieth week of gestation. Although the disease is primarily asymptomatic in mothers, their babies are almost always affected, especially if the infection occurs during the first 12 weeks of pregnancy (Lanzieri et al. 2020). Early complications include deafness, cataracts, heart defects, brain disorders, bone deformities, and liver or spleen damage in newborns (Lanzieri et al. 2020). Late-onset manifestations of CRS are diabetes, thyroid dysfunction, and visual or neurological abnormalities.