Role of Gut Microbiome in Autism Spectrum Disorder and Its Therapeutic Regulation

1Concerns over autism spectrum disorder (ASD) are alarming, as many people are being diagnosed every year. Statistics report that ASD affects 1 in 68 people worldwide. ASD is a highly prevalent neurodevelopmental disorder that affects normal brain development and is characterized by poor communication skills, poor reasoning, and repetitive and obstructive behavioral patterns. Doctors try to alleviate these conditions through behavioral therapies and specific therapeutic interventions. They often administer drugs such as Aripiprazole, Escitalopram, antidepressants, and drugs that affect the concentration of a neurotransmitter or chemical messenger called serotonin in the brain. Selective serotonin uptake inhibitors (SSRIs) may improve the communication, social skills, and adaptability of the patients. Previous studies have suggested that therapeutic interventions may also cause hyperactivity and aggression and side effects such as vomiting, irritability, increased appetites, weight gain, and sedation. Scientists have been trying to explore the molecular mechanisms behind the pathology of ASD to facilitate alternative treatments. Several evidence suggests that genetic factors such as chromosomal abnormalities and environmental factors like diet and stress are involved in the pathogenesis and advancement of ASD. Over many years, parents have reported that their children diagnosed with ASD also suffer from gastrointestinal (GI) symptoms like constipation, abdominal pain, diarrhea, and vomiting (;;;; Interestingly, accumulating research has demonstrated the gut–brain axis or multiple biochemical signaling pathways that take place between the gastrointestinal tract (GI tract) and the central nervous system (CNS), and its possible association with ASD. Mounting evidence explains that gut microbial dysbiosis is implicated in the pathogenesis of multiple diseases, including inflammatory bowel disease (IBS), coeliac disease (CD), allergy, asthma, metabolic syndrome, cardiovascular disease, obesity, and ASD. This mini-review focuses on the association between ASD and the gut microbial population. We also addressed the impact of unregulated antibiotic usage on the gut microbiome, the pathogenesis involved, and various potential microbial therapeutics such as fecal microbiota transplantation (FMT) for treating autistic patients.

2Recent studies on the gut–brain axis suggested that the gut contains millions of nerve cells, eventually forming an extensive network called the enteric nervous system (ENS). This enteric nervous system is also considered our second brain. The ENS and the central nervous system (CNS) are mainly connected via the vagus nerve and form the gut–brain axis. The communications within the gut–brain axis occur through the autonomic nervous system, enteric nervous system, neurotransmitters, hormones, and immune responses ( Figure 1 ). Neurotransmitters produced in the gut influence our emotions by regulating the gut–brain axis. Around 90% of neurotransmitters, such as serotonin, are produced in the gut. The human gut microbiome consists of trillions of bacterial cells, which is 10-fold more significant than human body cells. The average human gut contains 1 kg of diverse groups of bacteria that mostly perform beneficial activities often involved in metabolites production and transportation and maintenance of gut homeostasis. Earlier, the majority of microbiota cannot be classified by traditional cultural techniques. Researchers have overcome this limitation by using next-generation sequencing technology and metagenomic sequencing to identify unculturable microorganisms. Our intestines harbor numerous beneficial bacteria that generate many neurotransmitters and active metabolites in the gut by utilizing the consumed foods. Essential amino acid tryptophan found in food acts as a precursor of a neurotransmitter called serotonin. Over 1%–2% of dietary tryptophan is converted into serotonin. Beneficial gut bacteria such as Bifidobacterium infantis convert tryptophan into serotonin, regulating emotions and behavior. Clostridium sporogenes increases the production of tryptophan metabolites called indole-3-propionic acid (IPA), a bioactive molecule that increases the production of antioxidant and neuro-protectant molecules inside the gut ( Table 1 ). The gut can also be invaded by various pathogenic microbes, leading to the development of gastrointestinal problems following the presence of Clostridium bolteae. Interestingly, Clostridium bacteria in the colon indicate higher risk and severity of ASD. This specific strain of bacteria produces tetanus neurotoxin (TeNT), which passes through the vagus nerve to the CNS and blocks neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein, and precipitates a whole range of behavioral deficits. Li et al. have implied that the presence of Clostridium tetani can be used as an indicator for the ASD diagnosis ( Table 1 ).

3Numerous evidence explains that microbial colonization starts during prenatal development inside the mother’s womb, in the placenta, and amniotic fluid. Bacteria colonization in the maternal–fetal unit can have beneficial or adverse effects on pregnancy and/or fetal development. Colonization of some bacterial species such as Lactobacillus, Enterococcus, Streptococcus, Peptostreptococcus, Corynebacterium, Escherichia, and Staphylococcus in infants passed from the breast milk of lactating mothers. Pioneer microbial colonization inside the GI tract of preterm infants begins at birth. After 1 year in which a unique and complex microbiota develops, the composition of the microbiota stabilizes around the age of 2–3 years. The intrauterine period during pregnancy and postnatal period can provide a critical window for infant microbiome development, which has lifelong ramifications on overall health. Interestingly, the brain of neonates also grows from 36% to approximately 90% of its future adult volume until the age of 2. Thus, establishing a healthy microbial composition falls into the same critical time for brain development. A study with rat models has demonstrated that maternal separation from infant rats daily for 3 h from day 2 to 12 postnatal led to microbial dysbiosis. After being exposed to chronic stress, there was an elevation in the abundance of Bacteroides and Clostridium in rats and an increase in immune cells such as pro-inflammatory cytokines and chemokines (IL-6 and CCL2).

4The interval between conception and gestation is a crucial period for fetal neurodevelopment. During this time, several factors, such as an unhealthy diet, microbial infection, and metabolic stress, can lead to maternal microbiome dysbiosis that may influence the abnormal neurological development of offspring, leading to lifelong behavioral deficits.

5The study conducted by Vuong et al. in a mice model of three categories namely, antibiotic-treated (ABX) embryos and specific pathogen-free (SP) and germ-free (GF) mice mothers, demonstrated that colonization of Clostridia-dominant spore-forming bacteria in maternal microbiota leads to the reduction of fetal brain gene expression and thalamocortical axonogenesis connecting the thalamus and the cerebral cortex and impaired outgrowth of thalamic axons. Previous studies have shown that spore-forming Clostridia species downregulate NTNG1 expression and minimize netrin-G1a+ thalamocortical axons, leading to the impairment of thalamic axon outgrowth. Vuang and co-workers have demonstrated that colonization of Bacteroidetes (BD) in mother mice may significantly enhance netrin-G1a+ thalamocortical axons. The healthy microbial composition inside the maternal mice can be restored via fecal transplantation, increasing the relative abundance of Bacteroides species by 95%. The relative abundance of Clostridium difficile was reduced to 5%.

6In a mother’s intestinal tract, a specific bacterial consortium produces metabolites that influence the development of the fetal brain. Further experiments showed that the maternal gut microbiota promotes healthy brain development by regulating biochemical profiles and selecting metabolites in the fetal brains of developing offspring. During pregnancy, the gut microbiota modulates the bioavailability of many biochemicals, nutrients, and growth factors that circulate in the maternal blood, supporting offspring growth and proper fetal brain development. After analyzing the molecules in the mothers’ blood and their offspring’s blood and brain, they found that the levels of specific metabolites were reduced in GF and ABX pregnant mothers. SP mice, ABX, and GF mice were exposed to thalamic explants. The result showed that an increase in metabolites, such as trimethylamine-N-oxide (TMAO), N,N,N-trimethyl-5-aminovalerate (TMAV), imidazole propionate (IP), 3-indoxyl sulfate (3-IS), and hippurate (HIP), in embryos of SP mice compared to ABX and GF mice, may increase axon numbers via thalamocortical axonogenesis. Restoration of these metabolites into the microbiota-deficient mice mothers can potentially prevent the abnormalities in the development of neuronal connections and improve the behavior of the offspring. However, there might be increases in relationships with other neurons in the presence of the metabolites. Further studies are highly required to implement and analyze the result of this study in human maternal gut microbiota during pregnancy.

7Multiple studies have shown that babies born via vaginal delivery have more composition of healthy bacteria than the babies born via cesarean section delivery. Babies born via standard (vaginal) delivery are exposed to the mother’s vaginal bacteria, whereas children born via C-section delivery are remarkably exposed to the mother’s skin flora and environmental microorganisms. During the prenatal period, the fetus’ gut acquires a complex mixture of bacteria from mothers via breastfeeding exposure to vaginal and enteric microorganisms. At birth, the fetus’ microbiota is almost identical to the mother’s microbiome. At this stage, the abundance of lactobacilli became the pioneer colonizer of the infant’s gut. The subsequent colonization of aerobic or facultative anaerobic bacteria like enterobacteria, enterococci, and staphylococci are found. The colonization of these bacteria uses oxygen for their growth and makes a suitable habitat for the colonization of anaerobes, including Bifidobacterium, Clostridium, and Bacteroides, and later prominent phyla such as Firmicutes and Bacteroides, and Verrucomicrobia; less dominant microbiota such as Proteobacteria and aerobic Gram-negative bacteria are developing inside the gut of adult human. On the other hand, babies born via C-section were dominated by Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Streptococcus parasanguinis, Klebsiella oxytoca, Klebsiella pneumoniae, and Clostridium spp. (Clostridium perfringens, Clostridium difficile) ( Table 1 ); these microbes are predominant at the mother’s skin surface and hospital environment. Growing evidence displays that a globally increased cesarean section (CS) can also alter gut microbial composition in early infancy and might delay neurological adaptation in infants. Surprisingly, a current meta-analysis study has demonstrated that a child born via C-section delivery has a 23% risk of developing ASD compared with a child born via vaginal delivery. In a most significant multi-national population-based study, which consisted of 5 million births from Norway, Sweden, Denmark, Finland, and Western Australia, each participant was observed for 36–42 weeks. More than 31,000 cases of ASD were confirmed, which supported the hypothesis that birth delivery by C-section possesses a higher risk of ASD than vaginal delivery.

8From birth until about the age of 3, a human’s microbiome starts to develop. Using antibiotics during these formative years can disrupt the development of immune-mediated, metabolic, and neurological diseases. Antibiotics significantly alter the microbial composition by inhibiting the growth of pathogenic microbes. Early and uncontrolled doses of antibiotics may lead to loss of predominant microbial phyla, loss of diversity, change in metabolic activity, and colonization of pathogens. Antibiotic treatment during the 1–2 years of life can crucially impact the maturation of the immune system and have a detrimental effect on typical microbiota establishment with severe long-term consequences, such as inflammation, immune dysregulation, allergies, infections, and GI diseases such as Crohn’s, inflammatory bowel disease (IBD), constipation, and diarrhea. Further cohort studies performed using antibiotics during infancy and early childhood showed significant alterations in gut microbiota, which could be directly responsible for turning on or off specific genes. Early use and overuse of antibiotics lead to microbial dysbiosis and may turn on the autism gene. This affects the gut–brain axis by causing epigenetic modification, which potentially facilitates the pathogenesis of ASD.

9Early colonization of beneficial gut bacteria during infancy is essential for maintaining gut homeostasis. Using antibiotics in that period can increase gut microbiome dysbiosis inside the infant gastrointestinal tract. A recent study showed that 34.5% of autistic children were exposed to extensive and repeated broad-spectrum antibiotic treatments, which was more than six courses compared to a control group with more than six courses. Further investigations are required to understand the impact of antibiotics overuse during the first years of life on the gut–brain axis and its association with other health outcomes such as autism in later years.

10According to recent studies, dietary fiber significantly increases microbial diversity. In a dietary intervention, subjects were provided dietary fiber. After 2 weeks, there was a significant change in microbial diversity observed among 20% of the fiber consumers, with an increase in the abundance of fiber degrading beneficial bacteria such as Prevotellacopri.

11Microbial fermentation of dietary fibers by gastrointestinal bacteria, such as Firmicutes, Clostridia, Bacteroides, and Desulfovibrio, produce short-chain fatty acids (SCFAs) butyrate, acetate, and propionate. SCFA induces pyruvate dehydrogenase kinase (PDK), leading to the inactivation of the pyruvate dehydrogenase complex. As a result, cells cannot produce sufficient energy due to the cessation of pyruvate to acetyl-CoA conversion. To meet the energy requirement of the cell, colonocytes produce acetyl-CoA via β-oxidation of the SCFA. As the oxidative respiration increases in the cell, the oxygen concentration decreases, resulting in the stabilization of hypoxia-inducible factor-1α (HIF-1α). HIF-1α is a regulator of oxygen homeostasis controlled by the oxygen concentration inside the intestinal lumen. Kumar et al. have reported that hypoxia-inducible factor 1-alpha (HIF1α) prevents the transportation of gut microbiota and protects the microorganisms from host immune response.

12The importance of dietary fibers and their impact on the CNS has been demonstrated in several epidemiological studies. SCFA is a neuroactive compound produced by gastrointestinal bacteria, which leads to alteration of metabolic and immune system function and regulates the gene expression. Butyrate, one of the short-chain fatty acids (SCFAs) produced, can profoundly improve the function of the CNS by inhibiting histone deacetylases (HDACs). Histone deacetylases inhibition is necessary because it does not allow acetylation of histone protein, which eventually inhibits loosen up of the chromatin for acetyl groups to be added to N-terminal L-lysine residues by histone acetyltransferases (HATs) for transcription. Lack of histone acetyltransferases activity leads to transcriptional dysfunction, potentially causing many neurodegenerative diseases. HDAC inhibitors such as sodium butyrate (NaB) have become very promising and attractive therapeutic candidates for their ability to increase histone acetylation and promote the expression of genes. Conversely, a recent experiment showed that propionate injection inside the intracerebroventricular during development results in autistic-like behavior, and the same results were observed in a rodent model of autism. Excessive propionate can impact CNS function by crossing the gut–brain and blood–brain barriers, causing neuroinflammatory responses and behavioral alterations, leading to aggravation of neurodegenerative conditions like autism spectrum disorder. SCFAs can be both beneficial and detrimental to neurodegenerative disorders. Recent studies demonstrated that children with ASD have increased production of SCFAs, mainly indicated by the alteration in the fermentation of dietary fiber and abundance of SCFAs in ASD children compared to children without ASD. Therefore, these microbial fermentation products can also act as a biomarker for detecting ASD.

13Based on years of research, it has been projected that antibiotics applied for treatment impose a threat to the human gut microbiome due to antibiotic resistance within microbes and lack of specificity and efficacy. To overcome these challenges in developing modern medicines and reduce the dependency on antibiotics, microbial therapies are widely popularized for using beneficial microbes, naturally found in the human body. It has undergone several advancements like genetic engineering to enhance host–microbe interaction and increase treatment efficacy and effectiveness.

14In additive therapy, a cocktail of beneficial microbes is extracted from the healthy human body and introduced into fecal microbial transplantation (FMT), or through the consumption of probiotics. Probiotics are non-pathogenic beneficial microorganisms (such as Lactobacillus and Bifidobacterium) introduced into the body to restore the healthy composition of the gut microbiome. In FMT, the patient suffering from a gastrointestinal disorder is subjected to complete restoration of the intestinal microbial consortium. This alternative approach to probiotics and antibiotic treatment consists of bacteria, archaea, fungi, and viruses (bacteriophage), regulating gut microbial homeostasis and the host immune system. FMT therapy has been successfully implemented in several studies related to GI disorders like recurrent Clostridium difficile infections (rCDIs) and IBD, demonstrating effective results with satisfactory efficacy, and children showed significant improvement in GI symptoms.

15In subtractive therapy, engineered bacteriocins and bacteriophages that exert promising antimicrobial activity are used to target pathogens in the gut without causing harm to other microbes in the ecosystem. This is a much better option than antibiotic usage, which eventually develops antibiotic resistance.

16Bacteriocins are peptides synthesized in the ribosome, which target unwanted microbes in various ways: releasing toxins, cessation of respiratory mechanisms, disintegrating the membrane, and cell death. One recent study presented that Lactobacillus salivarius strain UCC118 produces broad-spectrum bacteriocin called Abp118, which acts against the food-poisoning pathogen Listeria monocytogenes. Another mice model study demonstrated that bacteriocins produced by Lactobacillus casei L26 LAFTI prevented the growth of specific strains of E. coli and L. monocytogenes and protected the intestinal wall. Probiotic strain Lactobacillus johnsonii LA1 releases a particular bacteriocin that inhibits the growth of ulcer-causing bacteria Helicobacter pylori. Research has also shown the effective use of bacteriocins against Clostridium, Enterococcus, Pediococcus, Lactobacillus, and Leuconostoc.

17On the other hand, Bacteriophages are viruses that are engineered to infect specific bacteria and kill the bacterial host in the process. Due to the rise in antibiotic resistance, bacteriophages have been proven to be effective in tackling bacterial infections. However, research has shown that both bacteriophage and bacterial populations can coexist inside the gut of the mice model, which is why more research is required to determine the appropriate conditions for effectiveness in the bacteriophage therapy.

18Conversely, modulatory therapy may significantly restore a healthy balance in the gut microbiome by changing the diet, exercise, and antibiotics. This favors the colonization of beneficial microbiota over pathogens. A healthy diet plays a crucial role in shaping the gut microbiome composition, depending on the amount, type, and balance of the important dietary macronutrients (carbohydrates, proteins, and fats) including micronutrients such as vitamin D. A study was conducted to demonstrate the influence of dietary carbohydrate intervention on the composition of intestinal microbiota among two groups of 171 rural African and 172 urban European children. For this particular study, these two groups were subjected to completely different types of dietary carbohydrates. The result showed higher dominance of Bacteroidetes (73%) in the microbiota of African children, whereas in the EU children, there was a 27% abundance of Bacteroidetes and 51% abundance of Firmicutes. Our gut comprises proteolytic bacteria such as Bacteroides species (especially Bacteroides fragilis) and Clostridium perfringens, propionibacteria, streptococci, bacilli, and staphylococci, those that convert protein into nitrogen and amino acids. Studies investigating the influence of dietary fats consumption showed that high-fat consumption reduces the concentration of short-chain fatty acid (butyrate) and the abundance of bifidobacteria. However, a high-fat diet reduces intestinal inflammation but causes an increase in plasma markers of inflammation and lipopolysaccharide circulation.

19In addition to internal and external factors affecting the efficacy of microbial therapy, exercise (an environmental factor) positively impacts the healthy composition of microbiota, which regulates gut homeostasis and maintains gut integrity. It enriches the microbial diversity leading to an increase in the Bacteroidetes–Firmicutes ratio, contributing to the reduction in metabolic disorders and gastrointestinal disorders by enhancing the production of bacterial metabolites like SCFA.

20Remarkably, up to 70% of children with ASD have impaired GI function. Clinicians working with such children increasingly assume a link between ASD and gut dysfunction. Studies conducted at McMaster University suggested a strong connection between the microbiome and behavioral disorders in mice. The investigators used a gut bacteria makeover to turn anxious mice into extroverted mice by transferring gut microbes from the former to the latter and vice versa. Within 3 weeks after the transplant, the anxious mice became more sociable with prompt responsiveness, while extroverted mice became more nervous with delayed responsiveness. This experiment suggested that gut microbiota dysbiosis may further facilitate the development of neurobehavioral disorders. However, studies examining connections between the gut microbiome and ASD in humans have begun recently.

21A better understanding of the gut–brain axis and the roles of gut bacteria may lead to the treatment of neurological disorders, including ASD, with fewer side effects. The exciting findings on the existence of a so-called “microbiota–gut–brain axis” support the hypothesis that the gut microbiota could trigger neuropsychiatric symptoms in ASD patients. Doctors usually recommend a plant-based diet, prebiotics, and probiotics to ASD patients. Prebiotic is a non-digestible food ingredient, which increases the abundance of commensal bacteria in the gut.

22In a recent case, doctors noticed that a child with symptoms of autism had large numbers of pathogenic bacteria like Clostridium tetani in his gut. Treatment with vancomycin caused immediate symptom reversal. Treatment with anti-Clostridium antibiotics in some autistic children has significantly reduced abnormal behavior. However, vancomycin treatment has a significant drawback, as it is not selective to favorable or harmful bacteria. Thus, more research should be conducted to find more specific targeted antibiotics and discover an alternative approach to reduce the use of vancomycin as a part of pre-treatment in Microbiota Transfer Therapy (MTT). Conversely, in the mouse model of autism, adding more symbiotic bacteria such as B. fragilis through fecal transplant profoundly ameliorates the ASD-associated symptoms. Fecal transplant treatment can potentially treat ASD patients by consuming beneficial bacteria. It has received many criticisms for using a small sample group, no replication, and not considering possible other variables.

23Over the years of intervention, significant improvements in GI and autism symptoms have been observed. A clinical trial of MTT was performed on 18 participants for 2 years, consisting of antibiotics, bowel cleanses, a stomach-acid suppressant, and fecal microbiota transplant. After 2 years of treatment, significant improvement in GI- and ASD-related symptoms and elevation of relative abundances of Bifidobacterium and Prevotella were observed among all 18 participants. After the treatment, most participants experienced an increase in microbial diversity, even after 2 years of therapy in typicallydeveloping children. This study has demonstrated the long-term efficiency of MTT for ASD patients suffering from GI disorders, and permanent change in the gut ecosystem created a favored condition for the survival of new healthy microbes. However, the limitation of the study was that in certain bacteria, short reads, including conserved regions of bacterial 16S rRNA, were targeted for amplicon sequencing for the taxonomic classifications.

24It is essential to address that MTT has significant drawbacks, and there are chances that potentially life-threatening pathogens from the donor might invade the recipient’s intestine during the treatment. In a particular incident, an immunocompromised patient suffered from severe illness, who eventually died after receiving multi-drug-resistant bacteria during microbiota transplantation. To avoid such an incident, proper microbial screening of the donor should be performed to identify multi-drug-resistant organisms.

25Recapitulating all mounting evidence elucidates the association between the gut–brain axis and ASD. Several factors leading to ASD were identified, mainly early colonization on infant health development and the effect of early microbiota dysbiosis in the gestation period, mode of delivery, uncontrolled usage of antibiotics, and stress. These factors eventually lead to gut microbiome dysbiosis and colonization of pathogenic microbes, which impact the CNS function by the production of neurotoxins. The presence of these pathogenic bacteria, such as Clostridium found in the colon of children, shows the possibility of developing ASD. Studies also indicated the importance of two SCFA produced from microbial formation of dietary fiber, including butyrate and propionate. Butyrate improves brain function by inhibiting histone deacetylases, whereas propionate impacts brain function, leading to changes in behavior and aggressiveness in ASD patients. According to many studies on the gut microbiome, MTT can potentially treat autism-like symptoms, including the restoration of healthy gut microbiome composition in the gestation period and early stage of infancy. Most studies have been conducted in experimental animal models. Some studies have small sample sizes, especially studies performed using human volunteers. The majority of them lacked randomization or control groups. Significant limitations in these studies eventually reduced the validity of the results. Recently, scientists have carried out several clinical trials to investigate the efficacy of MTT on children who have ASD, and the results obtained were satisfactory, showing remarkable improvement in GI symptoms with minimal adverse effects due to pre-treatment with vancomycin. An increasing number of news articles and science media are featuring the successes of microbiome research and its promising therapeutics regime. The general population is excited to learn that the gut microbiome is linked to the progression of various diseases and is also essential for healthy organ development. Based on the forecasted information regarding the success of microbial therapeutics in clinical trials, people are hopeful that this treatment will be a better alternative compared to conventional medicinal practices.

26In 2019, the Food and Drug Administration (FDA) recognized microbial transplant therapy and labeled it “fast-track” for ASD treatment after observing the successful clinical trials using long-term microbial transplant therapy on autistic children. Further research should be conducted to determine whether findings in animal studies and clinical trials can be successfully implemented with similar efficacy rates in different populations of ASD patients across various geographical locations and exhibit promising results among the patients. Furthermore, more follow-up studies are required with accurate methodologies and optimized dosage of antibiotics and microbial suspensions to increase the efficacy of the treatment to support the validity, reliability, and precision of past research. In addition, genomic analysis of the gut microbiome of the donor and recipient should further define specific strains, better pathways, and regimens to provide optimum supplements and treatment in this particular population. There is an increase in demand to find a substitute for microbial transplant therapy and reduce the dependency on the harness beneficial microbiota from a healthy human, which can be achieved by culturing a specific combination of bacteria for transplantation. Microbiome research has made much progress in recent years from animal studies to clinical trials, with satisfactory results and better efficacy in MTT treatment. In the next couple of years, FDA-approved pills, probiotics, and metabolites might be commercially available to establish a suitable bacterial composition to treat and regulate ASD. Our review investigations strongly imply exploring the underlying molecular mechanism of the gut microbiome in the pathogenesis and advancement of ASD and finding promising therapeutic agents/drugs that will deliver new hope for the treatment and management of ASD in the near future.

27MAT, AAM, SA, and NUE: wrote the manuscript draft, illustrated figure, and table. MAT and AAM: designed the review. HJC and JX: conceptualized and supervised the study. MMI, STH, and MAA, BRP: performed in searching works of literature. AAM, MAT and SAS: checked the manuscript the draft. All authors finally revised the paper and approved for final submission.

28This research was funded by the Korea Basic Science Institute (KBSI), grant C220000, C280320 and C210500.

29The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

30All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.