experimental (functional) analysis of behavior disorders

DOI: 10.1007/978-1-4614-3037-7_8
Corpus ID: 141193962

Experimental Functional Analysis

T. Vollmer , H. Roane , Amanda B. Rone
Published 2012

8 Citations

Functional assessment of challenging behavior, functional assessment of problematic forms of prelinguistic behavior, stereotypic movement disorder, conducting in-home functional analyses of aggression and self-injury exhibited by boys with fragile x syndrome, an evidence-driven, solution-focused approach to functional behavior assessment report writing., a contextual model of care for persons with dementia, treatment of problem behavior multiply maintained by access to tangible items and escape from demands, technology-aided interventions to reduce challenging behaviors for individuals with autism spectrum disorder, 65 references, the concept of automatic reinforcement: implications for behavioral research in developmental disabilities., functional analyses and treatment of precursor behavior., progressing from brief assessments to extended experimental analyses in the evaluation of aberrant behavior., reduction of multiple aberrant behaviors and concurrent development of self-care skills with differential reinforcement., an experimental analysis of aggression, an evaluation of the properties of attention as reinforcement for destructive and appropriate behavior., reducing behavior problems through functional communication training., identification of environmental determinants of behavior disorders through functional analysis of precursor behaviors., treatment of self-injury and hand mouthing following inconclusive functional analyses., functional analysis of problem behavior: a review., related papers.

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Experimental avoidance and behavioral disorders: a functional dimensional approach to diagnosis and treatment

Affiliation.

1 Department of Psychology, University of Nevada, Reno 89557-0062, USA.
PMID: 8991302
DOI: 10.1037//0022-006x.64.6.1152

Syndromal classification is a well-developed diagnostic system but has failed to deliver on its promise of the identification of functional pathological processes. Functional analysis is tightly connected to treatment but has failed to develop testable, replicable classification systems. Functional diagnostic dimensions are suggested as a way to develop the functional classification approach, and experiential avoidance is described as 1 such dimension. A wide range of research is reviewed showing that many forms of psychopathology can be conceptualized as unhealthy efforts to escape and avoid emotions, thoughts, memories, and other private experiences. It is argued that experiential avoidance, as a functional diagnostic dimension, has the potential to integrate the efforts and findings of researchers from a wide variety of theoretical paradigms, research interests, and clinical domains and to lead to testable new approaches to the analysis and treatment of behavioral disorders.

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Behav Anal Pract
v.1(1); Spring 2008

Clinical Application of Functional Analysis Methodology

Brian a iwata.

University of Florida

Claudia L Dozier

University of Kansas

Functional analysis (FA) methodology is a well-established standard for assessment in applied behavior analysis research. Although used less commonly in clinical (nonresearch) application, the basic components of an FA can be adapted easily in many situations to facilitate the treatment of problem behavior. This article describes practical aspects of FA methodology and suggests ways that it can be incorporated into routine clinical work.

Research methods used in applied behavior analysis provide an excellent model for practice, although standards for evaluating research are admittedly more stringent. The demonstration of experimental control is a good example—it is required in research but not often attempted in practice. Translation of research methodology into practical application often is a matter of what is useful yet feasible, and a demonstration of control, at least during assessment, is both. Most practitioners understand the value of knowing how problem behavior is maintained before attempting to reduce it; perhaps less clear is why practitioners should conduct a functional analysis (FA) when (a) information can be obtained from other sources and (b) practical constraints seem to preclude a thorough analysis. We address both of these issues and suggest ways to incorporate FA methodology into routine clinical assessments.

The term “functional analysis” refers to any empirical demonstration of a cause-effect relation ( Baer, Wolf, & Risley, 1968 ); its application with problem behavior is unique, however. A variety of reinforcement options are available when attempting to establish a new response because nonexistent target responses have no function. Although we may approach the treatment of problem behavior the same way—applying various sorts of contingencies and evaluating their effects, ongoing behavior does have a function based on its history of reinforcement. Thus, the consequences we use to reduce problem behavior must neutralize or compete with those that maintain it, and an FA allows us to identify sources of maintenance prior to treatment.

A great deal of research has shown that the same learning processes that account for the development of socially appropriate behavior—positive and negative reinforcement—are involved in the acquisition and maintenance of problematic behavior (see Iwata, Kahng, Wallace, & Lindberg, 2000 , for a more extended discussion). Self-injury, aggression, property destruction, and other harmful acts often produce a necessary reaction from caregivers to interrupt the behavior, which may be combined with other consequences (comfort, “redirection” to other activities, etc.) that may strengthen problem behavior through social-positive reinforcement. These behaviors also are sufficiently disruptive that they may terminate ongoing work requirements, thereby producing escape (social-negative reinforcement). Finally, some problem behaviors (self-injury and/or stereotypy) produce sensory consequences that are automatically reinforcing. Thus, the goal of an FA is to determine which sources of reinforcement account for problem behavior on an individual basis.

Sources of Information about Problem Behavior

A “functional behavioral assessment” consists of any formal method for identifying the reinforcers that maintain problem behavior. Informant responses to rating scales or questionnaires (also called indirect or anecdotal approaches) are easily obtained, which is why these methods are used most often by practitioners ( Desrochers, Hile, & Williams-Moseley, 1997 ; Ellingson, Miltenberger, & Long, 1999 ). Although indirect methods continue to be recommended ( Herzinger & Campbell, 2007 ), they have been shown repeatedly to be unreliable ( Arndorfer, Miltenberger, Woster, Rortvedt, & Gaffaney, 1994 ; Conroy, Fox, Bucklin, & Good, 1996 ; Duker, & Sigafoos, 1998 ; Newton & Sturmey, 1991 ; Sigafoos, Kerr, & Roberts, 1994 ; Sigafoos, Kerr, Roberts, & Couzens, 1993 ; Spreat & Connelly, 1996 ; Sturmey, 1994 ; Zarcone, Rodgers, Iwata, Rourke, & Dorsey, 1991 ) and, as a result, inadequate as the basis for developing an intervention program. Their use seems justifiable only when there are no opportunities whatsoever to collect direct-observation data, and these types of situations, in which client verbal report defines both the extent and cause of the initial problem, as well as when it is resolved, more closely resemble a traditional counseling context rather than the practice of behavior analysis.

The descriptive analysis ( Bijou, Peterson, & Ault, 1968 ), in which observational data are taken on the target behavior and the context in which it occurs, has a longstanding tradition in our field as the primary method for collecting baseline data and evaluating treatment effects. It is not, however, well suited to the identification of functional relations, a fact that was noted by Bijou et al.: “ . . . descriptive studies provide information only on events and their occurrence. They do not provide information on the functional properties of the events or the functional relationships among the events. Experimental studies provide that kind of information” (pp. 176–177). More specifically, descriptive analyses may not reveal differences among social contingencies (e.g., attention vs. escape) that maintain problem behavior ( Lerman & Iwata, 1993 ; Mace & Lalli, 1991 ), cannot detect extremely thin schedules of reinforcement ( Marion, Touchette, & Sandman, 2003 ), and may incorrectly suggest contingent attention as the source of maintenance because attention is a commonly observed consequence for problem behavior even though it may not be a reinforcer ( St. Peter et al., 2005 ). For these reasons, comparisons of outcomes from independent descriptive and functional analyses of problem behavior generally have shown poor correspondence ( Thompson & Iwata, 2007 ).

In light of limitations with both indirect and descriptive approaches, the functional or experimental analysis has emerged as the standard for assessment in clinical research. 1 For example, Kahng, Iwata, and Lewin (2002) examined trends in behavioral research on the treatment of self-injury over a 35-year period and noted a continuing increase in the number of studies incorporating FA methodology, whereas those using other methods have either greatly decreased (descriptive analyses) or ceased altogether (indirect methods).

Key Components of a Functional Analysis

Procedures used for conducting FAs have varied widely, to the point where qualitative and quantitative characteristics of assessment conditions, as well as experimental designs, have been modified to suit a wide range of applications (see Hanley, Iwata, & McCord, 2003 , for a review). Still, all methods share a common feature—observation of behavior under well-defined test versus control conditions. A test condition contains the variable (usually some combination of antecedent and consequent events) whose influence is being evaluated. Iwata, Dorsey, Slifer, Bauman, and Richman (1994/1982) described an initial set of test conditions to identify sources of reinforcement previously shown to maintain problem behavior: social-positive reinforcement (contingent-attention condition), social-negative reinforcement (escape-from-demands condition), and automatic reinforcement (alone condition). Variations of test conditions have included divided-attention ( Mace, Page, Ivancic, & O'Brien, 1986 ), access to tangible items ( Mace & West, 1986 ), and social avoidance ( Hagopian, Wilson, & Wilder, 2001 ). Appendix A contains a brief description of commonly used test conditions. Antecedent events are those in effect prior to the occurrence of problem behavior and serve as potential establishing operations or EOs ( Laraway, Snycerski, Michael, & Poling (2003 ). For example, in the test condition for attention, attention is withheld or is delivered to someone other than the client, either of which may increase the “value” of attention as a reinforcer. Consequent events are those that immediately follow behavior and may serve as reinforcers. The importance of a test condition is obvious; the control condition also is important to rule out the possibility that behavior observed under the test condition would have been seen regardless of what the condition contained.

As noted by Baer et al. (1968) , a functional analysis of a behavior consists of “ . . . a believable demonstration of the events that can be responsible for the occurrence or non-occurrence of that behavior” (pp. 93–94). From a research perspective, believability requires control over (a) measurement (dependent variable), (b) application of treatment (independent variable), and (c) potential sources of confounding. FAs reported in journals such as the Journal of Applied Behavior Analysis (JABA) typically meet this standard. The multielement design is the most efficient method for conducting multiple comparisons in an FA (see Figure 1 , Panel A, illustrating behavior maintained by escape). Because the rapidly alternating conditions of the multielement design sometimes result in discrimination failure, the reversal design (see Figure 1 , Panel B, illustrating maintenance by attention) or the pairwise, test-control design, which combines features of the multielement and reversal designs ( Figure 1 , Panel C, illustrating maintenance by automatic reinforcement), are used as alternatives.

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Variations of functional analysis designs (see text for details)

The standards for believability in practice are different yet may approximate those of research in many respects. For example, we expect objective measurement of target behaviors in routine clinical application even though assessment of observer reliability may be less than desirable (or nonexistent). In a similar way, we can incorporate the key components of an FA during assessment even though it may not meet the standards imposed on research because the essential feature—the controlled comparison—can be accommodated in many applied situations. When contingency-management programs are implemented to decrease the frequency of problem behavior, intervention usually is preceded by initial observation of clients and significant others in the setting in which treatment will occur and the collection of baseline data. Both of these provide an opportunity to conduct an FA because the only additional requirement is the inclusion of test and control conditions. Although practical constraints may preclude a demonstration of control similar to that seen in research reports, the methodology has been adapted for use under a number of limiting conditions.

Practical Constraints in the Implementation of Functional Analysis Methodology

The chief limitations of a typical FA include constraints on the time available for assessment, risk posed by severe problem behavior, and the inability to exert tight control over environmental conditions. Each of these has been addressed through several procedural variations, which are described below and outlined in Table 1 .

Summary of Functional Analysis (FA) Variations

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Limited Assessment Time

When contact with clients is limited, as in outpatient and consultation work, it may be impossible to obtain repeated measures across an extended series of assessment sessions. The “brief functional analysis” or BFA ( Northup et al., 1991 ) was developed exactly for these situations. It consists of a single exposure to 5-min test and control conditions, with replication of a key test condition if time permits, followed by a treatment “probe,” all of which can be accommodated within a 90-min time period (see Figure 1 , Panel D, illustrating behavior maintained by attention). Derby et al. (1992) summarized results obtained with the BFA for 79 outpatient cases and reported that they were able to identify the function(s) of problem behavior in approximately 50% of the cases. This finding is remarkable given that the assessment was completed in such a short period of time and under highly novel clinic conditions. Furthermore, the assessment provided evidence of an empirical functional relation (unlike that obtained from indirect or descriptive methods) in less time than what has been required to conduct many descriptive analyses.

Aside from the BFA, a typical, repeated-measures FA may be abbreviated through the use of single-function testing. The FA most often used in research attempts to identify which of several sources of reinforcement maintains problem behavior and therefore includes multiple test conditions. By contrast, when anecdotal report or informal observation strongly suggests a particular source of maintenance, an FA could consist of a single test condition versus a control (see Figure 1 , Panel E, illustrating behavior maintained by escape). Thus, preliminary information from rating scales and descriptive analyses, although unreliable or tentative, may enhance the efficiency of an FA. Positive results of a single-function test lead directly to treatment; only negative results require further analysis.

A second type of single-function test might be considered when behavior is presumed to be “self-stimulatory” in nature (i.e., maintained by automatic reinforcement), and consists of observing the individual during repeated “alone” sessions ( Vollmer, Marcus, Ringdahl, & Roane, 1995 ). Although this procedure does not involve a test-control comparison, it provides a simple way to verify that problem behavior persists in the absence of all social stimulation (and therefore is unlikely to be maintained by social reinforcement). By contrast, decreased responding across sessions suggests the possibility of extinction and the need to include test conditions for social contingencies (see Figure 1 , Panel F, illustrating two different outcomes in the alone condition–maintenance and extinction).

Potentially Dangerous Behavior

Behaviors such as severe self-injury or aggression are difficult to assess if they cannot be allowed to occur frequently. Although the descriptive analysis is appealing because it simply takes advantage of naturally occurring episodes, severe problem behavior often produces caregiver reactions (e.g., response interruption) that can bias interpretation. The challenge faced when conducting an FA is arranging conditions under which problem behavior may increase while at the same time minimizing risk. The most obvious strategy, in the case of self-injury, consists of having participants wear protective devices. Le and Smith (2002) observed, however, that protective equipment suppressed responding across all FA conditions. As an alternative, Smith and Churchill (2002) noticed that individuals who engaged in self-injury or aggression also engaged in other responses that reliably preceded the occurrence of problem behavior. Results of independent FAs of the “precursor” and target behaviors showed that both had the same functions and that occurrences of severe problem behavior were reduced during the FA of precursors, suggesting that an analysis of precursor behaviors might be helpful in reducing risk.

Another strategy consists of using a measure of responding that is not based on repeated occurrences of behavior. Response rate and duration are the typical measures in research, but latency to the first response also may be sensitive to the effects of contingencies. Thomason, Iwata, Neidert, and Roscoe (2008) , for example, conducted independent FAs of problem behavior during sessions in which response rates were measured and during sessions that were terminated following the first occurrence of a target response. Correspondence between results of the two assessments was observed in 9 out of 10 cases, and in every case the latency-based FA resulted in many fewer occurrences of problem behavior (see Figure 1 , Panel G, illustrating maintenance by attention. Note: shorter latency indicates responding earlier in a session; 5-min latency indicates that the response never occurred).

Limited Control over Environmental Conditions

Almost all FAs reported in research were conducted in settings that facilitated the environmental control needed to isolate the effects of independent variables. This raises the question of whether FAs can be applied under more naturalistic conditions in which the uncontrolled actions of bystanders may compromise results. In addition to conducting FAs in outpatient clinics, David Wacker's group at the University of Iowa has conducted a series of assessment-treatment studies in which FAs were conducted in homes (e.g., Wacker, Berg, Derby, Asmus, & Healey, 1998 ). Therapists “coached” parents to implement assessment conditions with their children, and procedures were implemented without any loss of precision. Extension to school settings has been shown in studies in which assessment conditions were embedded as probe trials during ongoing classroom routines across the school day ( Bloom, Iwata, Fritz, Roscoe, & Carreau, 2008 ; Sigafoos & Saggers, 1995 ). For example, a demand probe is conducted in an academic-work context and consists of a 1 min to 2 min control in which no work is presented, followed immediately by a 1 min to 2 min test in which work is presented as the EO and removed contingent on problem behavior (see Figure 1 , Panel H, illustrating maintenance by escape). Thus, it seems that the setting per se is not a limiting factor of the FA as long as confounding influences can be minimized for brief periods of time.

Other Suggestions for Implementation

Risk assessment.

When problem behavior results in injury to clients or others, more careful consideration of risk is needed than when conducting uncontrolled observations because the therapist explicitly arranges conditions under which problem behavior may increase. Documentation of past or potential risks of the behavior, informed consent, and modifications in assessment procedures (see previous comments on severe problem behavior) are strongly recommended in such cases.

Data Collection and Interpretation

Although a skilled therapist may be able to take data while conducting sessions, this practice is rarely used even in research. Thus, a therapist and observer are needed to conduct most sessions. If an observer is unavailable, sessions may be videotaped for later scoring. Traditional paper-and-pencil (data sheets) can be used for actual data recording. However, many inexpensive programs are available for recording data on laptop computers or PDAs and are highly recommended (see Kahng & Iwata, 1998 , for a general review and Sarkar et al., 2006 , for a recent example). Finally, because data interpretation is a subjective process, criteria for evaluating the results of single-subject designs may be helpful (see Fisher, Kelley, & Lomas, 2003 ; Hagopian et al., 1997 ).

Initial Case Selection

The most difficult problem faced by those first attempting to use FA methodology is the absence of a standard for comparison to establish the validity of assessment. That is, results of an assessment cannot be compared to those obtained by a more experienced clinician. Because some assessments may yield clear results only after several modifications, exceedingly complex cases (e.g., those suggesting multiple control or the influence of unusual combinations of events) are not ideal trial cases. A better strategy consists of selecting a case for which there is a strong (perhaps unanimous) suspicion that problem behavior is maintained by a particular social consequence. A positive test for the influence of that consequence provides a measure of face validity, whereas a negative test suggests the need to examine more closely the way in which assessment is conducted (a negative test also may simply reveal flaws in the information obtained initially). An accumulation of positive results, especially when they are combined with positive outcomes from function-based interventions, increases confidence that procedures are being implemented correctly and provides a basis for consideration of more complex cases.

Staff Training

FAs are more difficult to implement than other types of assessment because they require the ability to follow a prescribed sequence of interactions in a consistent manner. Although it can be argued that a behavior analyst who does not have the skills to conduct an FA also cannot implement any subsequent behavioral intervention, a more definitive reply is available by way of data. Results from several studies indicate that undergraduate students, teachers, and workshop participants all can acquire the skills to conduct FA sessions with a high degree of consistency following very brief training ( Iwata et al., 2000 ; Moore et al., 2002 ; Wallace, Doney, Mintz-Resudek, & Tarbox, 2004 ). Although actually designing an FA or modifying it if initial results are unclear requires greater skill, neither task should be particularly difficult for a supervising behavior analyst. One especially valuable training aid is video modeling. Observer training seldom is limited to verbal or written instruction, and the same applies to the implementation of FAs. Scripted, role-playing scenarios can be easily produced to demonstrate the correct presentation of antecedent and consequent events, and video samples of actual sessions can serve as the basis for performance feedback.

The use of FA methodology as an assessment tool was described over 25 years ago ( Iwata et al., 1994/1982 ). Since then, replication and extension have been reported in hundreds of published studies. Thus, the methodology is not new and has been adopted on a wide-scale basis in clinical research. It is unclear whether FA methodology has had a similar impact on practice because survey data ( Desrochers et al., 1997 ; Ellingson et al., 1999 ) suggest that psychologists and behavior analysts continue to rely more heavily on traditional forms of assessment such as the questionnaire and uncontrolled observation. One possible reason for limited extension from research to practice is that clinicians, having never been trained in the use of FA methodology, view it as impractical except for research purposes. An examination of current research, however, indicates that refinement has been aimed not only at improving control but also at adapting the methodology for real-world application. Procedural variations have been developed for limiting conditions faced by most clinicians, and we hope that this overview will encourage practitioners to adopt, whenever possible, experimental approaches to behavioral assessment.

In closing, it should be noted that medicine was once a profession in which treatment was prescribed based on causes inferred from patient report and observed symptoms. Claude Bernard, widely regarded as the father of modern medicine, suggested an alternative approach: “ . . . experimental analysis is our only means of going in search of the truth . . .” (1865/1927, p. 55). By incorporating experimental procedures into clinical practice, behavior analysis is uniquely positioned to make a similar contribution to the assessment and treatment of “psychological” disorders.

Functional Analysis Conditions

Test condition for maintenance by social-positive reinforcement.

a) Attention condition: Begin the session by informing the client that you are busy and “need to do some work.” Then move away and ignore all client behavior except as noted below.
b) “Divided-attention” variation: Begin the session in the same manner and then proceed to deliver attention to either another adult or to a peer of the client.
c) “Tangible” variation: Identify an item that is highly preferred by the client and allow the client free access to it just prior to the session. Begin the session by requesting and removing the item and then move away from client as in the attention condition.
a) Non-target behavior: If the target problem behavior does not occur (or if any behavior other than the target occurs), the antecedent event will remain in effect until the end of the session.
b) Problem behavior: If the target problem behavior occurs, deliver attention, usually in the form of a mild reprimand, a statement of concern, and some comforting physical contact (or response blocking). In the tangible variation, deliver the tangible item briefly (about 30 s). After delivering attention or the tangible item, reinstate the antecedent event.

Test Condition for Maintenance by Social-Negative Reinforcement

a) Task-Demand condition: Conduct repeated learning trials throughout the session using academic or vocational tasks that are appropriate to the client's skill level but that are somewhat effortful. Typically, a trial begins with an instruction, followed as needed by prompts consisting of a demonstration and then physical assistance.
b) Social-Avoidance variation: Initiate social interaction with the client at frequent intervals throughout the session. Do not conduct learning trials (academic or vocational) per se, but simply try to prompt some type of interaction by making comments about things in room, asking questions, etc.
a) Non-target behavior: Deliver praise following appropriate responses (compliance in the task-demand condition, any appropriate social response in the social-avoidance condition).
b) Problem behavior: If the target problem behavior occurs, immediately terminate the task (or ongoing interaction) and turn away from the client for about 30 s, then reinstate the antecedent condition.

Test Condition for Maintenance by Automatic-Positive Reinforcement

Antecedent event: This condition is designed to determine whether problem behavior will persist in the absence of stimulation; if so, it is not likely maintained by social consequences. Therefore, the condition is conducted ideally with the client alone in a relatively barren environment, and there is no programmed antecedent event.
Consequent event: None.

Control (Play) Condition

This condition is designed to eliminate or minimize the effects likely to be seen in the test conditions. Thus, it typically involves free access to preferred leisure items throughout the session, the frequent delivery of attention, and the absence of demands (Note: If social avoidance is suspected, attention will be deleted). Occurrences of problem behavior produce no consequences, other then the delay of attention for a brief period (5 s to10 s).

Preparation of this manuscript was supported in part by a grant from the Florida Agency on Persons with Disabilities. Reprints may be obtained from Brian Iwata, Psychology Department, University of Florida, Gainesville, Florida 32611.

1 Two criticisms have been raised about the status of FA methodology as a benchmark standard. First, the FA itself should be considered tentative pending comparison with another standard. This is true, but given the characteristics of a typical FA (repeated measures, control over dependent and independent variables, replication), it is not clear what a more precise standard would be. Second, although results of an FA reveal the effects of contingencies applied during assessment, it is not clear that the same contingencies influence behavior under typical, “real-world” conditions. This criticism also has some merit; however, results from hundreds of studies have shown that the contingencies identified in an FA are “close enough” to form the basis of highly effective treatment. Furthermore, the best way to verify the influence of a suspected “real-world” contingency would be to isolate its effects in an FA.

Arndorfer R. E, Miltenberger R. G, Woster S. H, Rortvedt A. K, Gaffaney T. Home-based descriptive and experimental analysis of problem behaviors in children. Topics in Early Childhood Special Education. 1994; 14 :64–87. [ Google Scholar ]
Baer D. M, Wolf M. M, Risley T. R. Some current dimensions of applied behavior analysis. Journal of Applied Behavior Analysis. 1968; 1 :91–97. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Greene H. C, translator. An introduction to the study of experimental medicine. NY: Macmillan; 1927. Trans. (Original work published 1865) [ Google Scholar ]
Bijou S. W, Peterson R. F, Ault M. H. A method to integrate descriptive and experimental field studies at the level of data and empirical concepts. Journal of Applied Behavior Analysis. 1968; 1 :175–191. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Bloom S. E, Iwata B. A, Fritz J. N, Roscoe E. M, Carreau A. B. Classroom application of a trial-based functional analysis. 2008. Manuscript submitted for publication. [ PMC free article ] [ PubMed ]
Conroy M. A, Fox J. J, Bucklin A, Good W. An analysis of the reliability and stability of the Motivation Assessment Scale in assessing the challenging behaviors of persons with developmental disabilities. Education and Training in Mental Retardation and Developmental Disabilities. 1996; 31 :243–250. [ Google Scholar ]
Derby K. M, Wacker D. P, Sasso G, Steege M, Northup J, Cigrand K, Asmus J. Brief functional assessment techniques to evaluate aberrant behavior in an outpatient setting: A summary of 79 cases. Journal of Applied Behavior Analysis. 1992; 25 :713–721. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Desrochers M. N, Hile M. G, Williams-Moseley T. L. Survey of functional assessment procedures used with individuals who display mental retardation and severe problem behaviors. American Journal on Mental Retardation. 1997; 101 :535–546. [ PubMed ] [ Google Scholar ]
Duker P. C, Sigafoos J. The Motivation Assessment Scale: Reliability and construct validity across three topographies of behavior. Research in Developmental Disabilities. 1998; 19 :131–141. [ PubMed ] [ Google Scholar ]
Ellingson S. A, Miltenberger R. G, Long E. S. A survey of the use of functional assessment procedures in agencies serving individuals with developmental disabilities. Behavioral Interventons. 1999; 14 :187–198. [ Google Scholar ]
Fisher W. W, Kelley M. E, Lomas J. E. Visual aids and structured criteria for improving visual inspection and interpretation of single-case designs. Journal of Applied Behavior Analysis. 2003; 36 :387–406. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Hagopian L. P, Fisher W. W, Thompson R. H, Owen-DeSchryver J, Iwata B. A, Wacker D. P. Toward the development of structured criteria for interpretation of functional analysis data. Journal of Applied Behavior Analysis. 1997; 30 :313–326. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Hagopian L. P, Wilson D. M, Wilder D. A. Assessment and treatment of problem behavior maintained by escape from attention and access to tangible items. Journal of Applied Behavior Analysis. 2001; 34 :229–232. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Hanley G. P, Iwata B. A, McCord B. E. Functional analysis of problem behavior: A review. Journal of Applied Behavior Analysis. 2003; 36 :147–186. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Herzinger C. V, Campbell J. M. Comparing functional assessment methodologies: A quantitative synthesis. Journal of Autism and Developmental Disorders. 2007; 37 :1430–1445. [ PubMed ] [ Google Scholar ]
Iwata B. A, Dorsey M. F, Slifer K. J, Bauman K. E, Richman G. S. Toward a functional analysis of self-injury. Journal of Applied Behavior Analysis. 1982; 27 :197–209. (Reprinted from Analysis and Intervention in Developmental Disabilities , 1982, 2, 3–20) [ PMC free article ] [ PubMed ] [ Google Scholar ]
Iwata B. A, Kahng S, Wallace M. D, Lindberg J.S. The functional analysis model of behavioral assessment. In: Austin J, Carr J. E, editors. Handbook of applied behavior analysis. Reno, NV: Context Press; 2000. pp. 61–89. (Eds.) [ Google Scholar ]
Iwata B. A, Wallace M. D, Kahng S, Lindberg J. S, Roscoe E. M, Conners J, Hanley G. P, Thompson R. T, Worsdell A. S. Skill acquisition in the implementation of functional analysis methodology. Journal of Applied Behavior Analysis. 2000; 33 :181–194. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Kahng S, Iwata B. A. Computerized systems for collecting real-time observational data. Journal of Applied Behavior Analysis. 1998; 31 :253–261. [ Google Scholar ]
Kahng S, Iwata B. A, Lewin A. B. Behavioral treatment of self-injury, 1964–2000. American Journal on Mental Retardation. 2002; 107 :212–221. [ PubMed ] [ Google Scholar ]
Laraway S, Snycerski S, Michael J, Poling A. Motivating operations and terms to describe them: Some further refinements. Journal of Applied Behavior Analysis. 2003; 36 :407–414. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Le D. D, Smith R. G. Functional analysis of self-injury with and without protective equipment. Journal of Developmental and Physical Disabilities. 2002; 14 :277–290. [ Google Scholar ]
Lerman D. C, Iwata B. A. Descriptive and experimental analyses of variables maintaining self-injurious behavior. Journal of Applied Behavior Analysis. 1993; 26 :293–319. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Mace F. C, Lalli J. S. Linking descriptive and experimental analyses in the treatment of bizarre speech. Journal of Applied Behavior Analysis. 1991; 24 :553–562. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Mace F. C, Page T. J, Ivancic M. T, O'Brien S. Analysis of environmental determinants of aggression and disruption in mentally retarded children. Applied Research in Mental Retardation. 1986; 7 :203–221. [ PubMed ] [ Google Scholar ]
Mace F. C, West B. J. Analysis of demand conditions associated with reluctant speech. Journal of Behavior Therapy & Experimental Psychiatry. 1986; 17 :285–294. [ PubMed ] [ Google Scholar ]
Marion S. D, Touchette P. E, Sandman C. A. Sequential analysis reveals a unique structure for self-injurious behavior. American Journal on Mental Retardation. 2003; 108 :301–313. [ PubMed ] [ Google Scholar ]
Moore J. W, Edwards R. P, Sterling-Turner H. E, Riley J, DuBard M, McGeorge A. Teacher acquisition of functional analysis methodology. Journal of Applied Behavior Analysis. 2002; 35 :73–77. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Newton J. T, Sturmey P. The Motivation Assessment Scale: Inter-rater reliability and internal consistency in a British sample. Journal of Mental Deficiency Research. 1991; 35 :472–474. [ PubMed ] [ Google Scholar ]
Northup J, Wacker D, Sasso G, Steege M, Cigrand K, Cook J, DeRaad A. A brief functional analysis of aggressive and alternative behavior in an outclinic setting. Journal of Applied Behavior Analysis. 1991; 24 :509–52. 2. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Sarkar A, Dutta A, Dhingra U, Verma P, Juyal R, Black R, Monon V, Kumar J, Sazawi S. Development and use of behavior and social interaction software installed on Palm handheld for observation of a child's social interactions with the environment. Behavior Research Methods. 2006; 38 (3):407–415. [ PubMed ] [ Google Scholar ]
Sigafoos J, Kerr M, Roberts D. Interrater reliability of the Motivation Assessment Scale: Failure to replicate with aggressive behavior. Research in Developmental Disabilities. 1994; 15 :333–342. [ PubMed ] [ Google Scholar ]
Sigafoos J, Kerr M, Roberts D, Couzens D. Reliability of structured interviews for the assessment of challenging behaviour. Behaviour Change. 1993; 10 :47–50. [ Google Scholar ]
Sigafoos J, Saggers E. A discrete-trial approach to the functional analysis of aggressive behaviour in two boys with autism. Australia & New Zealand Journal of Developmental Disabilities. 1995; 20 :287–297. [ Google Scholar ]
Spreat S, Connelly L. Reliability analysis of the Motivation Assessment Scale. American Journal on Mental Retardation. 1996; 100 :528–532. [ PubMed ] [ Google Scholar ]
Smith R. G, Churchill R. M. Identification of environmental determinants of behavior disorders through functional analysis of precursor behaviors. Journal of Applied Behavior Analysis. 2002; 35 :125–136. [ PMC free article ] [ PubMed ] [ Google Scholar ]
St. Peter C. C, Vollmer T. R, Bourret J. C, Borrero C. S. W, Sloman K. N, Rapp J. T. On the role of attention in naturally occurring matching relations. Journal of Applied Behavior Anlaysis. 2005; 38 :429–443. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Sturmey P. Assessing the functions of aberrant behaviors: A review of psychometric instruments. Journal of Autism and Developmental Disorders. 1994; 24 :293–304. [ PubMed ] [ Google Scholar ]
Thomason J. L, Iwata B. A, Neidert P. L, Roscoe E. M. Evaluation of response latency as the index of problem behavior during functional analyses. 2008. Manuscript submitted for publication. [ PMC free article ] [ PubMed ]
Thompson R. H, Iwata B. A. A comparison of outcomes from descriptive and functional analyses of problem behavior. Journal of Applied Behavior Analysis. 2007; 40 :333–338. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Vollmer T. R, Marcus B. A, Ringdahl J. E, Roane H. S. Progressing from brief assessments to extended experimental analyses in the evaluation of aberrant behavior. Journal of Applied Behavior Analysis. 1995; 28 :561–576. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Wacker D, Berg W, Derby K, Asmus J, Healey Evaluation and long-term treatment of aberrant behavior displayed by young children with disabilities. Journal of Developmental and Behavioral Pediatrics. 1998; 19 :260–266. [ PubMed ] [ Google Scholar ]
Wallace M. D, Doney J. K, Mintz-Resudek C. M, Tarbox R. S. F. Training educators to implement functional analyses. Journal of Applied Behavior Analysis. 2004; 37 :89–92. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Zarcone J. R, Rodgers T. A, Iwata B. A, Rourke D. A, Dorsey M. F. Reliability analysis of the Motivation Assessment Scale: A failure to replicate. Research in Developmental Disabilities. 1991; 12 :349–362. [ PubMed ] [ Google Scholar ]

Research on Challenging Behaviors and Functional Assessment

First Online: 27 March 2021

Cite this chapter

experimental (functional) analysis of behavior disorders

Matthew J. O’Brien 3 &
Nicole M. Hendrix 4

Part of the book series: Autism and Child Psychopathology Series ((ACPS))

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Various functional behavioral assessment (FBA) methods have been developed by researchers and employed by practitioners to better understand and treat challenging behavior from an operant perspective. Through decades of research, these methods have been extended to meet the needs of many populations, settings, and practitioners; however, research has also uncovered limitations to existing methods and led to new types and modifications that have increased the efficiency and precision of FBA, as well as its usability and acceptability by practitioners. This chapter provides an overview of recent research on FBA methods, with a particular focus on methodological advancements that have led to broader applications of FBA and improved treatment outcomes. Additionally, this chapter offers a glimpse into future research directions on FBA practice.

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Functional Behavior Assessment

Comparing Functional Behavior Assessment-Based Interventions and Non-functional Behavior Assessment-Based Interventions: A Systematic Review of Outcomes and Methodological Quality of Studies

Functional assessment of challenging behavior.

Alnemary, F., Wallace, M., Symon, J., & Barry, L. (2015). Using international videoconferencing to provide staff training on functional behavioral assessment. Behavioral Interventions, 30 (1), 73–86.

Article Google Scholar

American Psychological Association (APA). (2010). Ethical principles of psychologists and code of conduct . Washington, D.C.: Author.

Google Scholar

Anderson, C. M., & Long, E. S. (2002). Use of a structured descriptive assessment methodology to identify variables affecting problem behavior. Journal of Applied Behavior Analysis, 35 (2), 137–154.

Anderson, C. M., Rodriguez, B. J., & Campbell, A. (2015). Functional behavior assessment in schools: Current status and future directions. Journal of Behavioral Education, 24 (3), 338–371.

Anderson, C. M., & St. Peter, C. C. (2013). Functional analysis with typically developing children: Best practice or too early to tell?: In response to Hanley (2012). Behavior Analysis in Practice, 6 (2), 62–76.

Arron, K., Oliver, C., Moss, J., Berg, K., & Burbidge, C. (2011). The prevalence and phenomenology of self-injurious and aggressive behaviour in genetic syndromes. Journal of Intellectual Disability Research, 55 (2), 109–120.

Asmus, J., Vollmer, T., & Borrero, J. (2002). Functional behavioral assessment: A school-based model. Education and Treatment of Children, 25 (1), 67–90.

Barretto, A., Wacker, D. P., Harding, J., Lee, J., & Berg, W. K. (2006). Using telemedicine to conduct behavioral assessments. Journal of Applied Behavior Analysis, 39 (3), 333–340. https://doi.org/10.1901/jaba.2006.173-04

Bassingthwaite, B., Graber, J., Weaver, A., Wacker, D., White-Staecker, D., Bergthold, S., & Judkins, P. (2018). Using tele-consultation to develop independent skills of school-based behavior teams in functional behavior assessment. Journal of Educational and Psychological Consultation, 28 (3), 297–318.

Beaulieu, L., Van Nostrand, M., Williams, A., & Herscovitch, B. (2018). Incorporating interview-informed functional analyses into practice. Behavior Analysis in Practice, 11 (4), 385–389.

Beavers, G. A., Iwata, B. A., & Lerman, D. C. (2013). Thirty years of research on the functional analysis of problem behavior. Journal of Applied Behavior Analysis, 46 (1), 1–21.

Behavior Analysis Certification Board (BACB). (2014). Professional and ethical compliance code for behavior analysts . Littleton, CO: Author.

Behavior Analyst Certification Board. (2014). Applied behavior analysis treatment of autism spectrum disorder: Practice guidelines for healthcare funders and managers (2nd ed.). Littleton, CO: Author.

Benson, S., Dimian, A., Elmquist, M., Simacek, J., McComas, J., & Symons, F. (2018). Coaching parents to assess and treat self-injurious behaviour via telehealth. Journal of Intellectual Disability Research, 62 (12), 1114–1123.

Bice-Urbach, B. J., & Kratochwill, T. R. (2016). Tele-consultation: The use of technology to improve evidence-based practices in rural communities. Journal of School Psychology, 56 , 27–43. https://doi.org/10.1016/j.jsp.2016.02.001

Bijou, S. W., Peterson, R. F., & Ault, M. H. (1968). A method to integrate descriptive and experimental field studies at the level of data and empirical concepts. Journal of Applied Behavior Analysis, 1 (2), 175–191.

Blood, E., & Neel, R. S. (2007). From FBA to implementation: A look at what is actually being delivered. Education and Treatment of Children , 67–80.

Bloom, S. E., Iwata, B. A., Fritz, J. N., Roscoe, E. M., & Carreau, A. B. (2011). Classroom application of a trial-based functional analysis. Journal of Applied Behavior Analysis, 44 (1), 19–31.

Borlase, M. A., Vladescu, J. C., Kisamore, A. N., Reeve, S. A., & Fetzer, J. L. (2017). Analysis of precursors to multiply controlled problem behavior: A replication. Journal of Applied Behavior Analysis, 50 (3), 668–674.

Borrero, C. S., & Borrero, J. C. (2008). Descriptive and experimental analyses of potential precursors to problem behavior. Journal of Applied Behavior Analysis, 41 (1), 83–96.

Borrero, C. S., Vollmer, T. R., & Borrero, J. C. (2004). Combining descriptive and functional analysis logic to evaluate idiosyncratic variables maintaining aggression. Behavioral Interventions, 19 (4), 247–262.

Borrero, J. C., & Vollmer, T. R. (2002). An application of the matching law to severe problem behavior. Journal of Applied Behavior Analysis, 35 (1), 13–27.

Boyle, M. A., & Adamson, R. M. (2017). Systematic review of functional analysis and treatment of elopement (2000–2015). Behavior Analysis in Practice, 10 (4), 375–385.

Broussard, C. D., & Northup, J. (1995). An approach to functional assessment and analysis of disruptive behavior in regular education classrooms. School Psychology Quarterly, 10 (2), 151–164.

Carr, E. G. (1977). The motivation of self-injurious behavior: A review of some hypotheses. Psychological Bulletin, 84 (4), 800–816.

Caruthers, C. E., Lambert, J. M., Chazin, K. M., Harbin, E. R., & Houchins-Juarez, N. J. (2015). Latency-based FA as baseline for subsequent treatment evaluation. Behavior Analysis in Practice, 8 (1), 48–51.

Chitiyo, M., & Wheeler, J. J. (2009). Challenges faced by school teachers in implementing positive behavior support in their school systems. Remedial and Special Education, 30 (1), 58–63.

Conners, J., Iwata, B. A., Kahng, S., Hanley, G. P., Worsdell, A. S., & Thompson, R. H. (2000). Differential responding in the presence and absence of discriminative stimuli during multielement functional analyses. Journal of Applied Behavior Analysis, 33 (3), 299–308.

Cooper, L. J., Wacker, D. P., Sasso, G. M., Reimers, T. M., & Donn, L. K. (1990). Using parents as therapists to evaluate appropriate behavior of their children: Application to a tertiary diagnostic clinic. Journal of Applied Behavior Analysis, 23 (3), 285–296.

Council for Children with Behavioral Disorders: A Division of the Council for Exceptional Children: CCBD’S Position Summary on Physical Restraint and Seclusion Procedures in School Settings: Initially Approved by the Executive Committee on 5-17-09 Revised and Approved by the Executive Committee on 7-8-09. (2009). Beyond Behavior,19 (1), 40–41.

Couvillon, M. A., Bullock, L. M., & Gable, R. A. (2009). Tracking behavior assessment methodology and support strategies: A national survey of how schools utilize functional behavioral assessments and behavior intervention plans. Emotional and Behavioural Difficulties, 14 (3), 215–228.

Curtis, K. S., Forck, K. L., Boyle, M. A., Fudge, B. M., Speake, H. N., & Pauls, B. P. (2019). Evaluation of a trial-based interview-informed synthesized contingency analysis. Journal of Applied Behavior Analysis. https://doi.org/10.1002/jaba.618

Desrochers, M. N., Hile, M. G., & Williams-Moseley, T. L. (1997). Survey of functional assessment procedures used with individuals who display mental retardation and severe problem behaviors. American Journal of Mental Retardation, 101 (5), 535–546.

Dracobly, J., & Smith, R. (2012). Progressing from identification and functional analysis of precursor behavior to treatment of self-injurious behavior. Journal of Applied Behavior Analysis, 45 (2), 361–374.

Ducharme, J. M., & Shecter, C. (2011). Bridging the gap between clinical and classroom intervention: Keystone approaches for students with challenging behavior. School Psychology Review, 40 (2), 257–274.

Dufrene, B. A., Doggett, R. A., Henington, C., & Watson, T. S. (2007). Functional assessment and intervention for disruptive classroom behaviors in preschool and head start classrooms. Journal of Behavioral Education, 16 (4), 368–388.

Dufrene, B. A., Kazmerski, J. S., & Labrot, Z. (2017). The current status of indirect functional assessment instruments. Psychology in the Schools, 54 (4), 331–350.

Ellingson, S. A., Miltenberger, R. G., & Long, E. S. (1999). A survey of the use of functional assessment procedures in agencies serving individuals with developmental disabilities. Behavioral Interventions: Theory and Practice in Residential & Community-Based Clinical Programs, 14 (4), 187–198.

Embregts, P. J. C. M., Didden, R., Huitink, C., & Schreuder, N. (2009). Contextual variables affecting aggressive behaviour in individuals with mild to borderline intellectual disabilities who live in a residential facility. Journal of Intellectual Disability Research, 53 (3), 255–264.

Emerson, E. (2001). Challenging behaviour: Analysis and intervention in people with severe intellectual disabilities . Cambridge, UK: Cambridge University Press.

Book Google Scholar

Epstein, M., Atkins, M., Cullinan, D., Kutash, K., &Weaver, R. (2008). Reducing Behavior Problems in the Elementary School Classroom: A Practice Guide (NCEE #2008-012). Washington, D.C.: National Center for Education Evaluation and Regional Assistance, Institute of Education Sciences, U.S. Department of Education. Retrieved from http://ies.ed.gov/ncee/wwc/publications/practiceguides

Fahmie, T., Iwata, B., Harper, J., & Querim, A. (2013). Evaluation of the divided attention condition during functional analyses. Journal of Applied Behavior Analysis, 46 (1), 71–78.

Fahmie, T. A., & Iwata, B. A. (2011). Topographical and functional properties of precursors to severe problem behavior. Journal of Applied Behavior Analysis, 44 (4), 993–997.

Falcomata, T. S., Roane, H. S., Feeney, B. J., & Stephenson, K. M. (2010). Assessment and treatment of elopement maintained by access to stereotypy. Journal of Applied Behavior Analysis, 43 (3), 513–517. https://doi.org/10.1901/jaba.2010.43-513

Fisher, W. W., Greer, B. D., Romani, P. W., Zangrillo, A. N., & Owen, T. M. (2016). Comparisons of synthesized and individual reinforcement contingencies during functional analysis. Journal of Applied Behavior Analysis, 49 (3), 596–616.

Flanagan, T. F., & DeBar, R. M. (2018). Trial-based functional analyses with a student identified with an emotional and behavioral disorder. Behavioral Disorders, 43 (4), 423–435.

Flanagan, T. F., DeBar, R. M., Sidener, T. M., Kisamore, A. N., Reeve, K. F., & Reeve, S. A. (2019). Teacher-implemented trial-based functional analyses with students with emotional/behavioral disorders. Journal of Developmental and Physical Disabilities. https://doi.org/10.1007/s10882-019-09700-5

Floyd, R. G., Phaneuf, R. L., & Wilczynski, S. M. (2005). Measurement properties of indirect assessment methods for functional behavioral assessment: A review of research. School Psychology Review, 34 (1), 58–73.

Freeman, K. A., Anderson, C. M., & Scotti, J. R. (2000). A structured descriptive methodology: Increasing agreement between descriptive and experimental analyses. Education and Training in Mental Retardation and Developmental Disabilities, 35 , 55–66.

Frieder, J. E., Peterson, S. M., Woodward, J., Crane, J., & Garner, M. (2009). Tele-consultation in school settings: Linking classroom teachers and behavior analysts through web-based technology. Behavior Analysis in Practice, 2 , 32–39.

Fritz, J. N., Iwata, B. A., Hammond, J. L., & Bloom, S. E. (2013). Experimental analysis of precursors to severe problem behavior. Journal of Applied Behavior Analysis, 46 (1), 101–129.

Fryling, M. J., & Baires, N. A. (2016). The practical importance of the distinction between open- and closed-ended indirect assessments. Behavior Analysis in Practice, 9 (2), 146–151.

Galiatsatos, G. T., & Graff, R. (2003). Combining descriptive and functional analyses to assess and treat screaming. Behavioral Interventions, 18 (2), 123–138.

Greenberg, F., Guzzetta, V., de Oca-Luna, R. M., Magenis, R. E., Smith, A. C., Richter, S. F., … Lupski, J. R. (1991). Molecular analysis of the Smith-Magenis syndrome: A possible contiguous-gene syndrome associated with del(17)(p11.2). American Journal of Human Genetics, 49 (6), 1207–1218.

Greer, B., Mitteer, D., Briggs, A., Fisher, W., & Sodawasser, A. (2020). Comparisons of standardized and interview-informed synthesized reinforcement contingencies relative to functional analysis. Journal of Applied Behavior Analysis, 53 (1), 82–101.

Gresham, F. M. (2004). Current status and future directions of school-based behavioral interventions. School Psychology Review, 33 (3), 326–343.

Hagopian, L. P., Fisher, W. W., Thompson, R. H., Owen-DeSchryver, J., Iwata, B. A., & Wacker, D. P. (1997). Toward the development of structured criteria for interpretation of functional analysis data. Journal of Applied Behavior Analysis, 30 (2), 313–326.

Hagopian, L. P., Rooker, G. W., Jessel, J., & DeLeon, I. G. (2013). Initial functional analysis outcomes and modifications in pursuit of differentiation: A summary of 176 inpatient cases. Journal of Applied Behavior Analysis, 46 (1), 88–100.

Hall, S. S. (2005). Comparing descriptive, experimental and informant-based assessments of problem behaviors. Research in Developmental Disabilities, 26 (6), 514–526.

Hammond, J. L., Iwata, B. A., Rooker, G. W., Fritz, J. N., & Bloom, S. E. (2013). Effects of fixed versus random condition sequencing during multielement functional analyses. Journal of Applied Behavior Analysis, 46 (1), 22–30.

Hanley, G. P. (2010). Prevention and treatment of severe problem behavior. In E. Mayville & J. Mulick (Eds.), Behavioral foundations of autism intervention (pp. 233–256). New York, NY: Sloman Publishing.

Hanley, G. P. (2012). Functional assessment of problem behavior: Dispelling myths, overcoming implementation obstacles, and developing new lore. Behavior Analysis in Practice, 5 (1), 54–72.

Hanley, G. P., Iwata, B. A., & McCord, B. E. (2003). Functional analysis of problem behavior: A review. Journal of Applied Behavior Analysis, 36 (2), 147–185.

Hanley, G. P., Jin, C. S., Vanselow, N. R., & Hanratty, L. A. (2014). Producing meaningful improvements in problem behavior of children with autism via synthesized analyses and treatments. Journal of Applied Behavior Analysis, 47 (1), 16–36.

Harper, J., Iwata, B., & Camp, E. (2013). Assessment and treatment of social avoidance. Journal of Applied Behavior Analysis, 46 (1), 147–160.

Hartley, S. L., Sikora, D. M., & McCoy, R. (2008). Prevalence and risk factors of maladaptive behaviour in young children with autistic disorder. Journal of Intellectual Disability, 52 (10), 819–829.

Hausman, N., Kahng, S., Farrell, E., & Mongeon, C. (2009). Idiosyncratic functions: Severe problem behavior maintained by access to ritualistic behaviors. Education and Treatment of Children, 32 (1), 77–87.

Heath, H., & Smith, R. G. (2019). Precursor behavior and functional analysis: A brief review. Journal of Applied Behavior Analysis, 52 (3), 804–810.

Herman, C., Healy, O., & Lydon, S. (2018). An interview-informed synthesized contingency analysis to inform the treatment of challenging behavior in a young child with autism. Developmental Neurorehabilitation, 21 (3), 202–207.

Hodnett, J., Scheithauer, M., Call, N. A., Mevers, J. L., & Miller, S. J. (2018). Using a functional analysis followed by differential reinforcement and extinction to reduce challenging behaviors in children with Smith-Magenis syndrome. American Journal on Intellectual and Developmental Disabilities, 123 (6), 558–573.

Hoffmann, A. N., Bogoev, B. K., & Sellers, T. P. (2019). Using telehealth and expert coaching to support early childhood special education parent-implemented assessment and intervention procedures. Rural Special Education Quarterly, 38 (2), 95–106. https://doi.org/10.1177/8756870519844162

Individuals with Disabilities Education Act. (2004). Retrieved from http: idea.ed.gov /

Institute of Medicine (IOM). (2012). The role of telehealth in an evolving health care environment: Workshop summary . Washington, D.C.: National Academy Press.

Iwata, B. A., DeLeon, I. G., & Roscoe, E. M. (2013). Reliability and validity of the functional analysis screening tool. Journal of Applied Behavior Analysis, 46 (1), 271–284.

Iwata, B. A., Dorsey, M. F., Slifer, K. J., Bauman, K. E., & Richman, G. S. (1994). Toward a functional analysis of self-injury. Journal of Applied Behavior Analysis, 27 , 197–209. https://doi.org/10.1901/jaba.1994.27-197 . (Reprinted from Analysis and Intervention in Developmental Disabilities, 2, 3–20, 1982).

Jessel, J., Hanley, G. P., & Ghaemmaghami, M. (2016). Interview-informed synthesized contingency analyses: Thirty replications and reanalysis. Journal of Applied Behavior Analysis, 49 (3), 576–595.

Jessel, J., Ingvarsson, E., Metras, R., Whipple, R., Kirk, H., & Solsbery, L. (2018). Treatment of elopement following a latency-based interview-informed, synthesized contingency analysis. Behavioral Interventions, 33 (3), 271–283.

Jessel, J., Ingvarsson, E. T., Metras, R., Kirk, H., & Whipple, R. (2018). Achieving socially significant reductions in problem behavior following the interview-informed synthesized contingency analysis: A summary of 25 outpatient applications. Journal of Applied Behavior Analysis, 51 (1), 130–157.

Jessel, J., Metras, R., Hanley, G. P., Jessel, C., & Ingvarsson, E. T. (2020). Evaluating the boundaries of analytic efficiency and control: A consecutive controlled case series of 26 functional analyses. Journal of Applied Behavior Analysis, 53 (1), 25–43.

Johnson, A. H., Goldberg, T. S., Hinant, R. L., & Couch, L. K. (2019). Trends and practices in functional behavior assessments completed by school psychologists. Psychology in the Schools, 56 (3), 360–377.

Kahng, S., & Iwata, B. A. (1999). Correspondence between outcomes of brief and extended functional analyses. Journal of Applied Behavior Analysis, 32 (2), 149–160.

Kelley, M. E., LaRue, R., Roane, H. S., & Gadaire, D. M. (2011). Indirect behavioral assessments: Interviews and rating scales. In W. W. Fisher, C. C. Piazza, & H. S. Roane (Eds.), Handbook of applied behavior analysis (pp. 182–190). New York, NY: Guilford.

Knutsen, J., Wolfe, A., Burke, B., Hepburn, S., Lindgren, S., & Coury, D. (2016). A systematic review of telemedicine in autism spectrum disorders. Review Journal of Autism and Developmental Disorders, 3 (4), 330–344.

Kodak, T., Fisher, W. W., Paden, A., & Dickes, N. (2013). Evaluation of the utility of a discrete-trial functional analysis in early intervention classrooms. Journal of Applied Behavior Analysis, 46 (1), 301–306.

Kodak, T., Northup, J., & Kelley, M. E. (2007). An evaluation of the types of attention that maintain problem behavior. Journal of Applied Behavior Analysis, 40 (1), 167–171. https://doi.org/10.1901/jaba.2007.43-064

Laje, G., Morse, R., Richter, W., Ball, J., Pao, M., & Smith, A. C. (2010). Autism spectrum features in Smith–Magenis syndrome. American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 154 (4), 456–462.

Lambert, J. M., Staubitz, J. E., Roane, J. T., Houchins-Juárez, N. J., Juárez, A. P., Sanders, K. B., & Warren, Z. E. (2017). Outcome summaries of latency-based functional analyses conducted in hospital inpatient units. Journal of Applied Behavior Analysis, 50 (3), 487–494.

Lane, K. L., Oakes, W. P., Powers, L., Diebold, T., Germer, K., Common, E. A., & Brunsting, N. (2015). Improving teachers’ knowledge of functional assessment-based interventions: Outcomes of a professional development series. Education and Treatment of Children, 38 (1), 93–120.

Langdon, N. A., Carr, E. G., & Owen-DeSchryver, J. S. (2008). Functional analysis of precursors for serious problem behavior and related intervention. Behavior Modification, 32 (6), 804–827.

Langthorne, P., & McGill, P. (2011). Assessing the social acceptability of the functional analysis of problem behavior. Journal of Applied Behavior Analysis, 44 (2), 403–407.

LaRue, R. H., Lenard, K., Weiss, M. J., Bamond, M., Palmieri, M., & Kelley, M. E. (2010). Comparison of traditional and trial-based methodologies for conducting functional analyses. Research in Developmental Disabilities, 31 (2), 480–487.

Lee, J. F., Schieltz, K. M., Suess, A. N., Wacker, D. P., Romani, P. W., Lindgren, S. D., et al. (2015). Guidelines for developing telehealth services and troubleshooting problems with telehealth technology when coaching parents to conduct functional analyses and functional communication training in their homes. Behavior Analysis in Practice, 8 (2), 190–200. https://doi.org/10.1007/s40617-014-0031-2.

Leon, Y., Lazarchick, W., Rooker, G., & Deleon, I. (2013). Assessment of problem behavior evoked by disruption of ritualistic toy arrangements in a child with autism. Journal of Applied Behavior Analysis, 46 (2), 507–511.

Lerman, D. C., & Iwata, B. A. (1993). Descriptive and experimental analyses of variables maintaining self-injurious behavior. Journal of Applied Behavior Analysis, 26 (3), 293–319.

Lindgren, S., Wacker, D., Suess, A., Schieltz, K., Pelzel, K., Kopelman, T., … Waldron, D. (2016). Telehealth and autism: Treating challenging behavior at lower cost. Pediatrics, 137 , S167–S175. https://doi.org/10.1542/peds.2015-2851

Lindgren, S., Wacker, D., Schieltz, K., Suess, A., Pelzel, K., Kopelman, T., Lee, J., Romani, P., & O’Brien, M. (2020). A randomized controlled trial of functional communication training via telehealth for young children with autism spectrum disorder. Journal of Autism and Developmental Disorders 50 (12), 4449–4462.

Lloyd, B. P., Wehby, J. H., Weaver, E. S., Goldman, S. E., Harvey, M. N., & Sherlock, D. R. (2015). Implementation and validation of trial-based functional analyses in public elementary school settings. Journal of Behavioral Education, 24 , 167–195. https://doi.org/10.1007/s10864-014-9217-5

Lovaas, O. I., Freitag, G., Gold, V. J., & Kassorla, I. C. (1965). Experimental studies in childhood schizophrenia: Analysis of self-destructive behavior. Journal of Experimental Child Psychology, 2 (1), 67–84.

Mace, F. C., & Lalli, J. S. (1991). Linking descriptive and experimental analyses in the treatment of bizarre speech. Journal of Applied Behavior Analysis, 24 (3), 553–562.

Mace, F. C., & Mauk, J. E. (1995). Bio-behavioral diagnosis and treatment of self-injury. Mental Retardation and Developmental Disabilities Research Reviews, 1 (2), 104–110.

Machalicek, W., Lequia, J., Pinkelman, S., Knowles, C., Raulston, T., Davis, T., et al. (2016). Behavioral telehealth consultation with families of children with autism spectrum disorder. Behavioral Interventions, 31 (3), 223–250. https://doi.org/10.1002/bin.1450.

Machalicek, W., O’Reilly, M., Chan, J. M., Lang, R., Rispoli, M., Davis, T., et al. (2009). Using videoconferencing to conduct functional analysis of challenging behavior and develop classroom behavioral support plans for students with autism. Education and Training in Developmental Disabilities, 44 (2), 207–217.

Machalicek, W., O’Reilly, M. F., Rispoli, M., Davis, T., Lang, R., Franco, J. H., & Chan, J. M. (2010). Training teachers to assess the challenging behaviors of students with autism using video tele-conferencing. Education and Training in Autism and Developmental Disabilities, 45 (2), 203–215.

Martens, B., DiGennaro, F., Reed, D., Szczech, F., & Rosenthal, B. (2008). Contingency space analysis: An alternative method for identifying contingent relations from observational data. Journal of Applied Behavior Analysis, 41 (1), 69–81.

Martens, B. K., Baxter, E. L., McComas, J. J., Sallade, S. J., Kester, J. S., Caamano, M., … Pennington, B. (2019). Agreement between structured descriptive assessments and functional analyses conducted over a telehealth system. Behavior Analysis: Research andPractice, 19 (4), 343–356.

Matson, J. L., Kozlowski, A. M., Worley, J. A., Shoemaker, M. E., Sipes, M., & Horovitz, M. (2011). What is the evidence for environmental causes of challenging behaviors in persons with intellectual disabilities and autism spectrum disorders? Research in Developmental Disabilities, 32 (2), 693–698.

McCahill, J., Healy, O., Lydon, S., & Ramey, D. (2014). Training educational staff in functional behavioral assessment: A systematic review. Journal of Developmental and Physical Disabilities, 26 (4), 479–505.

McComas, J. J., Moore, T., Dahl, N., Hartman, E., Hoch, J., & Symons, F. (2009). Calculating contingencies in natural environments: Issues in the application of sequential analysis. Journal of Applied Behavior Analysis, 42 (2), 413–423.

McComas, J. J., Thompson, A., & Johnson, L. (2003). The effects of presession attention on problem behavior maintained by different reinforcers. Journal of Applied Behavior Analysis, 36 (3), 297–307.

McDonald, J., Moore, D. W., & Anderson, A. (2012). Comparison of functional assessment methods targeting aggressive and stereotypic behaviour in a child with autism. The Educational and Developmental Psychologist, 29 (1), 52–65.

McKerchar, P. M., & Thompson, R. H. (2004). A descriptive analysis of potential reinforcement contingencies in the preschool classroom. Journal of Applied Behavior Analysis, 37 (4), 431–444.

Mello, M., Urbano, R., Goldman, S., & Hodapp, R. (2016). Services for children with autism spectrum disorder: Comparing rural and non-rural communities. Education and Training in Autism and Developmental Disabilities, 51 (4), 355–365.

Monlux, K. D., Pollard, J. S., Rodriguez, A. Y. B., & Hall, S. S. (2019). Telehealth delivery of function-based behavioral treatment for problem behaviors exhibited by boys with fragile X syndrome. Journal of Autism and Developmental Disorders, 49 (6), 2461–2475.

Moreno, G., Wong-Lo, M., & Bullock, L. M. (2017). Investigation on the practice of the functional behavioral assessment: Survey of educators and their experiences in the field. International Journal of Emotional Education, 9 (1), 54–70.

Mueller, M. M., & Nkosi, A. (2007). State of the science in the assessment and management of severe behavior problems in school settings: Behavior analytic consultation to schools. International Journal of Behavioral Consultation and Therapy, 3 (2), 176–202.

Muething, C. S., Call, N. A., Lomas Mevers, J., Zangrillo, A. N., Clark, S. B., & Reavis, A. R. (2017). Correspondence between the results of functional analyses and brief functional analyses. Developmental Neurorehabilitation, 20 (8), 549–559.

Myers, S. M., & Johnson, C. P. (2007). Management of children with autism spectrum disorders.(Disease/disorder overview)(clinical report). Pediatrics, 120 (5), 1162–1182.

Nelson, J., Roberts, M., Rutherford, R., Mathur, S., & Aaroe, L. (1999). A statewide survey of special education administrators and school psychologists regarding functional behavioral assessment. Education & Treatment of Children, 22 (3), 267.

Northup, J., Wacker, D., Sasso, G., Steege, M., Cigrand, K., Cook, J., & DeRaad, A. (1991). A brief functional analysis of aggressive and alternative behavior in an outclinic setting. Journal of Applied Behavior Analysis, 24 (3), 509–522.

O’Reilly, M., Rispoli, M., Davis, T., Machalicek, W., Lang, R., Sigafoos, J., … Didden, R. (2010). Functional analysis of challenging behavior in children with autism spectrum disorders: A summary of 10 cases. Research in Autism Spectrum Disorders, 4 (1), 1–10.

Oakes, W. P., Schellman, L. E., Lane, K. L., Common, E. A., Powers, L., Diebold, T., & Gaskill, T. (2018). Improving educators’ knowledge, confidence, and usefulness of functional assessment-based interventions: Outcomes of professional learning. Education and Treatment of Children, 41 (4), 533–565.

Oliver, A. C., Pratt, L. A., & Normand, M. P. (2015). A survey of functional behavior assessment methods used by behavior analysts in practice. Journal of Applied Behavior Analysis, 48 (4), 817–829.

Oliver, C., Adams, D., Allen, D., Bull, L., Heald, M., Moss, J., … Woodcock, K. (2013). Causal models of clinically significant behaviors in Angelman, Cornelia de Lange, Prader–Willi and Smith–Magenis syndromes. International Review of Research in Developmental Disabilities, 44 , 167–211.

O’Neill, R. E., Bundock, K., Kladis, K., & Hawken, L. S. (2015). Acceptability of functional behavioral assessment procedures to special educators and school psychologists. Behavioral Disorders, 41 (1), 51–66.

Pence, S. T., Roscoe, E. M., Bourret, J. C., & Ahearn, W. H. (2009). Relative contributions of three descriptive methods: Implications for behavioral assessment. Journal of Applied Behavior Analysis, 42 (2), 425–446.

Poole, V. Y., Dufrene, B. A., Sterling, H. E., Tingstrom, D. H., & Hardy, C. M. (2012). Classwide functional analysis and treatment of preschoolers’ disruptive behavior. Journal of Applied School Psychology, 28 (2), 155–174.

Poppes, P., Van der Putten, A. J. J., & Vlaskamp, C. (2010). Frequency and severity of challenging behaviour in people with profound intellectual and multiple disabilities. Research in Developmental Disabilities, 31 (6), 1269–1275.

Powis, L., & Oliver, C. (2014). The prevalence of aggression in genetic syndromes: A review. Research in Developmental Disabilities, 35 (5), 1051–1071.

Radstaake, M., Didden, R., Oliver, C., Allen, D., & Curfs, L. M. (2012). Functional analysis and functional communication training in individuals with Angelman syndrome. Developmental Neurorehabilitation, 15 (2), 91–104.

Retzlaff, B., Fisher, W., Akers, J., & Greer, B. (2019). A translational evaluation of potential iatrogenic effects of single and combined contingencies during functional analysis. Journal of Applied Behavior Analysis, 53 (1), 67–81.

Rispoli, M., Neely, L., Healy, O., & Gregori, E. (2016). Training public school special educators to implement two functional analysis models. Journal of Behavioral Education, 25 (3), 249–274.

Roane, H., Lerman, D., Kelley, M., & Van Camp, C. (1999). Within-session patterns of responding during functional analyses: The role of establishing operations in clarifying behavioral function. Research in Developmental Disabilities, 20 (1), 73–89.

Roane, H. S., Fisher, W. W., Kelley, M. E., Mevers, J. L., & Bouxsein, K. J. (2013). Using modified visual-inspection criteria to interpret functional analysis outcomes. Journal of Applied Behavior Analysis, 46 (1), 130–146.

Romani, P., & Schieltz, K. (2017). Ethical considerations when delivering behavior analytic services for problem behavior via telehealth. Behavior Analysis: Research andPractice, 17 (4), 312–324.

Rooker, G., Iwata, B., Harper, J., Fahmie, T., & Camp, E. (2011). False-positive tangible outcomes of functional analyses. Journal of Applied Behavior Analysis, 44 (4), 737–745.

Rooker, G. W., DeLeon, I. G., Borrero, C. S., Frank-Crawford, M. A., & Roscoe, E. M. (2015). Reducing ambiguity in the functional assessment of problem behavior. Behavioral Interventions, 30 (1), 1–35.

Rooker, G. W., & Roscoe, E. M. (2005). Functional analysis of self-injurious behavior and its relation to self-restraint. Journal of Applied Behavior Analysis, 38 (4), 537–542. https://doi.org/10.1901/jaba.2005.12-05

Roscoe, E. M., Phillips, K. M., Kelly, M. A., Farber, R., & Dube, W. V. (2015). A statewide survey assessing practitioners’ use and perceived utility of functional assessment. Journal of Applied Behavior Analysis, 48 (4), 830–844.

Rose, J. C., & Beaulieu, L. (2019). Assessing the generality and durability of interview-informed functional analyses and treatment. Journal of Applied Behavior Analysis, 52 (1), 271–285. https://doi.org/10.1002/jaba.504

Rush, A. J., & Frances, A. J. (2000). Expert consensus guideline series: Treatment of psychiatric and behavioral problems in mental retardation. American Journal of Mental Retardation, 105 (3), 159–226.

Saini, V., Fisher, W. W., & Retzlaff, B. J. (2018). Predictive validity and efficiency of ongoing visual-inspection criteria for interpreting functional analyses. Journal of Applied Behavior Analysis, 51 (2), 303–320.

Saini, V., Fisher, W. W., Retzlaff, B. J., & Keevy, M. (2020). Efficiency in functional analysis of problem behavior: A quantitative and qualitative review. Journal of Applied Behavior Analysis. https://doi.org/10.1002/jaba.583

Saini, V., Ubdegrove, K., Biran, S., & Duncan, R. (2019). A preliminary evaluation of interrater reliability and concurrent validity of open-ended indirect assessment. Behavior Analysis in Practice. https://doi.org/10.1007/s4061

Santiago, J., Hanley, L., Moore, G., & Jin, P. (2016). The generality of interview-informed functional analyses: Systematic replications in school and home. Journal of Autism and Developmental Disorders, 46 (3), 797–811.

Schlichenmeyer, K., Roscoe, E., Rooker, G., Wheeler, E., & Dube, W. (2013). Idiosyncratic variables that affect functional analysis outcomes: A review (2001–2010). Journal of Applied Behavior Analysis, 46 (1), 339–348.

Sigafoos, J., & Saggers, E. (1995). A discrete-trial approach to the functional analysis of aggressive behaviour in two boys with autism. Australia and New Zealand Journal of Developmental Disabilities, 20 (4), 287–297.

Simacek, J., Dimian, A. F., &McComas, J. J. (2017). Communication intervention for young Skinner, B. F. (1938). The behavior of organisms: An experimental analysis . New York, London: D. Appletone-Century Company, Incorporated.

Skinner, B. (1938). The behavior of organisms: An experimental analysis/by B. F. Skinner. (The Century psychology series). New Jersey: Prentice-Hall.

Skinner, J. N., Veerkamp, M. B., Kamps, D. M., & Andra, P. R. (2009). Teacher and peer participation in functional analysis and intervention for a first grade student with attention deficit hyperactivity disorder. Education and Treatment of Children, 32 (2), 243–266. https://doi.org/10.1353/etc.0.0059.

Slaton, J., & Hanley, G. (2018). Nature and scope of synthesis in functional analysis and treatment of problem behavior. Journal of Applied Behavior Analysis, 51 (4), 943–973.

Slaton, J., Hanley, G., & Raftery, K. (2017). Interview-informed functional analyses: A comparison of synthesized and isolated components. Journal of Applied Behavior Analysis, 50 (2), 252–277.

Sloneem, J., Oliver, C., Udwin, O., & Woodcock, K. A. (2011). Prevalence, phenomenology, aetiology and predictors of challenging behaviour in Smith-Magenis syndrome. Journal of Intellectual Disability Research, 55 (2), 138–151.

Smith, R., & Churchill, R. (2002). Identification of environmental determinants of behavior disorders through functional analysis of precursor behaviors. Journal of Applied Behavior Analysis, 35 (2), 125–136.

Soke, G. N., Maenner, M. J., Christensen, D., Kurzius-Spencer, M., & Schieve, L. A. (2018). Prevalence of co-occurring medical and behavioral conditions/symptoms among 4-and 8-year-old children with autism spectrum disorder in selected areas of the United States in 2010. Journal of Autism and Developmental Disabilities, 48 (8), 2663–2676.

St. Peter, C. C., Vollmer, T. R., Bourret, J. C., Borrero, C. S., Sloman, K. N., & Rapp, J. T. (2005). On the role of attention in naturally occurring matching relations. Journal of Applied Behavior Analysis, 38 (4), 429–443.

Stokes, J. V., & Luiselli, J. K. (2009). Applied behavior analysis assessment and intervention for health: Threatening self-injury (rectal picking) in an adult with Prader-Willi syndrome. Clinical Case Studies, 8 (1), 38–47.

Strand, R. C. W., & Eldevik, S. (2018). Improvements in problem behavior in a child with autism spectrum diagnosis through synthesized analysis and treatment: A replication in an EIBI home program. Behavioral Interventions, 33 , 102–111. https://doi.org/10.1002/bin.1505

Sturmey, P. (1994). Assessing the functions of aberrant behaviors: A review of psychometric instruments. Journal of Autism and Developmental Disorders, 24 , 293–304.

Suess, A. N., Romani, P. W., Wacker, D. P., Dyson, S. M., Kuhle, J. L., Lee, J. F., … Waldron, D. B. (2014). Evaluating the treatment fidelity of parents who conduct in-home functional communication training with coaching via telehealth. Journal of Behavioral Education, 23 , 34–59. https://doi.org/10.1007/s10864-013-9183-3

Suess, A. N., Wacker, D. P., Schwartz, J. E., Lustig, N., & Detrick, J. (2016). Preliminary evidence on the use of telehealth in an outpatient behavior clinic. Journal of Applied Behavior Analysis, 49 (3), 686–692. https://doi.org/10.1002/jaba.305

Sullivan, A. L., Long, L., & Kucera, M. (2011). A survey of school psychologists’ preparation, participation, and perceptions related to positive behavior interventions and supports. Psychology in the Schools, 48 (10), 971–985.

Tarbox, J., Wilke, A. E., Najdowski, A. C., Findel-Pyles, R. S., Balasanyan, S., Caveney, A. C., … Tia, B. (2009). Comparing indirect, descriptive, and experimental functional assessments of challenging behavior in children with autism. Journal of Developmental and Physical Disabilities, 21 (6), 493–514.

Taylor, S. A., Phillips, K. J., & Gertzog, M. G. (2018). Use of synthesized analysis and informed treatment to promote school reintegration. Behavioral Interventions, 33 (4), 364–379. https://doi.org/10.1002/bin.1640

Thomason-Sassi, J. L., Iwata, B. A., Neidert, P. L., & Roscoe, E. M. (2011). Response latency as an index of response strength during functional analyses of problem behavior. Journal of Applied Behavior Analysis, 44 (1), 51–67.

Thompson, R. H., & Iwata, B. A. (2007). A comparison of outcomes from descriptive and functional analyses of problem behavior. Journal of Applied Behavior Analysis, 40 (2), 333–338.

Tiger, J. H., Fisher, W. W., Toussaint, K. A., & Kodak, T. (2009). Progressing from initially ambiguous functional analyses: Three case examples. Research in Developmental Disabilities, 30 (5), 910–926. https://doi.org/10.1016/j.ridd.2009.01.005

Tincani, M. J., Castrogiavanni, A., & Axelrod, S. (1999). A comparison of the effectiveness of brief versus traditional functional analyses. Research in Developmental Disabilities, 20 (5), 327–338.

Travis, R., & Sturmey, P. (2010). Functional analysis and treatment of the delusional statements of a man with multiple disabilities: A four-year follow-up. Journal of Applied Behavior Analysis, 43 (4), 745–749.

Tsami, L., Lerman, D., & Toper-Korkmaz, O. (2019). Effectiveness and acceptability of parent training via telehealth among families around the world. Journal of Applied Behavior Analysis, 52 (4), 1113–1129. https://doi.org/10.1002/jaba.645

Tunnicliffe, P., & Oliver, C. (2011). Phenotype–environment interactions in genetic syndromes associated with severe or profound intellectual disability. Research in Developmental Disabilities, 32 (2), 404–418.

Tuten, M. J., Jones, H., Ertel, J., Jakubowski, J., & Sperlein, J. (2006). Reinforcement-based treatment: A novel approach to treating substance abuse during pregnancy. Counselor Magazine, 7 (3), 22–29.

Virués-Ortega, J., & Haynes, S. N. (2005). Functional analysis in behavior therapy: Behavioral foundations and clinical application. International Journal of Clinical and Health Psychology, 5 (3), 567–587.

Vollmer, T. R., Marcus, B. A., Ringdahl, J. E., & Roane, H. S. (1995). Progressing from brief assessments to extended experimental analyses in the evaluation of aberrant behavior. Journal of Applied Behavior Analysis, 28 (4), 561–576.

Wacker, D., Berg, W., Harding, J., & Cooper-Brown, L. (2004). Use of brief experimental analyses in outpatient clinic and home settings. Journal of Behavioral Education, 13 (4), 213–226.

Wacker, D. P., Lee, J. F., Dalmau, Y. C. P., Kopelman, T. G., Lindgren, S. D., Kuhle, J., et al. (2013). Conducting functional analyses of problem behavior via telehealth. Journal of Applied Behavior Analysis, 46 (1), 31–46. https://doi.org/10.1002/jaba.29.

Wacker, D. P., Schieltz, K. M., Suess, A. N., Romani, P. W., Padilla Dalmau, Y. C., Kopelman, T. G., … Lindgren, S. D. (2016). Telehealth. In N. N. Singh (Ed.), Handbook of evidence-based practices in intellectual and developmental disabilities . https://doi.org/10.1007/978-3-319-26583-4_22

Chapter Google Scholar

Wallace, M. D., & Iwata, B. A. (1999). Effects of session duration on functional analysis outcomes. Journal of Applied Behavior Analysis, 32 (2), 175–183.

Wilder, D. A., Masuda, A., O’Connor, C., & Baham, M. (2001). Brief functional analysis and treatment of bizarre vocalizations in an adult with schizophrenia. Journal of Applied Behavior Analysis, 34 (1), 65–68.

Willner, P. (2015). Neurobiology of aggression. Journal of Intellectual Disability Research, 59 , 82–92. https://doi.org/10.1111/jir.12120

Zhang, Y., & Cummings, J. (2019). Supply of certified applied behavior analysts in the United States: Implications for service delivery for children with autism. Psychiatric Services (Washington, D.C.) , Appips201900058.

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Open access
Published: 07 August 2024

Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype

Christos Symeonides ORCID: orcid.org/0009-0009-9415-4097 1 , 2 , 3 na1 ,
Kristina Vacy ORCID: orcid.org/0009-0000-5330-5260 4 , 5 na1 ,
Sarah Thomson ORCID: orcid.org/0000-0002-5120-3997 4 ,
Sam Tanner ORCID: orcid.org/0009-0003-9363-0756 4 ,
Hui Kheng Chua ORCID: orcid.org/0000-0002-6047-4027 4 , 6 ,
Shilpi Dixit ORCID: orcid.org/0000-0003-4837-0548 4 ,
Toby Mansell ORCID: orcid.org/0000-0002-1282-6331 2 , 7 ,
Martin O’Hely ORCID: orcid.org/0000-0002-0212-1207 2 , 8 ,
Boris Novakovic ORCID: orcid.org/0000-0002-5623-9008 2 , 8 ,
Julie B. Herbstman 9 , 10 ,
Shuang Wang ORCID: orcid.org/0000-0002-1693-6888 9 , 11 ,
Jia Guo ORCID: orcid.org/0000-0002-9774-9856 9 , 11 ,
Jessalynn Chia 4 ,
Nhi Thao Tran ORCID: orcid.org/0000-0002-0396-9760 4 nAff28 ,
Sang Eun Hwang ORCID: orcid.org/0009-0009-7271-7493 4 ,
Kara Britt ORCID: orcid.org/0000-0001-6069-7856 12 , 13 , 14 ,
Feng Chen 4 ,
Tae Hwan Kim ORCID: orcid.org/0009-0000-6163-3483 4 ,
Christopher A. Reid 4 ,
Anthony El-Bitar 4 ,
Gabriel B. Bernasochi ORCID: orcid.org/0000-0002-3966-2074 4 , 15 ,
Lea M. Durham Delbridge ORCID: orcid.org/0000-0003-1859-0152 15 ,
Vincent R. Harley ORCID: orcid.org/0000-0002-2405-1262 12 , 16 ,
Yann W. Yap 6 , 16 ,
Deborah Dewey ORCID: orcid.org/0000-0002-1323-5832 17 ,
Chloe J. Love ORCID: orcid.org/0000-0002-2024-4083 8 , 18 ,
David Burgner ORCID: orcid.org/0000-0002-8304-4302 2 , 7 , 19 , 20 ,
Mimi L. K. Tang 2 , 15 ,
Peter D. Sly ORCID: orcid.org/0000-0001-6305-2201 8 , 21 , 22 ,
Richard Saffery ORCID: orcid.org/0000-0002-9510-4181 2 ,
Jochen F. Mueller ORCID: orcid.org/0000-0002-0000-1973 23 ,
Nicole Rinehart ORCID: orcid.org/0000-0001-6109-3958 24 ,
Bruce Tonge ORCID: orcid.org/0000-0002-4236-9688 25 ,
Peter Vuillermin ORCID: orcid.org/0000-0002-6580-0346 2 , 8 , 18 ,
the BIS Investigator Group ,
Anne-Louise Ponsonby ORCID: orcid.org/0000-0002-6581-3657 2 , 3 , 4 na2 &
Wah Chin Boon 4 , 26 na2

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Autism spectrum disorders
Epigenetics and behaviour

Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort ( n = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region ( P = 0.009) and aromatase gene methylation mediates ( P = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention.

Maternal diabetes-mediated RORA suppression in mice contributes to autism-like offspring through inhibition of aromatase

Sex differences in the effects of prenatal bisphenol A exposure on autism-related genes and their relationships with the hippocampus functions

Long term transcriptional and behavioral effects in mice developmentally exposed to a mixture of endocrine disruptors associated with delayed human neurodevelopment

Introduction.

Autism spectrum disorder (ASD or autism) is a clinically diagnosed neurodevelopmental condition in which an individual has impaired social communication and interaction, as well as restricted, repetitive behavior patterns 1 . The estimated prevalence of ASD is approximately 1–2% in Western countries 2 , with evidence that the incidence of ASD is increasing over time 3 . While increased incidence is partly attributable to greater awareness of ASD 4 , other factors including early life environment, genes and their interplay are important 5 . Strikingly, up to 80% of individuals diagnosed with ASD are male, suggesting sex-specific neurodevelopment underlies this condition 5 .

Brain aromatase, encoded by CYP19A1 and regulated via brain promoter I.f 6 , 7 , 8 converts neural androgens to neural estrogens 9 . During fetal development, aromatase expression within the brain is high in males 10 in the amygdala 11 , 12 . Notably, androgen disruption is implicated in the extreme male brain theory for ASD 13 , and postmortem analysis of male ASD adults show markedly reduced aromatase activity compared to age-matched controls. Furthermore, CYP19A1 aromatase expression was reduced by 38% in the postmortem male ASD prefrontal cortex 14 , as well as by 52% in neuronal cell lines derived from males with ASD 15 . Environmental factors, including exposure to endocrine-disrupting chemicals such as bisphenols, can disrupt brain aromatase function 16 , 17 , 18 .

Early life exposure to endocrine-disrupting chemicals, including bisphenols, has separately been proposed to contribute to the temporal increase in ASD prevalence 19 . Exposure to these manufactured chemicals is now widespread through their presence in plastics and epoxy linings in food and drink containers and other packaging products 20 . Although bisphenol A (BPA) has since been replaced by other bisphenols such as bisphenol S in BPA-free plastics, all bisphenols are endocrine-disrupting chemicals that can alter steroid signaling and metabolism 21 . Elevated maternal prenatal BPA levels are associated with child neurobehavioral issues 20 including ASD-related symptoms 22 , 23 , with many of these studies reporting sex-specific effects 20 , 22 , 23 , 24 . Furthermore, studies in rodents have found that prenatal BPA exposure is associated with gene dysregulation in the male hippocampus accompanied by neuronal and cognitive abnormalities in male but not female animals 20 , 23 , 24 . One potential explanation is that epigenetic programming by bisphenols increases aromatase gene methylation, leading to its reduced cellular expression 16 and a deficiency in aromatase-dependent estrogen signaling. If such is the case, it is possible that estrogen supplementation, such as with 10-hydroxy-2-decenoic acid (10HDA), a major lipid component of the royal jelly of honeybees, may be relevant as a nutritional intervention for ASD. Indeed, 10HDA is known to influence homeostasis through its intracellular effects on estrogen responsive elements that regulate downstream gene expression 25 , 26 , as well as its capacity to influence neurogenesis in vitro 27 .

Here, we have investigated whether higher prenatal BPA exposure leads to an elevated risk of ASD in males and explore aromatase as a potential underlying mechanism. We demonstrate in a preclinical (mouse) model that postnatal administration of 10HDA, an estrogenic fatty acid, can ameliorate ASD-like phenotypes in young mice prenatally exposed to BPA.

Human studies

We examined the interplay between prenatal BPA, aromatase function and sex in relation to human ASD symptoms and diagnosis in the Barwon Infant Study (BIS) birth cohort 28 . By the BIS cohort health review at 7-11 years (mean = 9.05, SD = 0.74; hereafter referred to as occurring at 9 years), 43 children had a pediatrician- or psychiatrist- confirmed diagnosis of ASD against the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria, as of the 30th of June 2023. ASD diagnosis was over-represented in boys with a 2.1:1 ratio at 9 years (29 boys and 14 girls; Supplementary Table 1 ). In BIS, the DSM-5 oriented autism spectrum problems (ASP) scale of the Child Behavior Checklist (CBCL) at age 2 years 29 predicted diagnosed autism strongly at age 4 and moderately at age 9 in receiver operating characteristic (ROC) curve analyses; area under the curve (AuC) of 0.92 (95% CI 0.82, 1.00) 30 and 0.70 (95% CI 0.60, 0.80), respectively. The median CBCL ASP score in ASD cases and non-cases at 9 years was 51 (IQR = 50, 58) and 50 (IQR = 50, 51), respectively. Only ASD cases with a pediatrician-confirmed diagnosis of ASD against the DSM-5, as verified by the 30th of June 2023, were included in this report. We thus examined both outcomes (ASP scale and ASD diagnosis) as indicators of ASD over the life course from ages 2 to 9 years (Supplementary Table 1 ). Quality control information for the measurement of BPA is presented in Supplementary Table 2 .

BPA effects on ASD symptoms at age 2 years are most evident in boys genetically predisposed to low aromatase enzyme activity

Of the 676 infants with CBCL data in the cohort sample, 249 (36.8%) had an ASP score above the median based on CBCL normative data (Supplementary Table 1 ). From a whole genome SNP array (Supplementary Methods), a CYP19A1 genetic score for aromatase enzyme activity was developed based on five single nucleotide polymorphisms (SNPs; rs12148604, rs4441215, rs11632903, rs752760, rs2445768) associated with lower estrogen levels 31 . Among 595 children with prenatal BPA and CBCL data, those in the top quartile of the genetic predisposition score, that is, children with three or more variants associated with lower levels of estrogens were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’ (Fig. 1 ). Regression analyses stratified by this genetic score and child’s sex were performed and an association between high prenatal BPA exposure (top quartile (>2.18 μg/L) and greater ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.56 (95% CI 1.13, 11.22); P = 0.03 (Supplementary Table 4 ). These findings were minimally altered following adjustment for additional potential confounders. Among males with low aromatase activity, the fraction with higher than median ASP scores attributable to high BPA exposure (the population attributable fraction) was 11.9% (95% CI 4.3%, 19.0%). These results indicate a link between low aromatase function and elevated ASP scores. A sensitivity analysis using an independent weighted CYP19A1 genetic score confirmed these findings. For the additional score, the Genotype-Tissue Expression (GTEx) portal was first used to identify the top five expression quantitative trait loci (eQTLs; rs7169770, rs1065778, rs28757202, rs12917091, rs3784307) for CYP19A1 in any tissue type that showed a consistent effect direction in brain tissue. A functional genetic score was then computed for each BIS participant by summing the number of aromatase-promoting alleles they carry across the five eQTLs, weighted by their normalized effect size (NES) in amygdala tissue. This score captures genetic contribution to cross-tissue aromatase activity with a weighting towards the amygdala, a focus in our animal studies. The score was then reversed so that higher values indicate lower aromatase activity and children in the top quartile were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’. Again, a positive association between prenatal BPA exposure and ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.74 (95% CI 1.12, 12.50); P = 0.03. Additional adjustment for individual potential confounders provided matched ORs between 3.13 to 3.85 (Supplementary Table 5 ).

Conditional logistic regression models were run where participants were matched on ancestry and time of day of urine collection and, for ASD diagnosis at 9 years, each case within these matched groups was individually matched to eight controls based on nearest date of and age at year 9 interview. BPA was classified in quartiles with the top quartile above 2.18 μg/L as high BPA exposure vs the other three quartiles. ‘Low aromatase enzyme activity’ means being in the top quartile and ‘high aromatase enzyme activity’ means being in the lower three quartiles of an unweighted sum of the following genotypes associated with lower estrogen levels 31 (participant given 1 if genotype is present, 0 if not): CC of rs12148604, GG of rs4441215, CC of rs11632903, CC of rs752760, AA of rs2445768. ‘Greater ASD symptoms’ represents a T-score above 50 (that is, above median based on normative data) on the DSM-5-oriented autism spectrum problems scale of the Child Behavior Checklist for Ages 1.5-5 (CBCL). Data are OR ± 95% CI. Source data are provided as a Source Data file. * Since there were only two ASD cases at age 9 in the girls with low aromatase enzyme activity group, the regression model was not run.

BPA effects on ASD diagnosis at 9 years are most evident in boys genetically predisposed to low aromatase enzyme activity

In subgroup analyses where we stratified by child’s sex and unweighted CYP19A1 genetic score, the results were consistent with those found at 2 years. A positive association between high prenatal BPA exposure and ASD diagnosis was only seen in males with low aromatase activity, with a matched OR of 6.24 (95% CI 1.02, 38.26); P = 0.05 (Supplementary Table 4 ). In this subgroup, the fraction of ASD cases attributable to high BPA exposure (the population attributable fraction) was 12.6% (95% CI 5.8%, 19.0%). In a sensitivity analysis where the weighted CYP19A1 genetic score was used, a similar effect size was observed in this subgroup; matched OR = 6.06 (95% CI 0.93, 39.43), P = 0.06 (Supplementary Table 4 ).

Higher prenatal BPA exposure predicts higher methylation of the CYP19A1 brain promoter PI.f in human cord blood

We investigated the link between BPA and aromatase further by evaluating epigenetic regulation of the aromatase gene at birth in the same BIS cohort. CYP19A1 (in humans; Cyp19a1 in the mouse) has eleven tissue-specific untranslated first exons under the regulation of tissue-specific promoters. The brain-specific promoters are PI.f 6 , 7 , 8 and PII 17 . For a window positioned directly over the primary brain promoter PI.f, higher BPA was positively associated with average methylation, mean increase = 0.05% (95% CI 0.01%, 0.09%); P = 0.009 (Fig. 2 ). Higher BPA levels predicted methylation across both PI.f and PII as a composite, mean increase per log 2 increase = 0.06% (95% CI 0.01%, 0.10%); P = 0.009. Methylation of a control window, comprising the remaining upstream region of the CYP19A1 promoter and excluding both PI.f and PII brain promoters, did not associate with BPA, P = 0.12. These findings persisted after adjustment for the CYP19A1 genetic score for aromatase enzyme activity. Thus, higher prenatal BPA exposure was associated with increased methylation of brain-specific promoters in CYP19A1 . Sex-specific differences were not observed. While these effects were identified in cord blood, methylation of CYP19A1 shows striking concordance between blood and brain tissue (Spearman’s rank correlation across the whole gene: ρ = 0.74 (95% CI 0.59, 0.84); over promoter PI.f window: ρ = 0.94 (95% CI 0.54, 0.99) 32 . Thus, prenatal BPA exposure significantly associates with disruption of the CYP19A1 brain promoter and hence likely the level of its protein product, aromatase.

Visualized using the coMET R package. A Association of individual CpGs along the region of interest with BPA exposure, overlaid with three methylation windows: a 2 CpG window positioned directly on promoter PII, and 7 and 15 CpG windows overlapping PI.f. The red shading reflects each CpG’s level of methylation (beta value). B The CYP19A1 gene, running right to left along chromosome 15, and the positions of both brain promoters. Orange boxes indicate exons. C A correlation matrix for all CpGs in this region. Highlighted in tan are the two CpGs located within the PII promoter sequence and the single CpG located within PI.f. For the 7 CpG window over promoter PI.f, higher BPA associated positively with methylation, mean increase = 0.05% (95% CI 0.01%, 0.09%); P = 0.009, after adjustment for relevant covariates including cell composition. The BPA-associated higher methylation of the brain promoter PI.f region remained evident when the window was expanded to 15 CpGs (mean increase = 0.06%, 95% CI [0.01%, 0.11%], P = 0.04). For PII, the BPA-associated mean methylation increase was 0.07%, 95% CI [-0.02%, 0.16%], P = 0.11). BPA also associated positively with methylation across both PI.f and PII as a composite, mean increase = 0.06% (95% CI 0.01%, 0.10%); P = 0.009. For the remainder of CYP19A1 , excluding both PI.f and PII brain promoters, there was no significant association, P = 0.12. Higher CYP19A1 brain promoter methylation leads to reduced transcription 17 . All statistical tests are two sided. Source data are provided as a Source Data file.

Replication of the association between higher BPA levels and hypermethylation of the CYP19A1 brain promoter

Previously, the Columbia Centre for Children’s Health Study-Mothers and Newborns (CCCEH-MN) cohort (Supplementary Table 3 ) found BPA increased methylation of the BDNF CREB-binding region of promoter IV both in rodent blood and brain tissue at P28 and in infant cord blood in the CCCEH-MN cohort 33 . In rodents, BDNF hypermethylation occurred concomitantly with reduced BDNF expression in the brain 33 . Re-examining the CCCEH-MN cohort, BPA level was also associated with hypermethylation of the aromatase brain promoter P1.f (adjusted mean increase 0.0040, P = 0.0089), replicating the BIS cohort finding.

Molecular mediation of higher BPA levels and hypermethylation of BDNF through higher methylation of CYP19A1

In BIS, we aimed to reproduce these BDNF findings and extend them to investigate aromatase methylation as a potential mediator of the BPA- BDNF relationship. A link between aromatase and methylation of the BDNF CREB-binding region is plausible given that estrogen (produced by aromatase) is known to elevate brain expression of CREB 34 , 35 . In BIS, male infants exposed to BPA (categorized as greater than 4 µg/L vs. rest, following the CCCEH-MN study) had greater methylation of the BDNF CREB-binding site (adjusted mean increase = 0.0027, P = 0.02). This was also evident overall (adjusted mean increase = 0.0023, P = 0.006), but not for females alone (adjusted mean increase = 0.0019, P = 0.13). We then assessed whether methylation of aromatase promoter P1.f mediates this association. In both cohorts, aromatase methylation was positively associated with BDNF CREB-binding-site methylation in males (BIS, adjusted mean increase = 0.07, P = 0.0008; CCCEH-MN, adjusted mean increase = 0.91, P = 0.0016). In the two overall cohorts, there was evidence that the effect of increased BPA on BDNF hypermethylation was mediated partly through higher aromatase methylation (BIS, indirect effect, P = 0.012; CCCEH-MN, indirect effect, P = 0.012).

Prenatal programming laboratory studies—BPA effects on cellular aromatase expression in vitro, neuronal development as well as behavioral phenotype in mice

Bpa reduces aromatase expression in human neuroblastoma sh-sy5y cell cultures.

To validate the findings of our human observational studies on BPA and aromatase expression, we began by studying the effects of BPA exposure on aromatase expression in the human neuroblastoma cell line SH-SY5Y (Fig. 3A ). Indeed, the aromatase protein levels more than halved in the presence of BPA 50 μg/L ( P = 0.01; Fig. 3B ) by Western Blot analysis.

A Western Blot (1 representative blot) demonstrates that increasing BPA concentrations reduced immunoblotted aromatase protein signals (green fluorescence, 55 kDa) in lysates from human-derived neuroblastoma SH-SY5Y cells. Each sample was normalized to its internal house keeping protein β-Actin (red fluorescence, 42 kDa). B Aromatase immunoblotted signals in SH-SY5Y cells treated with vehicle or BPA ( n = 3 independent experiments/group). Five-day BPA treatment of SH-SY5Y cells leads to a significant reduction in aromatase following 50 mg/L(MD = 89, t(6) = 4.0, P = 0.01) and 100 mg/L (MD = 85, t(6) = 3.9, P = 0.01) BPA treatment, compared to vehicle. C BPA treatment (50 µg/kg/day) of Cyp19 -EGFP mice at E10.5-E14.5 results in fewer EGFP+ neurons in the medial amygdala (MD = -5334, t(4) = 5.9, P = 0.004) compared to vehicle mice, n = 3 mice per treatment. Independent t -tests were used and where there were more than two experimental groups ( B ), P -values were corrected for multiple comparisons using Holm-Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: UT = untreated.

The effects of prenatal BPA exposure on aromatase-expressing neurons within the amygdala of male mice

There is a prominent expression of aromatase within cells of the male medial amygdala (MeA) 11 . To visualize aromatase-expressing cells, we studied genetically modified, Cyp19 -EGFP transgenic mice harboring a single copy of a bacterial artificial chromosome (BAC) encoding the coding sequence for enhanced green fluorescent protein (EGFP) inserted upstream of the ATG start codon for aromatase ( Cyp19a1 ) 11 (see Methods). As shown, EGFP (EGFP+) expression in male mice was detected as early as embryonic day (E) 11.5 (Supplementary Fig. 1 ), indicating that aromatase gene expression is detectable in early CNS development.

To study the effects of prenatal BPA exposure on brain development, pregnant dams were subject to BPA at a dose of 50 μg/kg/day via subcutaneous injection, or a vehicle injection during a mid-gestation window of E10.5 to E14.5, which coincides with amygdala development. This dose matches current USA recommendations 36 , 37 as well as the Tolerable Daily Intake (TDI) set by the European Food Safety Authority (EFSA) at the time that the mothers in our human cohort were pregnant 28 , 38 . In these experiments, we observed that prenatal BPA exposure led to a 37% reduction ( P = 0.004) in EGFP+ neurons in the MeA of male EGFP+ mice compared to control mice (Fig. 3C ). These results are consistent with our findings in SH-SY5Y cells that indicate that BPA exposure leads to a marked reduction in the cellular expression of aromatase.

Prenatal BPA exposure at mid-gestation influences social approach behavior in male mice

Next, we evaluated post-weaning social approach behavior (postnatal (P) days P21-P24) using a modified three-chamber social interaction test 39 (Fig. 4C ). As shown, male mice with prenatal exposure to BPA were found to spend less time investigating sex- and age-matched stranger mice, when compared with vehicle-treated males (with a mean time ± SEM of 101.2 sec ± 11.47 vs. 177.3 s ± 26.97, P = 0.0004; Fig. 4A ). Such differences were not observed for female mice prenatally exposed to BPA (Fig. 4A ). As a control for these studies, we found that the presence of the EGFP BAC transgene is not relevant to behavioral effects in the test (Supplementary Fig. 2 ), and the proportions of EGFP transgenic mice were not significantly different across BPA-exposed and vehicle-exposed cohorts.

Sociability is the higher proportion of time spent in the stranger interaction zone compared to the empty interaction zone. In the three chamber social interaction test ( A ) BPA-exposed mice ( n = 30, MD = 75 s, t(96) = 3.7 P = 0.0004) spent less time investigating the stranger mouse as compared with male control ( n = 21) mice. B Male ArKO ( n = 8, MD = 43 s, t(30) = 2.3, P = 0.03) mice also spent less time with the stranger compared to male WT littermates ( n = 9). C A schematic of the 3-Chamber Sociability Trial. Created with BioRender.com. D Male BPA-exposed mice ( n = 12, MD = 8.2, U = 11, P = 0.048.) spent more time grooming compared to control ( n = 5) mice. There were no differences between female BPA-exposed ( n = 9) and female control ( n = 6) mice. Independent t -tests were used P -values were corrected for multiple comparisons using Holm-Sidak. For ( C ), a Mann–Whitney U test was used. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: Veh = vehicle.

To determine if the effects of prenatal BPA exposure were developmentally restricted, we delivered subcutaneous injections (50 µg/kg/day) of BPA to pregnant dams at early (E0.5–E9.5), mid (E10.5–E14.5), and late (E15.5–E20.5) stages of gestation. From these experiments, we found that while male pups exposed to BPA in mid-gestation developed a social approach deficit, such behavioral impairments were not observed for early or late gestation BPA exposure (Supplementary Fig. 3 ). In addition, we performed experiments in which BPA was available to dams by voluntary, oral administration (50 µg/kg/day) during mid-gestation. As shown, a social approach deficit was again observed in male mice (Supplementary Fig. 4 ), consistent with results from prenatal (mid-gestation) BPA exposure by subcutaneous injections. Thus, we find that prenatal BPA exposure at mid-gestation (E10.5-E14.5) in mice leads to reduced social approach behavior in male, but not female offspring. Notably, the amygdala of embryonic mice undergoes significant development during mid-gestation 40 .

Aromatase knockout (ArKO) male mice have reduced social behavior

Having demonstrated that prenatal BPA exposure reduces aromatase expression in SH-SY5Y cells and affects the postnatal behavior of mice, we next asked if the aromatase gene ( Cyp19a1 ) is central to these phenotypes. To address this, we performed social approach behavioral testing (Supplementary Fig. 5 ) on aromatase knockout (ArKO) mice 41 which have undetectable aromatase expression. The social preference towards the stranger interaction zone compared to the empty zone was only evident for the wildtype ( P = 0.003 Fig. 4B ) but not the ArKO ( P = 0.45 Fig. 4B ). This male-specific social interaction deficit is similar to the BPA exposed pups. Further, postnatal estrogen replacement could reverse the ArKO reduction in sociability seen in males ( P = 0.03 Supplementary Fig. 5 ) resulting in a similar stranger-to-empty preference in the E2-treated ArKO as observed for wildtype. The female ArKO pups did not have a sociability deficit (Supplementary Fig. 5 ).

Further, we did not observe any behavioral differences between ArKO vs WT (or BPA exposed vs unexposed) mice of both sexes in Y-maze test. All groups were able to distinguish the novel arm from the familiar arm. All groups spent significantly more time in the novel arm compared to the familiar arm (Supplementary Fig. 6 ), excluding major short-term memory, motor and sensory intergroup difference contributions.

Prenatal exposure to BPA affects repetitive behavior in male mice

Using the water squirt test, we have previously reported that male ArKO, but not female ArKO mice displayed excessive grooming, a form of repetitive behavior, compared to WT mice 42 . Thus, we conducted the water squirt test on BPA-exposed mice to find that male but not female mice exhibited excessive grooming behavior ( P = 0.048; Fig. 4D). Thus, male prenatal BPA-exposed mice and ArKO mice, but not females, exhibited such repetitive behaviors compared to control mice.

The development of the MeA is altered in male ArKO mice as well as in prenatal BPA-exposed male mice

The development and function of the amygdala are highly relevant to human brain development and ASD 43 , 44 . Notably, the medial amygdala (MeA) is central to emotional processing 45 , and this tissue is a significant source of aromatase-expressing neurons. Given that aromatase function in the amygdala is significant for human cognition 46 and behavior 12 , 47 , and that aromatase is highly expressed in the mammalian MeA, as particularly observed in male mice 11 , we investigated changes to the structure and function of this brain region. We performed stereology analyses on cresyl violet (Nissl)-stained sections of male MeA, we observed a 13.5% reduction in neuron (defined by morphology, size, and presence of nucleolus) number. Compared to the vehicle-exposed males, BPA-exposed males had significantly reduced total neuron number (mean count of 91,017 ± SEM of 2728 neurons vs 78,750 ± SEM of 3322 neurons, P = 0.046; Supplementary Fig. 7 ).

We further examined the characteristics of cells within this amygdala structure in detail using Golgi staining (Fig. 5A, B ). We found that the apical and basal dendrites in the MeA were significantly shorter in male BPA-exposed mice vs. vehicle-treated mice (apical: 29.6% reduction, P < 0.0001; basal, P < 0.0001). This phenotype was also observed for male ArKO vs. WT mouse brains (apical 35.0% reduction, P < 0.0001; basal 31.9% reduction, P < 0.0001; Fig. 5A ). Dendritic spine densities of apical and basal dendrites of male ArKO mice, as well as male mice exposed to BPA, were also significantly reduced (KO vs WT apical, P = 0.01; KO vs WT basal, P < 0.0001; BPA treated vs vehicle treated apical P < 0.0001; BPA treated vs vehicle treated basal, P = 0.004; Fig. 5B ). The dendritic lengths (Fig. 5A ) and spine densities (Fig. 5B ) for apical and basal neurites within the MeA of female ArKO mice or BPA-exposed mice were not significantly different compared to control. Thus, in the context of aromatase suppression by prenatal BPA-exposure, or in ArKO mice lacking aromatase, we find that the apical and basal dendrite features within the MeA are affected in a sexually dimorphic manner.

A Golgi staining showed shorter apical and basal dendrites in male BPA-exposed (apical: n = 27β = −136 μ m, 95% CI [−189, −83], P = 6.0 × 10 −7 ; basal: n = 27 neurons β =-106, 95% CI [−147, −64], P = 5.1 × 10 −7 ) and ArKO mice (apical: n = 27 β = −194, 95% CI [−258, −130], P = 2.9 × 10 −9 ; basal: n = 27, β = −121, 95% CI [−143, −100], P = 1.2 × 10 −29 ) compared to male vehicle (apical n = 27, basal n = 27) or WT (apical n = 27, basal n = 29). Female BPA-exposed mice had longer basal dendrites vs. vehicle ( n = 26 neurons/group, β = 133, 95% CI [76, 191], P = 5.5 × 10 −6 ), while female ArKO mice had shorter basal dendrites vs. WT ( n = 22 neurons/group, β = −45, 95% CI [−91, −0.2], P = 0.049). Significant sex-by-BPA-treatment interaction effects were observed for apical ( P = 0.0002) and basal ( P = 3.0 × 10 −11 ) dendritic lengths, and a sex-by-genotype interaction for basal length ( P = 0.003) but not apical length ( P = 0.19). B Golgi staining showed male BPA-exposed (apical: n = 39, β = −7.0, 95% CI [−10.1, −4.0], P = 5.5 × 10 −6 ; basal: n = 90, β = −3.4, 95% CI [−5.7, −1.1], P = 0.004) and ArKO (apical: n = 51, β = −6.8, 95% CI [−12.2, −1.3], P = 0.01; basal: n = 97, β = −3.8, 95% CI [−5.4, −2.2], P = 5.2 × 10 −6 ) mice had lower spine densities on apical and basal dendrites vs. vehicle (apical n = 46, basal n = 106) or WT (apical n = 53, basal n = 109) mice. Female mice exhibited no spine density differences for BPA exposure (apical n = 74, basal n = 106) vs. vehicle (apical n = 61, basal n = 103) and ArKO (apical n = 94, basal n = 86) vs. WT (apical n = 88, basal n = 83). There was a significant sex-by-BPA-treatment interaction for apical spine density ( P = 0.0005) but not basal ( P = 0.99), and no significant sex-by-genotype interactions (apical: P = 0.08; basal: P = 0.19). For golgi staining experiments, 3 mice/group with 6–9 neuron measures/mouse. Spine count datapoints represent the number of spines on a single 10μm concentric circle. C c-Fos fluorescent immunostaining in adult male PD-MeA revealed fewer c-Fos+ve cells in BPA-exposed ( n = 3) vs. vehicle mice ( n = 3; mean difference MD = 3687, t (4) = 16.12, P < 0.0001) and ArKO ( n = 4) vs. WT mice ( n = 4; MD = −10237; t (4) = 6.48, P = 0.0002). Early postnatal estradiol restored ArKO c-Fos to WT levels ( n = 4; MD = −3112; t(4) = 1.97, P = 0.08). D Microelectrode array electrophysiology showed a lower rate of change in EPSP over 1-4 volts in male BPA-exposed mice ( n = 5 mice, n = 11 slices) vs. vehicle ( n = 7 mice, n = 12 slices; P = 0.02). Generalized estimating equations were used clustering by mouse ( A , B ) or voltage input ( D ) and assuming an exchangeable correlation structure. For ( C ), independent t -tests were used and where there were more than two experimental groups (ArKO analysis), P -values were corrected for multiple comparisons using Holm–Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to amygdala hypoactivation and alters behavioral response to a novel social stimulus

The amygdala, a social processing brain region, is hyporesponsive in ASD (see review ref. 48 ). A post-mortem stereology study reported that adolescents and adults diagnosed with ASD feature an ~15% decrease in the numbers of neurons within the amygdala 43 . Also, functional MRI studies report amygdala hypoactivation in participants with ASD compared to controls 49 . Given that the amygdala is a significant source of aromatase-expressing neurons, we next conducted a series of studies to explore how aromatase deficiency influences the male mouse amygdala, using a combination of c-Fos immunohistochemistry, Golgi staining of brain sections, as well as electrophysiological analyses.

To investigate amygdala activation responses after interacting with a stranger mouse, we performed c-Fos immunohistochemistry (a marker for neuronal activation 50 ; Supplementary Fig. 8 ). As shown, prenatal BPA-exposed mice featured 58% fewer c-Fos positive neurons than in the amygdala of vehicle-exposed mice brains ( P < 0.0001; Fig. 5C ). Similarly, we found that the MeA of ArKO mice had a marked deficit of 67% c-Fos-positive neurons when compared with WT ( P = 0.0002) mice, which was ameliorated by early postnatal estradiol replacement (Fig. 5C ). Therefore, prenatal BPA exposure or loss of aromatase expression in ArKO mice leads to amygdala hypoactivation.

Next, we measured the synaptic excitability (I/O curve) of the MeA using multiple electrode analysis, with excitatory postsynaptic potential (EPSP) output indicative of electrical firing by local neurons. As shown, compared to corresponding controls, we find that MeA excitability (I/O curve) is significantly reduced in male mice prenatally exposed to BPA as well as in male ArKO mice (Figs. 5 D and 9D ). As shown, at 4-volt input, BPA treatment resulted in a 22.8% lower ( P = 0.02) excitatory EPSP output than the vehicle treatment, while a 21% reduction ( P = 0.03) in signal was observed for male ArKO mice compared to male WT mice. Thus, prenatal BPA exposure leads to functional hypoactivation of the amygdala of male mice, and this pattern is also evident in male ArKO mice.

Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to abnormalities in neuronal cortical layer V as well as brain function

It has been reported that individuals with ASD show distinct anatomical changes within the somatosensory cortex, including in neurons of cortical layer V 51 . We previously reported that layer V within the somatosensory cortex is disrupted in ArKO mice 52 . Thus, we performed Golgi staining to study the apical and basal dendrites of neurons within layer V of the somatosensory cortex following prenatal BPA exposure, as well as in ArKO mice. As shown, we found that apical and basal dendrite lengths of layer V cortical neurons were significantly decreased in male mice prenatally exposed to BPA, compared with vehicle-treated mice (apical P = 0.04; basal P < 0.0001, Fig. 6A ). Such reductions in dendrites were also reported in male ArKO vs. WT mice (apical P < 0.0001; basal P = 0.02; Fig. 6A ). Furthermore, we found that dendritic spine densities on apical dendrites were also reduced (BPA-exposed mice vs. vehicle, P = 0.04; ArKO vs. WT mice, P = 0.01 (Fig. 6B ).

A Golgi staining showed shorter apical and basal dendrites in male BPA-exposed (apical: n = 36, β =-350μm, 95% CI [−679, −20], P = 0.04; basal: n = 36, β = −217, 95% CI [−315, −119], P = 1.4 × 10 −5 ) and ArKO mice (apical: n = 35, β = −541.9, 95% CI [−666, −417], P = 1.3 × 10 −17 ; basal: n = 35, β = −163, 95% CI [−308, −17], P = 0.02) compared to male vehicle (apical n = 35, basal n = 36) or WT(apical n = 36, basal n = 36). B Golgi staining showed male BPA-exposed (apical: n = 186, β = −4.7, 95% CI [−9.2, −0.2], P = 0.04; basal: n = 40 β = −6.7, 95% CI [−16, 2.8], P = 0.17) and ArKO (apical: n = 148 β = −4.4, 95% CI [−7.7, −1.0], P = 0.01; basal: n = 51 β = −5.2, 95% CI [−14.4, 4.1], P = 0.27) mice had lower spine densities on apical but not on basal dendrites vs. vehicle (apical n = 189, basal n = 56) or WT mice (apical n = 185, basal n = 55). For golgi staining experiments, 3 mice/group with 9–12 neuron measures/mouse. Spine count datapoints represents the number of spines on a single 10 μm concentric circle. C Representative photomicrographs of golgi stained cortical neurons, scale bar is 100μm. D Electrocorticograms (ECoG) revealed an increased in the average spectral power at 8 Hz in BPA-exposed ( n = 4; * a 8 Hz MD = −0.5; t(325) = 3.4 P = 0.01) mice and (E) 4–6 Hz in ArKO mice ( n = 4; * b 4 Hz MD = −0.2; t(120) = 4.3, P = 0.0006; * c 5 Hz MD = −0.2, t (120) = 6.1, P < 0.0001; * d 6 Hz MD = −0.2, t (120) = 5.2 P < 0.0001) vs. vehicle ( n = 7) or WT ( n = 4)mice. Generalized estimating equations were used clustering by mouse (Panels A, B) and assuming an exchangeable correlation structure. For ( D ) and(E) Independent t test were used used, P -values were corrected for multiple comparisons using Holm-Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

To explore the effects of reduced aromatase on cortical activity, we performed electrocorticography (ECoG) recordings from mice in both experimental models (Fig. 6C ). As shown, spectral analysis revealed an increased power in the range of 4–6 Hz for ArKO vs. WT mice (4 Hz P = 0.0006, 5 Hz P < 0.0001, 6 Hz P < 0.0001; Fig. 6D ) and at 8 Hz for BPA-exposed vs. vehicle mice ( P = 0.01; Fig. 6C ). These data indicate that BPA-exposure or loss of aromatase in ArKO mice affects cortical activity, a result which is reminiscent of cortical dysfunction evidenced by EEG recordings on human participants diagnosed with ASD 53 .

Molecular docking simulations indicate 10HDA is acting as a ligand at the same site as BPA on Estrogen Receptors α and β

It has been reported that BPA interferes with estrogen signaling through its competitive interaction and binding with estrogen receptors α (ERα) and β (ERβ) 54 . To explore this in the context of our findings, we used high-resolution in silico 3D molecular docking simulations to model the binding affinity of the natural ligand 17β-estradiol, the putative ligand BPA, as well as a putative therapeutic ligand of interest 10HDA with ERα (Protein Data Bank (PDB) ID: 5KRI) and ERβ (PDB ID: 1YYE). As shown, our spatial analysis indicated that all three ligands have robust binding affinity (Fig. 7 ; Supplementary Movie 1 ). However, while docking alignment revealed that the predicted fit for 10HDA is strikingly similar to that of 17β-estradiol 25 , 55 , BPA showed a greater mismatch (Fig. 7D ), consistent with previous reports that BPA is 1000-fold less estrogenic than the native ligand 56 . Thus, at least for ERα and Erβ, we find that 10HDA may be effective as a competitive ligand that could counteract the effects of BPA on estrogen signaling within cells.

In silico molecular docking analysis of estrogen receptor β (ERβ, Protein Data Bank (PDB) ID: 1YYE; encoded by the ES gene) using the DockThor platform, showing binding predictions for ( A ) the native ligand 17β-estradiol (E2), ( B ) bisphenol A (BPA), ( C ) Trans-10-hydroxy-2-decenoic acid (10HDA), and ( D ) E2 and BPA (left) and E2 and 10HDA (right) superimposed for spatial alignment comparison. While the molecular affinities of BPA and 10HDA for Erβ were comparable (−9.2 vs. −7.9, respectively), 10HDA aligns better with the binding conformation of the endogenous ligand E2, which activates the receptor. BPA is previously reported as sub-optimally estrogenic 106 — >1000-fold less compared to natural estradiol 54 , 56 —whereas 10HDA has an estrogenic role in nature 25 , 55 . Thus, 10HDA may compensate for E2 deficiency caused by a reduction in aromatase enzyme, and in competition with binding by BPA. Please see Supplementary Movie 1 for a video of the above molecular docking of ERβ with E2 superimposed with BPA and Supplementary Movie 2 for the above molecular docking of ERβ with E2 superimposed with 10HDA.

In vitro effects of BPA and 10HDA in primary fetal cortical cell cultures from male brains

Examining male fetal primary cortical culture, BPA alone shortened neurite lengths (Fig. 8A , BPA; quantified in Fig. 8B-C ) and decreased the spine density. BPA treatment reduced both neurite length ( P = 0.0004) and spine densities ( P < 0.0001; Fig. 8A , BPA; quantified in Fig. 8B–C ). Co-administration with 10HDA ameliorated these adverse effects of BPA (Fig. 8A , 10HDA + BPA; quantified in Fig. 8B, C ).

A Representative photomicrographs of primary cultures of embryonic (ED15.5) mouse cortical neurons, red staining is βIII tubulin and green is aromatase. Scale bar is 100μm. B Compared to the BPA group, the vehicle group (β = 79.9, 95% CI [36, 124], P = 0.0004) and BPA + 10HDA group (β = 174, 95% CI [102, 247], P = 2.4 × 10 −7 ) have significantly longer neurites. The BPA + 10HDA group (β = 94, 95% CI [8, 180], P = 0.03) has longer neurites compared to the vehicle group, and there is no difference between the vehicle and 10HDA groups. C Compared to the BPA group, the vehicle group(β = 16, 95% CI [11, 21], P = 1.7 × 10 −8 ) and BPA + 10HDA group (β = 30, 95% CI [21, 40], P = 8.4 × 10 −10 ) have significantly higher spine densities. The BPA + 10HDA group (β = 14, 95% CI [4, 24], P = 0.006) has a higher spine density compared to the vehicle group, and there is no difference between the vehicle and 10HDA groups. n = 10 neurons/group. Primary cortical cell culture was obtained from 12 male mouse embryoes. Spine count datapoints represent the number of spines on a single 10μm concentric circle. Generalized estimating equations were used clustering by mouse and assuming an exchangeable correlation structure. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

In vivo effects of 10HDA on BPA mouse model

Guided by our findings in cultured neurons, we next investigated the effects of postnatal 10HDA administration on mice prenatally exposed to BPA at mid-gestation, as follows. After weaning, pups (six litters, 3 weeks of age) were administered daily injections of 10HDA (0 and 500 μg/kg/day; dissolved in saline, i.p.) for 3 weeks, following which pups were assessed for behavioral phenotypes. Strikingly, 10HDA treatment significantly improved social interaction (Fig. 9A ). To determine whether the effect of 10HDA administration is permanent, all treatments were withdrawn for 3 months, and mouse behaviors were subsequently re-tested. Withdrawal of 10HDA treatment in BPA-exposed male mice resulted in a deficit in social interaction (Fig. 9B ), and this deficit was once again ameliorated by a subsequent 10HDA treatment (Fig. 9C ) at 5 months of age, in adulthood. Taken together, these data demonstrate that continuous, postnatal 10HDA administration is effective for ameliorating social interaction deficits in male mice following prenatal BPA exposure.

A 10HDA treatments increased social approach in males ( n = 10/group, MD = 41.14, U = 11, P = 0.03,) but not females ( n = 8/group), compared to saline controls. After 3 months of treatment withdrawal ( B ), male mice (saline n = 8, 10HDA n = 7) no longer spent more time interacting with strangers, compared to vehicle treatment. C When male mice ( n = 8/group) were subsequently treated with a second round of 10HDA, social approach behavior was once again significantly elevated (MD = 39.2, U = 5, P = 0.003), indicative of a rescue of this behavioral effect. D Compared to the WT Saline ( n = 10) group (β = 57.1, 95% CI [47.4, 66.8]), EPSP increases at a 21% lower rate with increasing input in the ArKO Saline ( n = 14) group (β = 45.1 μV, 95% CI [40.1, 50.1], P = 0.03). No differences in slope were detected when comparing the WT Saline group with each of the other two treatment (WT n = 18, KO n = 12) groups. Mann–Whitney U tests were used and for ( D ), Generalized estimating equations were used clustering by voltage input ( D ) and assuming an exchangeable correlation structure. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: Sal = saline, w/d = withdrawal.

Next, we wanted to determine if hypoactivity arising from the absence of aromatase in the amygdala may be influenced by 10HDA. To address this question, we studied ArKO mice using multiple-electrode analyses, following 3 weeks of treatment with 10HDA (500 μg/kg/day, i.p.). As shown in Fig, 9D , the electrical activity of the male ArKO amygdala treated with 10HDA was similar to male WT activity levels, whereas saline-treated male ArKO amygdala showed significantly lower activity ( P = 0.03) when stimulated by an input/output paradigm, suggesting that 10HDA treatment was effective to compensate the absence of aromatase. Therefore, we interpret these results to suggest that 10HDA restores signaling deficits arising from aromatase deficiency. Given that prenatal BPA exposure suppresses aromatase, 10HDA supplementation may be relevant to aromatase-dependent signaling in that context as well.

Transcriptomic studies of the fetal brain cortex and cortical cell cultures

MiSeq Next-Gen Sequencing was performed on the transcriptome libraries generated from the brain cortex of the E16.5 fetuses after maternal mid-gestation BPA or vehicle exposure. The action of 10HDA was analyzed by RNAseq of transcriptome libraries from total RNA extracted from primary mouse fetal cortical cultures treated with vehicle or 10HDA. Firstly, pathway analysis of the RNAseq data was performed for Gene Ontology (GO) categories using the clusterProfileR R package. No individual pathways in the BPA analysis survived correction for multiple comparisons using an agnostic (non-candidate) approach. Further candidate investigation using the binomial test showed a significant inverse effect of BPA and 10HDA on pathways previously linked to autism 57 , with 10HDA treatment counteracting the effects of BPA on these pathways (Supplementary Fig. 9 ). Based on our Golgi staining experimental findings relating to altered dendrite morphology, we further assessed the category “dendrite extension” as a candidate pathway. Genes in this pathway were downregulated by BPA (Supplementary Figs. 10 A, 9A) and upregulated by 10HDA (Supplementary Figs. 9B , 10B ). More broadly, Fisher’s exact test showed a significant BPA-associated down-regulation ( P = 0.01), and 10HDA-associated up-regulation ( P = 0.0001), of pathways with the terms “axon” and “dendrite”. Notably, the majority (82%; 9 of the top 11 available) mid gestational biological processes whose activity is overrepresented in induced pluripotent stem cells of autism cases vs. controls 57 were impacted by BPA, and in the opposite direction to 10HDA ( P = 0.03; Supplementary Fig. 9 ).

Next, we performed pathway enrichment analysis, also using a candidate pathway approach, of the RNAseq data using Ingenuity (Fig. 10 ). Strikingly, the effects of BPA and 10HDA on gene expression were diametrically opposed across many functional domains (Fig. 10 ). For example, the canonical pathways “Synaptogenesis Signaling pathway” and “CREB signaling” were downregulated by BPA but upregulated by 10HDA. Similarly, key brain functions, e.g., growth of neurites and neural development were down regulated by BPA and reciprocally upregulated by 10HDA (Fig. 10 ). Taken together, prenatal BPA exposure is detrimental to gene expression through a mechanism that may be ameliorated by postnatal 10HDA administration. The full list of differentially expressed genes can be found in Supplementary Dataset 1 .

The Canonical pathways and Disease and Function—Brain pathway databases were selected in Ingenuity. Several key signaling pathways and brain functions were downregulated ( Z -score less than zero) by BPA and also upregulated ( Z -score greater than zero) by 10HDA. 10HDA downregulated four brain disorder related pathways—hyperactive behavior, seizures, seizure disorder and behavioral deficit. Colored boxes indicate significant ( P < 0.05, Fisher’s Exact Test with Benjamani-hochberg) changes in z -score. Boxes shaded in gray indicate non-significant gene expression changes ( P = 0.05 or greater). Source data are provided as a Source Data file.

Here, we report that prenatal BPA exposure leads to ASD endophenotypes in males, and that this involves the actions of the aromatase gene, as well as its functions in brain cells. Our multimodal approach, incorporating both human observational studies and preclinical studies with two mouse models, offer significant insight into how prenatal programming by BPA disrupts aromatase signaling to cause anatomical, neurological, as well as behavioral changes reminiscent of ASD in males.

In the Barwon Infant Study (BIS) human birth cohort the adverse effect of high prenatal BPA exposure on ASD symptoms (ASP score) at age 2, and clinical ASD diagnosis at age 9, was particularly evident among males with a low aromatase enzyme activity genetic score. Further, studying cord blood gene methylation as an outcome we have demonstrated that BPA exposure specifically methylated the offspring CYP19A1 brain promoter in the BIS cohort, replicated in the CCCEH-MN cohort. Previously, in a meta-analysis of two epigenome-wide association studies (EWAS), two CpGs in the region around the brain promoters PI.f and PII were significantly associated ( P < 0.05) with ASD in both EWAS 58 . Past work 16 , and our findings of BPA reduced aromatase expression in a neuronal cell culture and reduced aromatase-eGFP expression in mouse brain, are consistent with the brain-specific suppression of aromatase expression. We also demonstrated that BPA exposure led to a reduction in steady-state levels of aromatase in a neuronal cell line. Further, we replicated past work that higher prenatal BPA levels are associated with BDNF hypermethylation 33 , previously demonstrated to be associated with lower BDNF expression in males 33 .

We find that prenatal BPA exposure at mid-gestation in mice induces ASD-like behaviors in male but not female offspring, concomitant with cellular, anatomical, functional, and behavioral changes (Supplementary Fig. 11 and Supplementary Fig. 12 ). We found that these features were also observed in male ArKO mice, and this is important because aromatase expression is disrupted in BPA-treated male mice. In the Y-maze test, both the ArKO and BPA offspring did not show any differences with the respective control indicating that there are no major memory, sensory or motor issues in these animals. Given the distinct parallels between the effects of BPA that suppresses aromatase, as well as ArKO mice that lack aromatase, we surmise from our studies that BPA disrupts aromatase function to influence the male mouse brain which manifests as: (i) reduced excitatory postsynaptic potentials in the amygdala, (ii) reduced neuron numbers as well as dendritic lengths and spine densities for neurons within the MeA, (iii) altered cortical activity as recorded by ECoG concomitant with decreased dendritic length and spine density in layer IV/V somatosensory cortical neurons, as well as (iv) enhanced repetitive behaviors and reduced social approach to a stranger. These results in mice are consistent with studies with human participants that report abnormal neuronal structure in these comparative regions within the brains of individuals with ASD 51 . Furthermore, we investigated our gene expression dataset and found that the majority of the top biological processes over-represented in cells derived from ASD cases compared to non-cases in a human pluripotent stem cell analysis with a focus on mid gestational brain development 57 were impacted in opposite directions by BPA compared to 10HDA in our gene expression studies on the male mouse brain (Supplementary Fig. 9 ). Of note, the sexually dimorphic effect we report is consistent with work of others demonstrating that prenatal BPA exposure of rodents led to dysregulation of ASD-related genes with neuronal abnormalities, and learning and memory problems only in males 59 .

In our investigations of BPA, we recognized that 10HDA may be a suitable compound as a ligand in the context of brain ER signaling 27 because of its positive effect on gene expression through stimulation of estrogen responsive DNA elements 25 and its role in neurogenesis 27 —characteristics that altogether may compensate for a relative lack of aromatase-generated neural estrogens. Administration of 10HDA alongside BPA protected neuronal cells in culture from the adverse sequelae observed for BPA alone at the same dose. Three weeks of daily postnatal 10HDA treatment significantly enhanced the sociability of the male BPA-exposed mice and dendrite morphology in primary cell culture. The adverse decrease in dendrite lengths and spine densities of the BPA-exposed mice was also corrected by 10HDA administration (Fig. 8 ). Furthermore, postnatal 10HDA treatment restored amygdala electrical activity in the ArKO mice, indicating that 10HDA likely acts downstream of, rather than directly upon, the aromatase enzyme, given that ArKO mice lack functional aromatase. Transcriptomic analyses revealed that 10HDA upregulated, whereas BPA downregulated, gene expression for fetal programming such as for synaptogenesis and growth of neurites. Some of these pathways could be activated by factors downstream of aromatase, such as 17β-estradiol (Supplementary Fig. 13 ). In this study, the ArKO model was useful because it provided an estrogen deficient comparison 41 . We were able to demonstrate that early postnatal E2 administration restored both MeA neural activation and social preference behavior in the ArKO males.

The molecular docking simulations indicate that ERα and ERβ both comprise docking sites for 10HDA and BPA, however, 10HDA is strongly estrogenic 25 , 55 while BPA is greater than 1000-fold less potent than natural estrogen 54 . Such differences in binding are likely relevant to the diverse transcriptomic effects observed in the cells we analyzed by RNAseq.

Strengths of this study include the multimodal approach to test the hypothesis of the interplay of BPA, male sex, and aromatase suppression. In our human epidemiological studies, extensive information was available to allow confounding to be accounted for using matched analyses for the BPA-ASD cohort finding, and findings persisted after adjustment for further individual confounders. Using a modern causal inference technique, molecular mediation 60 we demonstrate in both birth cohorts that aromatase gene promoter I.f methylation underlies the known effect of higher prenatal BPA on BDNF hypermethylation. Other key features that support an underlying causal relationship include: the consistency of the findings across studies in this program (Supplementary Fig. 11 ); and the consistency with which our experimental laboratory work maps to prior studies of people with ASD (summarized in Supplementary Fig. 12 ) in relation to neuronal and structural abnormality in the amygdala 43 and abnormality in amygdala connectivity 44 , and resting-state cortical EEG 53 . Our findings are also consistent with past work indicating reduced prefrontal aromatase levels in individuals with ASD at postmortem 14 , 15 . The finding that BPA-associated gene methylation patterns in the BIS cohort were not sex-specific but that BPA-associated ASD symptoms and clinical diagnosis were more evident in males with a low genetic aromatase score would be consistent with the male vulnerability to BPA reflecting not differential epigenetic programming, but a greater vulnerability to reduced aromatase function in the developing male brain. This is reinforced by the ArKO model which resulted in an ASD-like phenotype in males not females. We have provided experimental evidence not only on the adverse neurodevelopment effects of BPA, but also experimental evidence of the alleviation of the behavioral, neurophysiological, and neuroanatomical defects following postnatal treatment with 10HDA. A human randomized controlled prevention trial that achieved bisphenol A elimination during pregnancy, with a resultant reduction in ASD among male offspring, would be a useful next step to provide further causal evidence of BPA risks but the feasibility and ethics of such an undertaking would be considerable. We demonstrate that postnatal administration of 10HDA may be a potential therapeutic agent that counteracts the detrimental impacts on distinct gene expression signatures directly impacted by prenatal BPA exposure. Furthermore, 10HDA may ameliorate deficits in ArKO mice which further suggests its utility as a replacement therapy for aromatase deficiency.

Two limitations of our human study were that BPA exposure was measured in only one maternal urine sample at 36 weeks, and that the assay may have low sensitivity 61 . We partially redressed the latter by focusing on categorical BPA values, as recommended 61 , and undertook a matched ASD analysis where determinants for BPA variation, such as the urine collection time of day, were matched to reduce misclassification. Also, functional gene expression studies were unavailable for human samples in our study, but whilst the misclassification introduced by a reliance on a SNP based score would likely lead to an underestimation, an effect was still found among males with a low genetic aromatase score. It would be useful in further studies to consider altered aromatase function with a combined epigenetic-genetic score to reflect environment-by-epigenetic and genetic determinants of low aromatase function. Direct brain EWAS measures were not available, but for the key brain promoter PI.f region of CYP19A1 , the brain-blood correlation is very high: Spearman’s rho= 0.94, 95% CI [0.80, 0.98] 32 . Although ASD symptoms (ASP score) at 2 years were based on parent report, we have previously reported that a higher ASP score was predictive of later ASD diagnosis by age 4 30 . ASD diagnosis at age 9 was verified to meet DSM-5 criteria by pediatrician audit of medical records, thereby reducing diagnostic misclassification.

In our preclinical studies, we performed the 10HDA studies and some of the BPA mechanistic studies only on male animals because our extensive laboratory and human studies, with more than 25 analyses, demonstrated that BPA exposure had significantly more adverse effects in males than females. In addition, we performed the RNASeq on the cortex of fetal mice exposed to BPA in vivo, whereas 10HDA was performed in vitro on cortical primary cell culture. This would likely increase the variability between the BPA RNASeq and the 10HDA RNASeq, yet many of the same pathways were impacted but in opposing directions. Furthermore, the changes we observed in our RNAseq data could be due to changes in the cell type or cell state. This could be clarified by future single cell RNAseq experiments now that this specific issue has been identified.

The BPA exposure of mouse dams under our experimental conditions (50 µg/kg bodyweight) matches the current Oral Reference Dose set by the United States Environmental Protection Agencyc, the current safe level set by the U.S. Food and Drug Administration (FDA) 37 , as well as the Tolerable Daily Intake set by the European Food Safety Authority 38 at the time that the mothers in our human cohort were pregnant 28 . The EFSA set a new temporary TDI of 4 µg/kg bodyweight in 2015 62 and, in December 2021, recommended further reducing this by five orders of magnitude to 0.04 ng/kg 63 ; although this was subsequently revised to 0.2 ng/kg in EFSA's scientific opinion published in 2023 64 . Therefore, the timing of this new evidence is particularly pertinent and provides direct human data to support the reduced TDI.

Consistent with typical human exposure in other settings 20 , BPA exposure in our birth cohort was substantially lower than the above, and yet we see adverse effects. Assuming fractional excretion of 1 65 and average daily urine output of 1.6 L 65 , the median urinary bisphenol concentration of 0.68 µg/L—for which we see increased odds of ASD diagnosis—equates to a total daily intake of just 13 ng/kg, given a mean maternal bodyweight of 80.1 kg at time of urine collection. Notably, while we find an adverse association at 13 ng/kg, we do not have sufficient participants with lower exposure to evaluate a safe lower limit of exposure below this. Our findings in cell culture, with concentration 5 µg/L, parallels these human findings in terms of dose response. Although there are limitations in translating concentrations across body compartments without a stronger understanding of pharmacokinetics of BPA, 5 µg/L corresponds to the 90th and 95th percentile of BPA in urine in our human cohort, and allowing for a standard factor of 10 for variability in human sensitivity used when setting TDIs 38 indicates relevance down to at least 0.5 µg/L, below the median urine concentration. Our findings in laboratory animal studies, with exposure of 50 µg/kg bodyweight, are a little higher, as they were designed to correspond to the then current recommendations 36 , 37 , 38 , but implications nevertheless have relevance within the range of exposure in our cohort. Allowing for standard factors of 10 for interspecies variability 38 and variability in human sensitivity 38 , our animal study findings support a TDI at 500 ng/kg or below, which corresponds to the upper 0.5% of our human cohort. The findings of the human study, also allowing for a factor of 10 for variability in human sensitivity 38 , therefore, support a TDI at or below 1.3 ng/kg.

Despite bans on its use in all infant products by the European Union in 2011 and the U.S. FDA in 2012, BPA remains widespread in the environment 66 . The main source of human exposure to BPA is dietary contamination 68 . Bisphenols are used in the production of common food contact materials, and migrate from those materials during use 69 , including polycarbonate food and beverage containers and the epoxy linings of metal food cans, jar lids, and residential drinking water storage tanks and supply systems 64 . Additional sources of exposure include BPA-based dental composites and sealant epoxies, as well as thermal receipts 64 . BPA levels in pregnant women have previously been reported to be higher for young mothers, smokers, lower education, and lower income 70 . A substantial proportion of ASD cases might be prevented at the population level if these findings were causal and prenatal maternal BPA exposure were reduced. Here, exposures in the top quartile of BPA (>2.18 ug/L) correspond to a population attributable fraction (PAF) for males with low aromatase of 12.6% (95% CI 5.8%, 19.0%) although this estimate is imprecise as it is based on low case numbers. The only other available study with data on BPA exposure (>50 ug/L) and ASD provides an estimate in all children of 10.4% 67 . These studies have misclassification issues (e.g., a single urine measure for BPA and, in the Stein et al. study, an ASD diagnosis derived from health care sources 67 ) but these misclassifications are likely non-differential and thus would bias findings towards the null. Additionally, we need to consider that the above findings of RfD/TDI and PAF are based on BPA alone. Factoring in that most exposures occur as part of a chemical mixture adds additional concern 71 . For example prenatal valproic acid exposed mice (an established ASD mouse model) also have a lower brain aromatase expression 72 .

In summary, this multimodal program of work has shown an adverse effect of higher maternal prenatal BPA on the risk of male offspring ASD by a molecular pathway of reduced aromatase function, which plays a key role in sex-specific early brain development. Overall, these findings add to the growing evidence base of adverse neurodevelopmental effects from bisphenol and other manufactured chemical exposure during pregnancy. The case is compelling and supports broader evidence on the need to further reduce BPA exposure, especially in pregnancy. We also envision that our findings will contribute to new interventions for the prevention and/or amelioration of ASD targeting this specific pathophysiological pathway and we have identified one possible neuroprotective agent—10HDA—that has strong laboratory support. This agent now warrants further study, including human safety and efficacy evaluation.

The human Barwon Infant Study cohort study was approved by the Barwon Health Human Research Ethics Committee, and families provided written informed consent. Parents or guardians provided written informed consent at prenatal recruitment and again when the child was 2 years of age. The human Columbia Center for Children’s Environmental Health Mothers and Newborn cohort study was approved by the Institutional Review Boards of Columbia University and the Centers for Disease Control and Prevention, and all participants in the study provided informed consent. All procedures involving mice were approved by the Florey Institute of Neuroscience and Mental Health animal ethics committee and conformed to the Australian National Health and Medical Research Council code of practice for the care and use of animals for scientific purposes and All experiments were designed to minimize the number of animals used, as well as pain and discomfort. This work adheres to the ARRIVE essential 10 guidelines.

The Barwon Infant Study birth cohort

Participants.

From June 2010 to June 2013, a birth cohort of 1074 mother–infant pairs (10 sets of twins) were recruited using an unselected antenatal sampling frame in the Barwon region of Victoria, Australia 28 . Eligibility criteria, population characteristics, and measurement details have been provided previously 28 ; 847 children had prenatal bisphenol A measures available (Supplementary Table 1 ).

Bisphenol A measurement

We used a direct injection liquid chromatography tandem mass spectrometry (LC-MS/MS) method, as previously described in detail 73 . In summary, a 50 µL aliquot of urine was diluted in milli-Q water and combined with isotopically-labeled standards and b-glucuronidase (from E. Coli -K12). Samples were incubated for 90 min at 37 °C to allow for enzymatic hydrolysis of bisphenol conjugates before quenching the reaction with 0.5% formic acid. Samples were centrifuged before analysis, which was performed using a Sciex 6500 + QTRAP in negative electrospray ionization mode. The BPA distribution and quality control attributes for the application of this method to the Barwon Infant Study (BIS) cohort are shown in Supplementary Table 2 .

Child neurodevelopment

Between the ages of seven and ten, a health screen phone call was conducted to gather information on autism spectrum disorder (ASD) diagnoses and symptomology. Out of the 868 individuals who responded to the health screen, 80 had an ASD diagnosis reported by their parents/guardians or were identified as potentially having ASD. The parent-reported diagnoses were confirmed by pediatric audit of the medical documentation to verify an ASD diagnosis as per DSM-5 guidelines. Participants that had a parent-reported diagnosis and then a verified pediatrician diagnosis by 30 June 2023 and whose diagnoses occurred before the date of their 9-year health screen were included as ASD cases in this study’s analyses ( n = 43). Participants were excluded if (i) their parent/guardian responded with ‘Yes’ or ‘Under Investigation’ to the question of an ASD diagnosis on the year-9 health screen but their diagnosis was not verified by 30 June 2023 ( n = 26), or (ii) they had a verified diagnosis of ASD by 30 June 2023 but their date of diagnosis did not precede the date of their year-9 health screen ( n = 15). The DSM-5-oriented autism spectrum problems (ASP) scale of the Child Behavior Checklist for Ages 1.5-5 (CBCL) administered at 2-3 years was also used as an indicator of autism spectrum disorder.

Whole genome SNP arrays

Blood from the umbilical cord was gathered at birth and then transferred into serum coagulation tubes (BD Vacutainer). Following this, the serum was separated using centrifugation as described elsewhere 74 . Genomic DNA was extracted from whole cord blood using the QIAamp DNA QIAcube HT Kit (QIAGEN, Hilden, Germany), following manufacturer’s instructions. Genotypes were measured by Erasmus MC University Medical Center using the Infinium Global Screening Array-24 v1.0 BeadChip (Illumina, San Diego, CA, USA). The Sanger Imputation Service (Wellcome Sanger Institute, Hinxton, UK) was used for imputing SNPs not captured in the initial genotyping using the EAGLE2 + PBWT phasing and imputation pipeline with the Haplotype Reference Consortium reference panel 75 . Detailed methods are provided elsewhere 76 .

Genome-wide DNA methylation arrays and analysis methods can be found in the Supplementary Methods.

Center for Children’s Environmental Health (CCCEH) epigenetic investigations

The study participants consisted of mothers and their children who were part of the prospective cohort at the Columbia Center for Children’s Environmental Health Mothers and Newborn (CCCEH-MN) in New York City (NYC). They were enrolled between the years 1998 and 2003, during which they were pregnant. The age range for these women was between 18 and 35, and they had no prior history of diabetes, hypertension, or HIV. Furthermore, they had not used tobacco or illicit drugs and had initiated prenatal care by the 20th week of their pregnancy. Every participant gave informed consent, and the research received approval from the Institutional Review Boards at Columbia University as well as the Centers for Disease Control and Prevention (CDC) 33 .

Epigenetic methods have been previously described 77 . Briefly, DNA methylation was measured in 432 cord blood samples from the CCCEH-MN cohort using the 450 K array (485,577 CpG sites) and in 264 MN cord blood samples using the EPIC array (866,895 CpG sites) (Illumina, Inc., San Diego, CA, USA).

BPA measures in the CCCEH were based on spot urine samples collected from the mother during pregnancy (range, 24–40 weeks of gestation; mean, 34.0 weeks) 33 , 78 .

Other statistical analysis

Maternal urinary BPA concentrations were corrected for specific gravity to control for differences in urine dilution. Given a high proportion of the sample (46%) had BPA concentrations that were not detected or below the limit of detection (LOD), a dichotomous BPA exposure variable was formed using the 75th percentile as the cut-point. Dichotomizing the measurements in this way is also likely to give similar results regardless of whether indirect or direct analytical methods were used 79 . This is desirable since indirect methods might be flawed and underestimate human exposure to BPA 61 .

To evaluate whether autism spectrum problems at 2 years could be used as a proxy for later ASD diagnosis, receiver operating characteristic (ROC) curve analyses were used. CBCL ASP at age 2 years predicted diagnosed autism strongly at age 4 and moderately at age 9 with an area under the curve of 0.92 (95% CI 0.82, 1.00) and 0.70 (95% CI 0.60, 0.80), respectively.

According to the normative data of the CBCL, T-scores greater than 50 are above the median. Due to a skewed distribution, ASP measurements were dichotomized using this cut point, which has respective positive and negative likelihood ratios of 2.68 and 0.00 in the prediction of verified ASD diagnosis at 4 years and 1.99 and 0.49 in the prediction of verified ASD diagnosis at 9 years.

A CYP19A1 genetic score for aromatase enzyme activity was developed based on five genotypes of single nucleotide polymorphisms (CC of rs12148604, GG of rs4441215, CC of rs11632903, CC of rs752760, AA of rs2445768) that are associated with sex hormone levels 31 . Participants were classified as ‘low activity’ if they were in the top quartile, that is, they had three or more genotypes associated with lower levels of estrogen and as ‘high activity’ otherwise. Conditional logistic regression model analyses investigating the association between prenatal BPA levels and (i) early childhood ASP scores and (ii) verified ASD diagnosis at 9 years were conducted in the full sample, repeated after stratification by child’s sex (assigned at birth based on visible external anatomy), and repeated again after further stratifying by the CYP19A1 genetic score. Matching variables included child’s sex (in the full sample analysis only), ancestry (all four grandparents are Caucasian vs not) and time of day of maternal urine collection (after 2 pm vs before). Within these matched groups, we additionally matched age-9 ASD cases and non-cases based on the date of the health screen and child’s age at the health screen using the following procedure. Each case was matched to a single non-case based on nearest date of and age at health screen. Once all cases had one matched non-case, a second matched non-case was allocated to each case, and so on until all cases had 8 matched non-cases (8 was the most possible in the boys with high aromatase activity sub-sample and so this number was used across all sub-samples). The order by which cases were matched was randomly determined at the start of each cycle.

The guidelines for credible subgroup investigations were followed 80 . Only two categorical subgroup analyses were conducted, and these were informed a priori by previous literature and by initial mouse study findings. The adverse BPA effects in males with low aromatase enzyme activity (as inferred from the CYP19A1 genetic score) were expected to be of higher magnitude, based on the prior probabilities from the laboratory work. A systematic approach was used to evaluate non-causal explanations and build evidence for causal inference, considering pertinent issues such as laboratory artefacts that are common in biomarker and molecular studies 81 .

A second CYP19A1 genetic score was developed for use in sensitivity analyses. The Genotype-Tissue Expression (GTEx) portal was used to identify the top five expression quantitative trait loci (eQTLs) for aromatase in any tissue type that showed a consistent effect direction in brain tissue. A functional genetic score was then computed for each BIS participant by summing the number of aromatase-promoting alleles they carry across the five eQTLs (AA of rs7169770, CC of rs1065778, AA of rs28757202, CC of rs12917091, AA of rs3784307), weighted by their normalized effect size (NES) in amygdala tissue. The score was then reversed so that higher values indicate lower aromatase activity. The score thus captures genetic contribution to reduced cross-tissue aromatase activity with a weighting towards the amygdala, a focus in our animal studies. The variable was dichotomized using the 75th percentile as the cut-point and the above stratified analyses were repeated with this new weighted score replacing the original, unweighted score.

For the human epigenetic investigations, we used multiple linear regression and mediation 60 approaches. As in past work 33 , BPA was classified as greater than 4 μg/L vs less than 1 μg/L in the CCCEH-MN cohort. A comparable classification was used for the BIS cohort, with greater than 4 μg/L vs the rest. In both cohorts the regression and mediation analyses were also adjusted for sex, gestational age, self-reported ethnicity, and cord blood cell proportions. In the BIS cohort, ethnicity was defined as all four grandparents are Caucasian vs not (see Table S3 ). For the CCCEH-MN cohort, ethnicity was defined as Dominican vs African American 33 . We used statistical software packages R v3.6.3 82 and Stata 15.1 83 .

LABORATORY STUDIES

Shsy-5y cell culture study, bpa treatment on aromatase expression in cell culture.

Human neuroblastoma SHSY-5Y cells were chosen because they were known to express aromatase and SH-SY5Y have been used in ASD research 84 . SHSY-5Y cells (CRL-2266, American Type Culture Collection, Virginia, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (10313-021, Gibco-life technologies, New York (NY), USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (12003C-500 mL, SAFC Biosciences, Kansas, USA), 1% penicillin streptomycin (pen/strep) (15140-122, Gibco-life technologies, NY, USA) and 1% L-Glutamine (Q) (25030-081, Gibco-life technologies, NY, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 . SHSY-5Y cells were grown in 175 cm 2 cell culture flasks (T-175) (353112, BD Falcon, Pennsylvania, USA). Cells were passaged when the seeding density of the T-175 flasks was reached (roughly 80-90% confluence). Cells were passaged by aspirating media from flasks and flasks were then washed once with 10 mL of DPBS (14190-136 Gibo-life technologies, NY, USA) to remove the FBS (inhibits the actions of trypsin). Next, cells were incubated with trypsin (2 mL/T-175 flask) at 37 °C for 5 min to detach cells from the flask wall. To prevent further action of trypsin, media (8 mL/T-175 flask) was added, and contents were pipetted up and down to disperse cell clumps. The cell suspension was then transferred to a 15 mL centrifuge tube (430791, Corning CentriStar, Massachusetts, USA) and centrifuge (CT15RT, Techcomp, Shanghai, China) for 5 min at 1000 RPM at room temperature (RT). The media was then aspirated from these tubes and the cell pellet resuspended in 1 mL of media. Cell viability counts were performed using a hemocytometer (Hausser Scientific, Pennsylvania, USA) to determine the number of live versus dead cells in solution. Two μL of cell suspension was diluted with media (98 μL) and then trypan blue (100 μL) (T8154, Sigma-Aldrich Co., St. Louis, MO, USA) (which labeled dead cells) in a sterile microcentrifuge tube (MCT-175-C-S, Axygen, California, USA). Ten μL of this solution was loaded into the hemocytometer and imaged using a light microscope (DMIL LED, Leica, Germany). Dead cells appeared blue under the microscope because these cells take up the dye whereas live cells were clear (i.e., unstained). Cells were counted in the outer four squares located in each chamber (two chambers, eight squares), with their dimensions known. The average of the eight counts was multiplied by the dilution factor and by 104, yielding the concentration of cells/mL solution. Average cell counts were plotted against treatment groups using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA).

Bisphenol A (BPA) (239658-50 G, Sigma-Aldrich Co., St. Louis, MO, USA) was used for cell treatment. Prior to treatment, stock solutions of each drug were prepared as stated below. BPA was dissolved in pure ethanol (EA043-2.5 L, Chem supply, South Australia, Australia) and the final concentration of the stock solutions was 0.0435 g/mL. Cells in T-175 flasks were randomly assigned to receive treatment with BPA at a dosage of 100 μg/L, 50 μg/L, 25 μg/L or 0 μg/L (vehicle). There was also a no treatment (no vehicle added) flask.

Cell treatment, protein assay, SDS-Page, and western blotting methods can be found in the Supplementary Methods.

Animal studies

Two colonies of mice, maintained at the Florey Institute, were used in this study. The Aromatase knockout (ArKO) mouse model and the Aromatase-enhanced fluorescent green protein (Cyp19-EGFP) transgenic mouse model. Animals were monitored daily except for weekends. If animals showed general clinical signs, an animal technician or a vet was consulted for advice and euthanasia performed as required.

Mice were maintained under specific pathogen-free (SPF) conditions on a 12 h day/night cycle, with ad libitum water and soybean-free food (catalog number SF06-053, Glen Forrest Stockfeeders, Glen Forrest, Western Australia, Australia). Facial tissues were provided for nesting material, and no other environmental enrichment was provided. The room temperature ranged from 18 °C-23 °C and the humidity ranged from 45%-55%.

The sex of mice was determined by SRY genotyping if fetal, otherwise sex was determined by examining the anogenital region around PND9 and again at weaning. Sex was confirmed by inspecting the gonads during dissection. Cyp19 -EGFP mice were toe and tail clipped for identification and genotyping at PND9, ArKO mice were ear notched and tailed clipped at two weeks of age. The oligonucleotide sequences (custom oligos, Geneworks, Australia) for ArKO, GFP and SRY genotyping can be found in Supplementary Data File 2 .

Aromatase knockout (ArKO) mouse model

The ArKO mouse is a transgenic model having a disruption of the Cyp19a1 gene. Exon IX of the Cyp19a1 gene was replaced with a neomycin-resistant cassette 41 . Homozygous Knockout (KO) and wild-type (WT) offspring were bred by mating heterozygous (het) ArKO parents and then PCR genotyped. ArKO mice were backcrossed onto a C57BL/6 J background strain, >10 generations (obtained from Animal Resources Centre, Western Australia) and the colony maintained at the Florey institute.

Aromatase-enhanced fluorescent green protein ( Cyp19 -EGFP) transgenic mouse model

The Cyp19-EGFP mouse model (backcrossed onto the FVBN background strain >10 generations, obtained from Animal Resources Centre, Western Australia) is a transgenic model having a bacterial artificial chromosome containing the full length of the Cyp19a1 gene with an Enhanced Green Fluorescent Protein (EGFP) gene inserted upstream of the ATG start codon 11 . Thus, EGFP expression is an endogenous marker for Cyp19a1 expression. This allows for the visualization and subsequent localization of EGFP as the marker for aromatase without the use of potentially nonspecific aromatase antibodies 11 . We have previously characterized this transgenic model and its brain expression of EGFP 11 . Based on our characterization studies, this transgenic model does not have phenotypes that are significantly different to wildtype mice.

Early postnatal 17β-estrodiol treatment

Mice were allocated into three groups: (1) WT mice receiving a sham implantation; (2) ArKO mice receiving a sham implantation; and (3) ArKO mice undergoing implantation with a 17β-estradiol pellet (sourced from Innovative Research America). This estradiol pellet was designed to release 0.2 mg of 17β-estradiol steadily over a period of 6 weeks. A corresponding sham pellet, identical in size but devoid of E2, was implanted in the control groups.

The implantation procedure was carried out on postnatal day 5. For anesthesia, mice were exposed to 2% isoflurane (IsoFlo, Abbott Laboratories, VIC, Australia) within an induction chamber. The efficacy of anesthesia was confirmed by the lack of response to foot-pinch stimuli. During the surgical procedure, mice were maintained on a heated pad to regulate body temperature. A small, 5 mm incision was made in the dorsal region for the subcutaneous insertion of the pellet, preceded by an injection of Bupivacane in the same area. Following the implantation, the incision was carefully sutured. Post-surgery, mice were placed in a thermal cage (Therma-cage, Manchester, UK) for recovery and monitoring until they regained consciousness and could be returned to their respective litters. Any mice exhibiting complications such as opened stitches were excluded from the study.

BPA injection administration treatment

Plugged FVBN dams were randomly assigned, blocking by weight gain at E9.5 and litter/cage where applicable, to receive daily scruff subcutaneous injections (24 G x 1”, Terumo, Somerset, New Jersey, USA) of BPA (239658-50 G, Sigma-Aldrich Co., St Louis, MO, USA) in ethanol and peanut oil (Coles, Victoria, Australia), either between E0.5-E9.5, E10.5-E14.5 or E15.5-birth at a dosage of 50 μg/kg (deemed as the safe consumption level by the Food and Drug Administration, FDA) 37 or 0 μg/kg (vehicle) of maternal body weight. The injection volume was 1.68 μL/g bodyweight. Mice were weighed directly before each injection. BPA and vehicle exposed litters did not differ in litter size (Supplementary Fig. 14 ).

10HDA injection administration treatment

Cyp19 -EGFP or ArKO mice were randomly assigned by blocking on sex and litter to receive daily intraperitoneal injections (31 G x 1”, Terumo, Somerset, New Jersey, USA) of 500 μg/kg 10HDA (Matreya, USA) in saline or vehicle saline for 21 consecutive days. The injection volume was 2.1 μL/g bodyweight. Mice were weighed directly before each injection.

BPA oral administration treatment

Plugged FVBN dams were exposed to jelly at E9.5. The jelly contained 7.5% Cottee’s Raspberry Cordial (Coles, Victoria, Australia) and 1% bacteriological agar (Oxoid, Australia) in milli-Q water. The pH was increased to between 6.5-7.5 with a pallet of NaOH to allow the jelly to set. Dams were then randomly assigned, blocking by weight gain at E9.5 and litter/cage where applicable, to receive a daily dose of jelly, which contained either ethanol or BPA dissolved in ethanol, at a dosage of 50 μg/kg or 0 μg/kg (vehicle) of maternal body weight. Dams received doses between E10.5-E14.5, and only dams that were observed to have consumed all the jelly each day were included in the study.

Behavioral paradigms

Three-chamber social interaction test.

The three-chamber social interaction test is extensively used to investigate juvenile and adult social interaction deficits, including in sociability 85 , 86 . BPA exposed pups were habituated in the experimental room on P21, directly after weaning. Following a two-to-three-day habituation, testing was conducted from P24 to P27, as only a maximum of ten mice could be tested during the light phase per day. ArKO mice treated with estrogen or sham pallet began habituation at PND28-29, with testing at PND31-33. Both male and female mice were tested. Testing was performed in a dedicated room for mouse behavior studies; no other animals were present in the room at the time of acclimatization and testing. The temperature of the room was maintained at approximately 21 °C.

The test apparatus, a three-chambered clear plexiglass, measuring 42 cm x 39 cm x 11 cm, had two partitions creating a left, right (blue zones), and center chamber (green zone) in which mice could freely roam via two 4 cm x 5 cm openings in the partitions (Supplementary Fig. 15 ). The two side chambers contained two empty wire cages. A 1 cm wide zone in front of each wire cage was defined as the interaction zone (yellow zones). The chamber was set on a black table for white mice, and on a white covering for black mice to aide tracking.

Each test consisted of two consecutive 10-min trials, a habituation trial (T1) and a sociability trial (T2). T1 allowed the test mouse to habituate, and any bias for either empty interaction zone was noted. For T2, a C57BL/6 J novel stranger mouse matched with the test mouse for age and gender was introduced into the cage on the opposite side to which the test mouse demonstrated an interaction zone bias. Thus, any evidence of sociability is bolstered as interaction zone bias would have to be overcome.

For each trial, the test mouse began in the center chamber, and its activity, both body center point, and nose point was tracked and quantified by TopScan Lite (Clever Sys Inc., Reston VA, USA). In this study, the key measure extracted was the average duration of the nose point in each interaction zone.

Social approach and sociability were analyzed. We define social approach as the time the test mouse’s nose point was tracked in the stranger cage interaction zone. Sociability is the higher proportion of time the test mouse to spends with the nose point in the stranger cage interaction zone compared to the empty cage interaction zone.

Details on the Y-maze and grooming methods can be found in the Supplementary Methods.

Golgi staining

Mice had not undergone any behavioral testing. For Golgi staining and analysis, Wild Type (WT) and Knockout (ArKO) and Cyp19 -EGFP littermate males (aged P65-P70); one mouse from n = 3 litters for each genotype) were deeply anesthetized with isopentane rapidly decapitated and fresh whole brain tissues were collected. Brains were first washed with milli-Q water to remove excess blood and then directly placed in the solution obtained from the FD Rapid GolgiStain TM Kit (FD Neuro-Technologies, Inc., MD, USA). Brains were stored at room temperature in the dark and the solutions were replaced after 24 hours, and the tissues were kept in the solution for two weeks. After two weeks, tissues were transferred into solution C for a minimum of 48 hours at room temperature. For sectioning, brains were frozen rapidly by dipping into isopentane pre-cooled with dry ice, and 100 µm thick coronal sections were cut at -22 °C and mounted on 1% gelatin-coated slides. The sections were then air dried in the dark at room temperature. When sections were completely dry, slides were further processed and rinsed with distilled water and placed in the solution provided in the kit for 10 min and washed again with distilled water followed by dehydration for 5 min each in 50%, 75%, 95%, and 100 % ethanol. Sections were further processed in xylene and mounted with Permount.

Neuron Tracing

Neuron tracing was conducted on the amygdala and somatosensory cortex of BPA-exposed mice (exposed ED10.5-14.4) and untreated ArKO mice. Neuron tracing in the amygdala was conducted in both male and female mice, and in the somatosensory cortex, only in male mice. Stained slides were coded to ensure that morphological analysis was conducted by an observer who was blind to the animals’ treatment. Morphological analysis followed a previously described protocol 87 with the following modifications: layer V pyramidal cells of the somatosensory cortex, which were fully impregnated and free of neighboring cells or cellular debris, were randomly selected for analysis (Supplementary Fig. 16 ). Golgi-stained coronal sections containing medial amygdala and somatosensory cortical area were visualized under Olympus BX51 microscope. Neuronal tracing was carried out with the help of Neurolucida and Neuroexplorer software (MicroBright Field Inc., Williston, USA). Up to three pyramidal cells in the MeA and four pyramidal cells in the somatosensory cortex per section over 3 sections (9 (MeA) and 12 (cortex) cells per animal respectively) were sampled 88 , 89 . For Sholl analysis 90 , concentric circles were placed at 10 µm intervals starting from the center of the cell body and the parameters i) total dendritic length (sums of the length of individual branches) of apical and basal dendrites of pyramidal cells and ii) number of spines (protrusions in direct contact with the primary dendrite) and their density (number of spines per 10 µm) were recorded.

Neuron selection criteria: Neurons were selected based on the following criteria. They had to be fully stained, and the cell body had to be in the middle third of the section thickness. The dendrites of the neuron had to be unobscured by the other nearby neuron. Also, neurons had to possess tapering of the majority of the dendrites towards their ends. Representative images of neurons from vehicle and BPA-exposed adult mice can be found in Supplementary Fig. 17 .

Visualizing c-Fos activation to conspecific exposure (amygdala)

Stranger exposure paradigm procedure.

Cyp19-EGFP mice of both sexes as well as male ArKO mice, together with male WT littermates were utilized in this study. Mice had not undergone any other behavior testing. All test mice were acclimatized to the testing room in individual cages for three nights prior to testing. All mice were age P24 on the day of testing, which was performed between 10 am-2 pm. Testing was performed in a dedicated room for mouse behavior studies and no other animals were present in the room at the time of acclimatization and testing. The temperature of the room was maintained at approximately 21 °C.

On the day of testing, each mouse cage containing the isolated test mouse was placed on a stage (a trolley). The lid containing food and water was removed and immediately following, a sex-/age-matched C57Bl/6 J stranger mouse or a novel object (new 1 mL syringe) was placed into the cage and a clean, empty lid was placed on the top. New gloves were used to handle each syringe to avoid transferring another mouse’s olfactory signature to it. The 10 min trial began as soon as the cage lid was shut. After 10 min had elapsed, the stranger or the novel object was removed, the test mouse with home cage was returned to its original location with the original cage lid with food and water for 2 hours prior to perfusion. Once it was established that there was a difference in c-fos expression between stranger exposure and novel object exposure in the medial amygdala, BPA and vehicle exposed (ED10.5-14.5) Cyp19-EGFP mice as well as estrogen and sham pallet treated ArKO and WT mice were exposed to an age and sex matched stranger as described above. C-fos expression was quantified in male mice only.

Histology and stereological analysis methods can be found in the Supplementary Methods.

Neuron count brain collection, staining, brain region delineation and stereology can be found in the Supplementary Methods.

Electrophysiological studies

Microelectrode array electrophysiology.

Male mice aged 8 weeks weighing between 15 and 20 g were used for this study. They had not undergone any behavioral testing prior to electrophysiology. We studied synaptic activity parameters such as the Input/Output (I/O) curve. Stimulation of the glutamatergic synapses terminate in the basolateral amygdala (BLA) and the basomedial amygdala (BMA), which were integrated with multiple inputs that compute to produce an output (field excitatory postsynaptic potential, fEPSP). I/O curve serves as an index of synaptic excitability of large neuronal populations. Mice were anesthetized with isoflurane (IsoFlo TM , Abbott Laboratories, Victoria, Australia) and decapitated. The whole brains were quickly removed and placed in ice-cold, oxygenated (95% O 2 , 5% CO 2 ) cutting solution (composition in mmol/L: 206 sucrose, 3 KCl, 0.5 CaCl 2 , 6 MgCl 2 -H 2 O, 1.25 NaH 2 PO 4 , 25 NaHCO 3, and 10.6 D-glucose). Coronal brain amygdala slices (300 µm) were prepared with a VT 1200 S tissue slicer (Leica) and quickly transferred to 34 o C carbogen bubbled artificial CSF (aCSF) (composition in mmol/L: 126 NaCl, 2.5 KCl, 2.4 CaCl 2 , 1.36 MgCl 2 -H 2 O, 1.25 NaH 2 PO 4 , 25 NaHCO 3, and 10 D-glucose) for 30 min. After further recovery of 1 h equilibrium in oxygenated aCSF at room temperature, the slices were transferred to a submission recording chamber, an MEA chip with 60 electrodes spaced 200 μm apart (60 MEA 200/30 iR-Ti: MCS GnbH, Reutlingen, Germany). The slice was immobilized with a harp grid (ALA Scientific Instruments, New York, USA) and was continuously perfused with carbogenated aCSF (3 mL/min at 32 °C). fEPSPs produced in BLA and BMA were by stimulation of a randomly chosen electrode surrounding the target area with a biphasic voltage waveform (100 μs) at intermediated voltage intensity. The electrode could only be chosen if it produced a fair number of fEPSPs in the surrounding recording electrodes. The width of the EPSP wave ranged from 20 to 30 ms was selected. We chose slices where BLA and BMA were greatly represented according to Allen Mouse Brain Atlas 91 . Care was taken to choose the stimulating electrode in the same region from one slice to the other. The peak-to-peak amplitude of fEPSP in BLA and BMA was recorded by a program of LTP-Director and analyzed using LTP-Analyzer (MCS GnbH, Reutlingen, Germany).

Electrocorticogram (ECoG)

Electrocorticogram recordings.

Male mice aged 8 weeks were used for this study. Mice had not undergone any behavioral testing prior to ECoG recording. For ECoG, surgeries were performed as previously described 92 . Mice were anesthetized with 1–3% isoflurane and two epidural silver ‘ball’ electrodes implanted on each hemisphere of the skull. Electrodes were placed 3 mm lateral of the midline and 0.5 mm, caudal from bregma. A ground electrode was placed 2.5 mm rostral from bregma and 0.5 mm lateral from the midline. Mice were allowed to recover for at least 48 hours after surgery. ECoGs were continuously recorded in freely moving mice for a 4–6-hour period during daylight hours following a standard 30-min habituation period. Signals were band-pass filtered at 0.1 to 40 Hz and sampled at 1 kHz using the Pinnacle EEG/EMG tethered recording system (Pinnacle Technology Inc, KS). Power spectrums were calculated using Hann window with a resolution of 1 Hz using Sirenia Pro analysis software (Pinnacle Technology Inc) on stable 30-min periods of ECoG recordings.

Primary Cortical Cultures

Neuroprotective effect of 10hda against injury induced by bpa on embryonic mouse cortical neurons.

Primary cortical neurons were obtained from male Cyp19 -EGFP mouse embryos at gestational day 15.5. Embryos were genotyped for SRY to determine sex, and only male embryos were used. Cells were seeded in 24-well plates containing 12 mm glass coverslips, coated with 100 µg/mL poly D-lysine to a density of ~0.45 x 10 6 cells/well and incubated in a humidified CO 2 incubator (5% CO 2 , 37 °C). Cells were pre-treated with vehicle (DMSO), 1 mM 10HDA (Matreya, PA,USA), 25 nM BPA and 1 mM 10HDA with 25 nM BPA. For each group, 10 neurons were measured, and the experiments were duplicated. Each replicate was from a separate culture.

Cells were fixed in 4% paraformaldehyde and stained with mouse anti-βIII tubulin monoclonal primary antibody (1:1000; cat #ab41489, Abcam, United Kingdom) and goat anti-mouse secondary antibody, Alexa Fluor 488, (1:2000; cat#A11017; Invitrogen, USA) to label neuronal cells. Aromatase was stained using Rabbit anti-aromatse Antibody (1:2000 cat# A7981; Sigma Aldrich, St. Louis, MO, USA) and donkey anti-rabbit Alexa594 (1:2000; cat# A-21207; Invitrogen, USA). Cell nucleus was stained with Hoechst 33258 solution (Sigma 94403 (2 µg/mL)). Images were captured using an Olympus IX51 microscope (X40 objective). Neurites were quantified using Neurolucida and Neuroexplorer software (MicroBright Field Inc, Williston, USA) as described in the neuron tracing section.

RNA extraction

Total RNA was extracted using PARIS kit (cat#: AM1921, Invitrogen™PARIS™ Kit) according to the protocol supplied by the manufacturer. cDNA libraries were generated using the SureSelect.

Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing kit (Agilent Technologies, CA, USA), according to manufacturer’s instructions.

In vivo effects of BPA on Fetal brain cortical RNA seq

Pregnant Cyp19-EGFP dams were injected subcutaneously with BPA or vehicle ED10.5-14.5 as described in previous section, and culled on ED15.5 by isoflurane overdosed. Fetuses were harvested and placed in chilled PBS. Embryo brain cortical tissue was dissected from fetuses, snap frozen in liquid nitrogen and stored in −80°C until RNA extraction. The sex of fetuses was determined by visual assessment of the gonads and Sry (a male-specific gene) genotyping. Each RNA seq run, 6 cDNA libraries (derived from total RNA samples with 3 biological samples per group), were analyzed by MidSeq Nano run, 50 bp, Single end read on the Illumina platform. Because of undetectable levels of Cyp19a1 RNA in the fetal brain, Cyp19a1 RNA levels were not included. This is consistent in that Aromatase+ cells represent <0.05% of neurons in the adult mouse brain 93 . Subsequent in vivo transcriptomic analyses were completed in males only. Read quality was then assessed with FastQC. The sequence reads were then aligned against the Mus musculus genome (Build version GRCm38). The Tophat aligner (v2.0.14) was used to map reads to the genomic sequences. Sequencing data were then summarized into reads per transcript using Feature counts 94 . The transcripts were assembled with the StringTie tool v1.2.4 using the reads alignment with Mus_musculus.GRCm38 and reference annotation based assembly option (RABT) using the Gencode gene models for the mouse GRCm38/mm10 genome build. Normalisation and statistical analysis on the count data were executed using EdgeR (version edgeR_3.14.2 in R studio, R version 3.14.2). The data were scaled using trimmed mean of M-values (TMM) 95 and differentially expressed genes between all treatment group (Benjamini–Hochberg false discovery rate >0.1). Differentially expressed genes (DEGs) were identified by comparing mice exposed to 50 μg/kg/day BPA with those exposed to the vehicle.

In vitro effects of 10HDA in primary cell culture RNASeq

Primary brain cortical neurons were obtained from C57BL/6 mouse embryos at GD 15.5. Neuronal cell cultures were treated with vehicle (DMSO) or 1 mM 10HDA (Matreya, PA,USA) as described above. The libraries were sequenced with 50 bp single end reads using an Illumina Hiseq and read quality assessed using FastQC. Untrimmed reads were aligned to mouse mm10 genome using Subjunc aligner (version 1.4.4) within the Subread package 96 . Sequencing data were then summarized into reads per transcript using Feature counts 94 and the Gencode gene models for the mouse GRCm38/mm10 genome build (August 2014 freeze) 97 . Normalisation and statistical analysis on the count data were executed using EdgeR (version edgeR_3.4.2 in R studio, R version 3.0.2) 98 after removing features with less than 10 counts per million for at least 3 of the samples. The data were scaled using trimmed mean of M-values (TMM) 95 and differentially expressed genes between all treatment group (Benjamini–Hochberg false discovery rate >0.1). Annotation was added using the ensemble mouse gene annotation added using bioMart package 99 . Differentially expressed genes were identified by comparing cells exposed to 10HDA with those exposed to the vehicle.

Pathway analysis

The BPA and 10HDA differential expression data for enriched pathways were analyzed using Ingenuity (QIAGEN) and tested against the Canonical Pathway Library, Brain Diseases and Functions Library and the Brain Disorders pathway libraries. We included the top 8 Canonical pathways. Then we included only pathways which were p < 0.05 in both the BPA and the 10HDA data for the Brain Diseases and Functions pathway libraries, and included all P -values for the Brain Disorders pathway library ( p > 0.05 are in gray).

An additional analysis of the gene expression data was performed using the c lusterProfileR R package 100 , which provides a range of statistical tests to detect pathways from a query gene set. The test used here was Gene Set Enrichment Analysis (GSEA) 101 , and the genes were tested against the Gene Ontology pathway database (specifically, GO: Biological Process) 102 .

Computational Molecular Docking

The DockThor molecular docking platform 103 was used to assess binding affinities between estrogen receptor beta (encoded by ESR2 gene) and the ligands 17-beta estradiol (E2; the native ligand), BPA, and 10HDA.DockThor takes as input 3D molecular structures for a putative receptor-ligand pair and employs a genetic-algorithm-based optimization strategy to identify optimal binding position within a specified search region. The crystal structures of estrogen receptor alpha (Erα) and beta (Erβ) were sourced from the Protein Data Bank (PDB) with respective PDB IDs: Erα - 5KRI and Erβ - 1YYE. For each ligand, the search grid was restricted to the known estrogen receptor beta ligand-binding domain, centered at x = 30, y = 35, z = 40, with total grid size of x = 25, y = 28 and z = 22. Default settings were used for the optimization procedure.

Statistical analysis

Researchers were blind to treatment during the conduct of the experiment and the outcome assessment but not during statistical analysis.

Mean, standard deviation (SD), and standard error of the mean (SEM) were calculated with GraphPad Prism version 9.4 (GraphPad Software).

Data were tested for equal variances and normality using the Shapiro Wilk test. As electrophysiology, Golgi staining and primary cell culture experiments utilized several data points per animal, observations were not independent, and this non-independence was accounted for in our analyses. We used generalized estimating equations (GEEs) in R version 4.1.2. in a marginal modeling approach that estimates population-averaged effects while treating the covariance structure as a nuisance. We specified the covariance structure as exchangeable (that is, assumed equal correlation between pairs of measurements on the same animal). Given the small number of clusters (i.e. animals), bootstrapped standard errors were estimated using 200 repeats to maintain a conservative type 1 error rate 104 . An interaction term was added to the amygdala Golgi staining study to assess a sex * genotype or sex * BPA exposure interaction.

Where data were normally distributed, parametric tests were conducted. For more than two groups, a one-way ANOVA was conducted with Holm-Sidak post hoc FDR correction, with alpha set to 0.05. Otherwise, unpaired two-tailed Student’s t -tests were used to compare two variables. In cases where normality was not assumed, a Mann-Whitney (comparing two groups) or Kruskal-Wallis with Dunn’s post hoc (comparing three or more groups) was used. In the case of the three-chamber data, a two-way mixed ANOVA was used to assess group x cage side interaction (stranger cage interaction zone vs empty cage interaction zone) with post hoc testing adjusted by the Holm-Sidak method.

Comparisons made are indicated on the Figure legends, and p -values < 0.05 were considered significant. All tests were two-sided (two-tailed) where applicable.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The BIS data including all data used in this paper are available under restricted access for participant privacy. Access can be obtained by request through the BIS Steering Committee by contacting Anne-Louise Ponsonby, The Florey institute of Neuroscience and Mental Health, [email protected]. Requests to access cohort data will be responded to within two weeks. Requests are then considered on scientific and ethical grounds and, if approved, provided under collaborative research agreements. Deidentified cohort data can be provided in Stata or CSV format. Additional project information, including cohort data description and access procedure, is available at the project’s website https://www.barwoninfantstudy.org.au . Source data underlying Figs. 1 – 6 , 8 – 10 and Supplementary Figs. 2 , 3 – 7 , 14 have been provided as a Source Data file with this paper. The RNAseq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus 105 and are accessible through GEO Series accession numbers; fetal brain expression with and without prenatal BPA exposure, GSE266401 and primary cortical culture treated with and without 10HDA, GSE266400 Source data are provided with this paper.

American Psychiatric Association aAPAD-TF. Diagnostic and Statistical Manual of Mental Disorders: DSM-5 (American Psychiatric Association, 2013).

Maenner, M. et al. Prevalence of autism spectrum disorder among children aged 8 years—autism and developmental disabilities monitoring network, 11 sites, United States, 2016. MMWR Surveill. Summ. 69 , 1–12 (2020).

Article PubMed PubMed Central Google Scholar

Li, Q. et al. Prevalence of autism spectrum disorder among children and adolescents in the United States From 2019 to 2020. JAMA Pediatrics 176 , 943–945 (2022).

Atladottir, H. O. et al. The increasing prevalence of reported diagnoses of childhood psychiatric disorders: a descriptive multinational comparison. Eur. Child Adolesc. Psychiatry 24 , 173–183 (2015).

Article PubMed Google Scholar

Lai, M.-C. & Lombardo, M. V. Baron-Cohen S. autism. Lancet 383 , 896–910 (2014).

Boon, W. C., Chow, J. D. & Simpson, E. R. The multiple roles of estrogens and the enzyme aromatase. Prog. Brain Res. 181 , 209–232 (2010).

Article CAS PubMed Google Scholar

Harada, N. & Honda, S.-i Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice. J. Steroid Biochem Mol. Biol. 95 , 49–55 (2005).

Tan, W., Zhu, Z., Ye, L. & Leung, L. K. Methylation dictates PI.f-specific CYP19 transcription in human glial cells. Mol. Cell Endocrinol. 452 , 131–137 (2017).

Roselli, C. E., Liu, M. & Hurn, P. D. Brain aromatization: classic roles and new perspectives. Semin Reprod. Med. 27 , 207–217 (2009).

Article CAS PubMed PubMed Central Google Scholar

Ruiz-Palmero, I. et al. Oestradiol synthesized by female neurons generates sex differences in neuritogenesis. Sci. Rep. 6 , 31891 (2016).

Article ADS CAS PubMed PubMed Central Google Scholar

Stanić, D. et al. Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors α and β, and androgen receptors. PLoS One 9 , e90451 (2014).

Article ADS PubMed PubMed Central Google Scholar

Takahashi, K. et al. Association between aromatase in human brains and personality traits. Sci. Rep. 8 , 16841 (2018).

Baron-Cohen, S. The extreme male brain theory of autism. Trends Cogn. Sci. 6 , 248–254 (2002).

Crider, A., Thakkar, R., Ahmed, A. O. & Pillai, A. Dysregulation of estrogen receptor beta (ERβ), aromatase (CYP19A1), and ER co-activators in the middle frontal gyrus of autism spectrum disorder subjects. Mol. Autism 5 , 46–46 (2014).

Sarachana, T. & Hu, V. W. Genome-wide identification of transcriptional targets of RORA reveals direct regulation of multiple genes associated with autism spectrum disorder. Mol. Autism 4 , 14 (2013).

Santangeli, S. et al. Effects of BPA on female reproductive function: the involvement of epigenetic mechanism. Gen. Comp. Endocrinol. 245 , 122–126 (2017).

Ali, A. A. et al. Developmental vitamin D deficiency increases foetal exposure to testosterone. Mol. Autism 11 , 96 (2020).

Andrade, A. J., Grande, S. W., Talsness, C. E., Grote, K. & Chahoud, I. A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): non-monotonic dose-response and low dose effects on rat brain aromatase activity. Toxicology 227 , 185–192 (2006).

Moosa, A., Shu, H., Sarachana, T. & Hu, V. W. Are endocrine disrupting compounds environmental risk factors for autism spectrum disorder? Hormones Behav. 101 , 13–21 (2018).

Article CAS Google Scholar

Ejaredar, M., Lee, Y., Roberts, D. J., Sauve, R. & Dewey, D. Bisphenol A exposure and children’s behavior: a systematic review. J. Expo. Sci. Environ. Epidemiol. 27 , 175–183 (2017).

Chen, D. et al. Bisphenol analogues other than BPA: environmental occurrence, human exposure, and toxicity—a review. Environ. Sci. Technol. 50 , 5438–5453 (2016).

Article ADS CAS PubMed Google Scholar

Hansen, J. B. et al. Prenatal exposure to bisphenol A and autistic- and ADHD-related symptoms in children aged 2 and 5 years from the Odense Child Cohort. Environ. Health 20 , 24 (2021).

Lim, Y. H. et al. Prenatal and postnatal bisphenol A exposure and social impairment in 4-year-old children. Environ. Health 16 , 79 (2017).

Ferri, S. L., Abel, T. & Brodkin, E. S. Sex differences in autism spectrum disorder: a review. Curr. Psychiatry Rep. 20 , 9 (2018).

Suzuki, K. M. et al. Estrogenic activities of fatty acids and a sterol isolated from royal jelly. Evid. Based Complement. Alternat. Med. 5 , 295–302 (2008).

Pirgon, O., Atar, M., Çiriş, M. & Sever, M. Effects of royal jelly supplementation on growth plate zones and longitudinal growth in young rats. Mellifera 19 , 1–13 (2019).

Google Scholar

Hattori, N., Nomoto, H., Fukumitsu, H., Mishima, S. & Furukawa, S. Royal jelly and its unique fatty acid, 10-hydroxy-trans-2-decenoic acid, promote neurogenesis by neural stem/progenitor cells in vitro. Biomed. Res. 28 , 261–266 (2007).

Vuillermin, P. et al. Cohort profile:the Barwon Infant Study. Int J. Epidemiol. 44 , 1148–1160 (2015).

Achenbach T. M. & Rescorla L. A. Manual for the ASEBA Preschool Forms & Profiles (University of Vermont, Research Center for Children, Youth, & Families, 2000).

Pham, C. et al. Early life environmental factors associated with autism spectrum disorder symptoms in children at age 2 years: a birth cohort study. Autism.: Int. J. Res. Pract. 26 , 1864–1881 (2022).

Article Google Scholar

Kidokoro, K. et al. Association between CYP19A1 polymorphisms and sex hormones in postmenopausal Japanese women. J. Hum. Genet. 54 , 78–85 (2009).

Braun, P. R. et al. Genome-wide DNA methylation comparison between live human brain and peripheral tissues within individuals. Transl. Psychiatry 9 , 47 (2019).

Kundakovic, M. et al. DNA methylation of BDNF as a biomarker of early-life adversity. Proc. Natl Acad. Sci. USA 112 , 6807–6813 (2015).

Panickar, K. S., Guan, G., King, M. A., Rajakumar, G. & Simpkins, J. W. 17beta-estradiol attenuates CREB decline in the rat hippocampus following seizure. J. Neurobiol. 33 , 961–967 (1997).

Solum, D. T. & Handa, R. J. Estrogen regulates the development of brain-derived neurotrophic factor mRNA and protein in the rat hippocampus. J. Neurosci. 22 , 2650–2659 (2002).

EPA USEPAU. Bisphenol A; CASRN 80-05-7. Integrated Risk Information System (IRIS) Chemical Assessment Summary (National Center for Environmental Assessment, 1988).

U.S. Food & Drug Administration. Bisphenol A (BPA): Use in Food Contact Application (U.S. Food & Drug Administration, 2023). [cited 2024 Jul]. Available from: https://www.fda.gov/food/food-additives-petitions/bisphenol-bpa-use-food-contact-application .

Authority, E. F. S. Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to 2, 2‐BIS (4‐HYDROXYPHENYL) PROPANE. EFSA J. 5 , 428 (2007).

Semple, B. D., Dixit, S., Shultz, S. R., Boon, W. C. & O’Brien, T. J. Sex-dependent changes in neuronal morphology and psychosocial behaviors after pediatric brain injury. Behav. Brain Res. 319 , 48–62 (2017).

Soma, M. et al. Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J. Comp. Neurol. 513 , 113–128 (2009).

Fisher, C. R., Graves, K. H., Parlow, A. F. & Simpson, E. R. Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc. Natl Acad. Sci. USA 95 , 6965–6970 (1998).

Hill, R. A. et al. Estrogen deficient male mice develop compulsive behavior. Biol. Psychiatry 61 , 359–366 (2007).

Avino, T. A. et al. Neuron numbers increase in the human amygdala from birth to adulthood, but not in autism. Proc. Natl Acad. Sci. USA 115 , 3710–3715 (2018).

Cai, S., Wang, X., Yang, F., Chen, D. & Huang, L. Differences in brain structural covariance network characteristics in children and adults with autism spectrum disorder. Autism Res. 14 , 265–275 (2021).

Keshavarzi, S., Sullivan, R. K. P., Ianno, D. J. & Sah, P. Functional properties and projections of neurons in the medial amygdala. J. Neurosci. 34 , 8699–8715 (2014).

Alia-Klein, N. et al. Human cognitive ability is modulated by aromatase availability in the brain in a sex-specific manner. Front Neurosci. 14 , 565668 (2020).

Biegon, A. et al. Relationship of estrogen synthesis capacity in the brain with obesity and self-control in men and women. Proc. Natl Acad. Sci. USA 117 , 22962–22966 (2020).

Sato, W. & Uono, S. The atypical social brain network in autism: advances in structural and functional MRI studies. Curr. Opin. Neurol. 32 , 617–621 (2019).

Herrington, J. D., Miller, J. S., Pandey, J. & Schultz, R. T. Anxiety and social deficits have distinct relationships with amygdala function in autism spectrum disorder. Soc. Cogn. Affect Neurosci. 11 , 907–914 (2016).

Krukoff, T. L. C-fos expression as a marker of functional activity in the brain. in Cell Neurobiology Techniques. Neuromethods . Vol. 33 213–230 (Humana Press, 1999).

Stoner, R. et al. Patches of disorganization in the neocortex of children with autism. N. Engl. J. Med. 370 , 1209–1219 (2014).

Anthoni, H. et al. The aromatase gene CYP19A1: several genetic and functional lines of evidence supporting a role in reading, speech and language. Behav. Genet. 42 , 509–527 (2012).

Wang, J. et al. Resting state EEG abnormalities in autism spectrum disorders. J. Neurodev. Disord. 5 , 24 (2013).

Matuszczak, E., Komarowska, M. D., Debek, W. & Hermanowicz, A. The impact of Bisphenol A on fertility, reproductive system, and development: a review of the literature. Int. J. Endocrinol. 2019 , 4068717 (2019).

Moutsatsou, P. et al. Fatty acids derived from royal jelly are modulators of estrogen receptor functions. PLoS ONE 5 , e15594 (2010).

Rubin B. S. Bisphenol A: An endocrine disruptor with widespread exposure and multiple effects. J. Steroid. Biochem. Mol. Biol . 127 , 27–34 (2011).

DeRosa, B. A. et al. Convergent pathways in idiopathic autism revealed by time course transcriptomic analysis of patient-derived neurons. Sci. Rep. 8 , 8423 (2018).

Andrews, S. V. et al. Case-control meta-analysis of blood DNA methylation and autism spectrum disorder. Mol. Autism 9 , 40 (2018).

Thongkorn, S. et al. Sex differences in the effects of prenatal bisphenol A exposure on autism-related genes and their relationships with the hippocampus functions. Sci. Rep. 11 , 1241 (2021).

VanderWeele, T. J. Explanation in causal inference: developments in mediation and interaction. Int J. Epidemiol. 45 , 1904–1908 (2016).

PubMed PubMed Central Google Scholar

Gerona, R., vom Saal, F. S. & Hunt, P. A. BPA: have flawed analytical techniques compromised risk assessments? Lancet Diabetes Endocrinol. 8 , 11–13 (2020).

Bolognesi, C. et al. Scientific Opinion on the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs: Executive summary. efsa J. (2015).

Authority E. F. S. Re-evaluation of the Risks to Public Health Related to the Presence of Bisphenol A (BPA) in foodstuffs (European Food Safety Authority, 2021).

EFSA Panel on Food Contact Materials E, et al. Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs. EFSA J. 21 , e06857 (2023).

Gao, H. et al. Cumulative risk assessment of phthalates associated with birth outcomes in pregnant Chinese women: a prospective cohort study. Environ. Pollut. 222 , 549–556 (2017).

Welch, C., Mulligan, K. & Does Bisphenol, A. Confer risk of neurodevelopmental disorders? what we have learned from developmental neurotoxicity studies in animal models. Int J. Mol. Sci. 23 , 2894 (2022).

Stein, T. P., Schluter, M. D., Steer, R. A., Guo, L. & Ming, X. Bisphenol A exposure in children with autism spectrum disorders. Autism Res. 8 , 272–283 (2015).

Rudel, R. A. et al. Food Packaging and Bisphenol A and Bis(2-Ethyhexyl) Phthalate Exposure: Findings from a Dietary Intervention. Environ. Health Perspect. 119 , 914–920 (2011).

Geueke, B. et al. Systematic evidence on migrating and extractable food contact chemicals: Most chemicals detected in food contact materials are not listed for use. Criti. Rev. Food Sci. Nutrition 63, 9425–9435 (2022).

Arbuckle, T. E. et al. Exposure to free and conjugated forms of bisphenol a and triclosan among pregnant women in the MIREC cohort. Environ. Health Perspect. 123 , 277–284 (2015).

Caporale, N. et al. From cohorts to molecules: adverse impacts of endocrine disrupting mixtures. Science 375 , eabe8244 (2022).

Hameed, R. A., Ahmed, E. K., Mahmoud, A. A. & Atef, A. A. G protein-coupled estrogen receptor (GPER) selective agonist G1 attenuates the neurobehavioral, molecular and biochemical alterations induced in a valproic acid rat model of autism. Life Sci. 328 , 121860 (2023).

Heffernan, A. L. et al. Harmonizing analytical chemistry and clinical epidemiology for human biomonitoring studies. A case-study of plastic product chemicals in urine. Chemosphere 238 , 124631 (2020).

Burugupalli, S. et al. Ontogeny of circulating lipid metabolism in pregnancy and early childhood—a longitudinal population study. Elife 11 , e72779 (2022).

McCarthy, S. et al. A reference panel of 64,976 haplotypes for genotype imputation. Nat. Genet. 48 , 1279–1283 (2016).

Ponsonby, A. L. et al. Prenatal phthalate exposure, oxidative stress-related genetic vulnerability and early life neurodevelopment: a birth cohort study. Neurotoxicology 80 , 20–28 (2020).

Wang, Y. et al. A methodological pipeline to generate an epigenetic marker of prenatal exposure to air pollution indicators. Epigenetics 17 , 32–40 (2022).

Perera, F. et al. Prenatal bisphenol a exposure and child behavior in an inner-city cohort. Environ. Health Perspect. 120 , 1190–1194 (2012).

Hunt, P. A., Vom Saal, F. S., Stahlhut, R. & Gerona, R. BPA and risk assessment—Authors’ reply. Lancet Diabetes Endocrinol. 8 , 271–272 (2020).

Burke James, F., Sussman Jeremy, B., Kent David, M. & Hayward Rodney, A. Three simple rules to ensure reasonably credible subgroup analyses. BMJ 351 , h5651 (2015).

Ponsonby, A.-L. Reflection on modern methods: building causal evidence within high-dimensional molecular epidemiological studies of moderate size. Int J. Epidemiol. 50 , 1016–1029 (2021).

Team RC. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2013).

StataCorp. Stata Statistical Software: Release 14 (StataCorp LP, 2015).

Thongkorn, S. et al. Investigation of autism-related transcription factors underlying sex differences in the effects of bisphenol A on transcriptome profiles and synaptogenesis in the offspring hippocampus. Biol. Sex. Differ. 14 , 8 (2023).

Nadler, J. J. et al. Automated apparatus for quantitation of social approach behaviors in mice. Genes, brain, Behav. 3 , 303–314 (2004).

Moy, S. S. et al. Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes, Brain, Behav. 3 , 287–302 (2004).

Champagne, D. L. et al. Maternal care and hippocampal plasticity: evidence for experience-dependent structural plasticity, altered synaptic functioning, and differential responsiveness to glucocorticoids and stress. J. Neurosci. 28 , 6037–6045 (2008).

Wang, I. T., Reyes, A. R. & Zhou, Z. Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation. Neurobiol. Dis. 58 , 3–12 (2013).

Kelly, E. A., Opanashuk, L. A. & Majewska, A. K. The effects of postnatal exposure to low-dose bisphenol-A on activity-dependent plasticity in the mouse sensory cortex. Front. Neuroanat. 8 , 117 (2014).

Sholl, D. A. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87 , 387–406 (1953).

ADS CAS PubMed PubMed Central Google Scholar

Allen Institute for Brain Science AMBA. https://mouse.brain-map.org/static/atlas (2004).

Tan, H. O. et al. Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy. Proc. Natl Acad. Sci. USA 104 , 17536–17541 (2007).

Unger, E. K. et al. Medial amygdalar aromatase neurons regulate aggression in both sexes. Cell Rep. 10 , 453–462 (2015).

Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30 , 923–930 (2013).

Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11 , R25 (2010).

Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41 , e108 (2013).

Harrow, J. et al. GENCODE: producing a reference annotation for ENCODE. Genome Biol. 7 , S4.1–9 (2006).

Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26 , 139–140 (2010).

Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat. Protoc. 4 , 1184–1191 (2009).

Wu, T. et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation 2 , 100141 (2021).

CAS PubMed PubMed Central Google Scholar

Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102 , 15545–15550 (2005).

The Gene Ontology Consortium. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 47 , D330–d338 (2019).

Santos, K. B., Guedes, I. A., Karl, A. L. M. & Dardenne, L. E. Highly flexible ligand docking: benchmarking of the DockThor Program on the LEADS-PEP protein–peptide data set. J. Chem. Inf. Model 60 , 667–683 (2020).

Leyrat, C., Morgan, K. E., Leurent, B. & Kahan, B. C. Cluster randomized trials with a small number of clusters: which analyses should be used? Int J. Epidemiol. 47 , 321–331 (2018).

Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30 , 207–210 (2002).

Xiao, X. et al. Bisphenol AP is anti-estrogenic and may cause adverse effects at low doses relevant to human exposure. Environ. Pollut. 242 , 1625–1632 (2018).

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Acknowledgements

The authors thank the BIS participants for their generous contribution to this project. The authors also thank current and past cohort staff. The establishment work and infrastructure for the BIS was provided by the Murdoch Children’s Research Institute, Deakin University, and Barwon Health, supported by the Victorian Government’s Operational Infrastructure Program. We thank all the children and families participating in the study, and the BIS fieldwork team. We acknowledge Barwon Health, Murdoch Children’s Research Institute, and Deakin University for their support in the development of this research. We thank Dr Shanie Landen for statistical advice, and Alex Eisner for independent statistical review of the analyses in the manuscript. We thank Soumini Vijayay and Kristie Thompson for human BPA lab measurement and Dr Steve Cheung for assistance preparing the primary cortical culture. We thank Chitra Chandran, Georgia Cotter, Stephanie Glynn, Oliver Wood and Janxian Ng for manuscript preparation. Manuscript editor Julian Heng (Remotely Consulting, Australia) provided professional editing of this article. This multimodal project was supported by funding from the Minderoo Foundation. Funding was also provided by the National Health and Medical Research Council of Australia (NHMRC), the NHMRC-EU partnership grant for the ENDpoiNT consortium, the Australian Research Council, the Jack Brockhoff Foundation, the Shane O’Brien Memorial Asthma Foundation, the Our Women’s Our Children’s Fund Raising Committee Barwon Health, The Shepherd Foundation, the Rotary Club of Geelong, the Ilhan Food Allergy Foundation, GMHBA Limited, Vanguard Investments Australia Ltd, and the Percy Baxter Charitable Trust, Perpetual Trustees, Fred P Archer Fellowship; the Scobie Trust; Philip Bushell Foundation; Pierce Armstrong Foundation; The Canadian Institutes of Health Research; BioAutism, William and Vera Ellen Houston Memorial Trust Fund, Homer Hack Research Small Grants Scheme and the Medical Research Commercialisation Fund. This work was also supported by Ms. Loh Kia Hui. This project received funding from a NHMRC-EU partner grant with the European Union’s Horizon 2020 Research and Innovation Programme, under Grant Agreement number: 825759 (ENDpoiNTs project). This work was also supported by NHMRC Investigator Fellowships (GTN1175744 to D.B., APP1197234 to A.-L.P., and GRT1193840 to P.S.). The study sponsors were not involved in the collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.

Author information

Nhi Thao Tran

Present address: The Ritchie Centre, Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, Australia

These authors contributed equally: Christos Symeonides, Kristina Vacy.

These authors jointly supervised this work: Anne-Louise Ponsonby, Wah Chin Boon.

Authors and Affiliations

Minderoo Foundation, Perth, Australia

Christos Symeonides

Murdoch Children’s Research Institute, Parkville, Australia

Christos Symeonides, Toby Mansell, Martin O’Hely, Boris Novakovic, David Burgner, Mimi L. K. Tang, Richard Saffery, Peter Vuillermin, Fiona Collier, Anne-Louise Ponsonby, Sarath Ranganathan, Lawrence Gray & Anne-Louise Ponsonby

Centre for Community Child Health, Royal Children’s Hospital, Parkville, Australia

Christos Symeonides, Sarath Ranganathan & Anne-Louise Ponsonby

The Florey Institute of Neuroscience and Mental Health, Parkville, Australia

Kristina Vacy, Sarah Thomson, Sam Tanner, Hui Kheng Chua, Shilpi Dixit, Jessalynn Chia, Nhi Thao Tran, Sang Eun Hwang, Feng Chen, Tae Hwan Kim, Christopher A. Reid, Anthony El-Bitar, Gabriel B. Bernasochi, Anne-Louise Ponsonby, Anne-Louise Ponsonby & Wah Chin Boon

School of Population and Global Health, The University of Melbourne, Parkville, Australia

Kristina Vacy

The Hudson Institute of Medical Research, Clayton, Australia

Hui Kheng Chua & Yann W. Yap

Department of Pediatrics, The University of Melbourne, Parkville, Australia

Toby Mansell & David Burgner

School of Medicine, Deakin University, Geelong, Australia

Martin O’Hely, Boris Novakovic, Chloe J. Love, Peter D. Sly, Peter Vuillermin, Fiona Collier & Lawrence Gray

Columbia Center for Children’s Environmental Health, Columbia University, New York, NY, USA

Julie B. Herbstman, Shuang Wang & Jia Guo

Department of Environmental Health Sciences, Columbia University, New York, NY, USA

Julie B. Herbstman

Department of Biostatistics, Columbia University, New York, NY, USA

Shuang Wang & Jia Guo

Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia

Kara Britt & Vincent R. Harley

Breast Cancer Risk and Prevention Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia

Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia

Faculty Medicine, Dentistry & Health Sciences, University of Melbourne, Parkville, Australia

Gabriel B. Bernasochi, Lea M. Durham Delbridge, Mimi L. K. Tang, Leonard C. Harrison & Sarath Ranganathan

Sex Development Laboratory, Hudson Institute of Medical Research, Clayton, Australia

Vincent R. Harley & Yann W. Yap

Departments of Paediatrics and Community Health Sciences, The University of Calgary, Calgary, Canada

Deborah Dewey

Barwon Health, Geelong, Australia

Chloe J. Love, Peter Vuillermin, Fiona Collier & Lawrence Gray

Department of General Medicine, Royal Children’s Hospital, Parkville, Australia

David Burgner

Department of Pediatrics, Monash University, Clayton, Australia

Child Health Research Centre, The University of Queensland, Brisbane, Australia

Peter D. Sly

WHO Collaborating Centre for Children’s Health and Environment, Brisbane, Australia

Queensland Alliance for Environmental Health Sciences, The University of Queensland, Brisbane, Australia

Jochen F. Mueller

Monash Krongold Clinic, Faculty of Education, Monash University, Clayton, Australia

Nicole Rinehart

Centre for Developmental Psychiatry and Psychology, Monash University, Clayton, Australia

Bruce Tonge

School of BioSciences, Faculty of Science, The University of Melbourne, Parkville, Australia

Wah Chin Boon

Walter and Eliza Hall Institute, Parkville, Australia

Leonard C. Harrison

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the BIS Investigator Group

, Toby Mansell
, Martin O’Hely
, David Burgner
, Mimi L. K. Tang
, Peter D. Sly
, Richard Saffery
, Jochen F. Mueller
, Peter Vuillermin
, Fiona Collier
, Anne-Louise Ponsonby
, Leonard C. Harrison
, Sarath Ranganathan
& Lawrence Gray

Contributions

Conceptualization—laboratory experiments: W.C.B., N.R., B.T., L.M.D.D. Laboratory experiments and analysis: W.C.B., K.V., S.D., H.K.C., J.C., F.C., CR, T.K., G.B.B., A.E.-B., S.E.H., N.T.T., K.B. Supervision of lab data collection: W.C.B., K.V., S.D., C.R. Laboratory statistical analysis: W.C.B., K.V., F.C., C.R., S.Th., V.H., Y.W.Y. Design and conduct of the Barwon Infant Study: C.S., A.-L.P., P.V., D.B., P.S., C.L., M.L.K.T., BIS Investigator Group. Design, conduct and analysis of the CCCEH-MN study: J.B.H., S.W., J.G. Design and conduct of BPA study measures in BIS: J.M., C.S., A.-.L.P. Design, conduct, and analysis of gene methylation studies: S.Ta., B.N., T.M., R.S., D.D., A.-L.P. Human studies statistical analysis: C.S., S.Th., A.-.L.P., S.Ta., K.V., M.O.H. Writing—reports and original draft: C.S., K.V., S.Th., S.Ta., A.-.L.P., W.C.B. Writing—editing: all authors. Results interpretation: all authors. Kara Britt did the laboratory experiment—estrogen pellet implantation.

Corresponding author

Correspondence to Wah Chin Boon .

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Competing interests.

W.C.B. is a co-inventor on ‘Methods of treating neurodevelopmental diseases and disorders’, USA Patent No. US9925163B2, Australian Patent No. 2015271652. This has been licensed to Meizon Innovation Holdings. A.-L.P. is a scientific advisor and W.C.B. is a board member of the Meizon Innovation Holdings. The remaining authors declare no competing interests.

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Symeonides, C., Vacy, K., Thomson, S. et al. Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype. Nat Commun 15 , 6367 (2024). https://doi.org/10.1038/s41467-024-48897-8

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DOI : https://doi.org/10.1038/s41467-024-48897-8

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The elevated blood pressure (BP) and lower cardiac autonomic modulation (CAM) are associated with higher morbidity mortality risk among older adults. Although exercise is an important intervention for cardiovascular promotion, it is unclear whether combat sports training could benefit cardiovascular outcomes as much as autonomic in this population. This study compared the effects of 12 weeks of Muay Thai (MT) training against functional training (FT) on CAM and hemodynamic parameters in older adults.

The sample consisted of 50 older adults (41 women; 66.0 ± 5.3 years old), who were equaly randomized into FT ( n = 25) and MT ( n = 25) intervention groups. CAM was measured by 30-min rest heart rate variability. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and resting heart rate (RHR) were measured using an automatic oscillometric device. Pulse pressure (PP) and the double product (DP) were also calculated. The interventions were carried out three times a week, with 60-min length per session, during 12 consecutive weeks. The intensity of the interventions was measured using the subjective perception of exertion scale and by accelerometer. Two-factor repeated measures analysis of covariance was used for groups comparison, considering intervention group and body mass as factors. The 95% confidence interval of the difference (95%CIdif) was also calculated and the effect size was measured using partial eta squared (η 2 p ).

CAM indices did not show significant changes across moments and intervention groups. In hemodynamic parameters, only in DBP was there an effect of the moment (F 1,39 = 8.206; P = 0.007; η 2 p = 0.174, large) and interaction effect between group*moment (F 1,39 = 7.950; P = 0.008; η 2 p = 0.169, large). Specifically, the MT group at the post-training moment showed lower DBP ( P = 0.010; 95%CI dif = -13.3; -1.89) in relation to the FT group. Furthermore, the MT group showed a decrease in DBP during training ( P = 0.002; 95%CI dif = -10.3; -2.6). Also, an increase in training intensity was also found over the 12 weeks in FT, with no difference between the groups.

After 12 weeks of MT practice there was a reduction in DBP compared to FT in older adults.

Trial registration

NCT03919968 Registration date: 01/02/2019.

Peer Review reports

Introduction

The autonomic nervous system plays an important role in cardiac and vascular adaptations [ 1 ]. Its behavior can be tracked through the analysis of cardiac autonomic modulation, which reflects global, sympathetic or parasympathetic modulations [ 2 ]. This indicator is important, since low cardiac autonomic modulation has been associated with a higher prevalence of mortality [ 3 ]. One of the characteristics that can contribute to the decrease in cardiac autonomic modulation is aging. A study of South Africans over the age of fifty found that low cardiac autonomic modulation was associated with a greater likelihood of mortality [ 4 ]. Furthermore, aging has been associated with other cardiovascular risk factors such as high blood pressure [ 5 , 6 ].

Low cardiac autonomic modulation and hypertension are also associated with worse quality of life, in addition to being associated with increased drug treatment [ 7 , 8 , 9 ]. In this sense, health promotion strategies that can increase cardiac autonomic modulation and reduce blood pressure values should be encouraged. Among the different types of strategies for this purpose, physical activity stands out. Some studies have shown positive effects of physical activity on cardiovascular parameters in older adults [ 10 , 11 , 12 ] and cardiac autonomic modulation is greater in active older adults when compared to insufficiently active older adults [ 13 ].

An alternative form of physical activity with this objective may be combat sports, which are intermittent and can help improve cardiorespiratory fitness [ 14 , 15 ]. Furthermore, combat sports, as long as they are adapted to the characteristics of older adults, following basic training principles (for example, progressive overload), with moderate to vigorous intensity and a frequency of two or three weekly 60-min sessions, improve physical health, functional, physiological and psycho-emotional of older adults [ 16 ]

Among the combat sports modalities is Muay Thai (MT), a percussion modality with an intermittent or interval character [ 17 ], which has already been shown to be beneficial in the pediatric population on body composition [ 18 ], aerobic fitness [ 19 ] and cardiovascular parameters, both acutely [ 20 ] and after 16 weeks of training [ 21 , 22 ]. Better physical fitness of healthy women practicing MT compared to non-practitioners was also observed [ 23 ]. This type of training is an alternative for practicing physical activity in older adults that should be tested, as they are generally carried out in groups and the exercises are divided into pairs, which can promote an increase in older adults’ adherence to exercise, since only 22% of them have participated in regular activities twice a week [ 24 ]. Even so, interval or intermittent training has been a tool that promotes improvement in the control of cardiac autonomic modulation [ 25 ], especially those of higher intensity [ 26 ].

Another training modality which has this intervallic character, and has also been developed with older adults is Functional Training (FT). FT is similar to the daily activities of the older adults, seeking adaptations that are more comfortable and effective to improve physical capabilities and health functionality in general [ 27 , 28 , 29 , 30 , 31 ]. FT appears to promote beneficial effects on cardiac autonomic modulation, characterized by increased parasympathetic modulation, in postmenopausal women [ 32 ]. Choi et al. [ 33 ] analyzed the effects of 12 weeks of FT on blood pressure and pulse pressure in healthy older adults, and observed a reduction in these variables in the group that practiced FT when compared to the control group.

A systematic review [ 34 ] indicated that studies on combat sports and the cardiovascular health of older adults mostly used a cross-sectional design. In turn, longitudinal studies on this topic compared an intervention group with a non-intervention group (control) [ 34 ]. Therefore, there is a need for studies in which intervention using combat sports modalities is compared with more traditional approaches. Another review [ 35 ] highlights that, despite the important findings of some studies on the effects of combat sports on cardiac autonomic modulation in older adults, the lack of randomization may be a limiting factor related to the composition of the sample, indicating the need for studies with this care in their designs. A short review presented the great difficulty of measuring training intensity with combat sports modalities due mainly to physical confrontation, intervals and plyometrics [ 36 ] therefore, studies are needed to better determine the external (e.g., via accelerometry) and internal (e.g., via subjective perception of effort) intensity of combat sports sessions.

Therefore, the present study aimed to explore these gaps of: i) not using just a control group; ii) low methodological rigor when detailing the intervention, not randomizing the sample, and the majority with cross-sectional studies; iii) more modalities and practice possibilities for the elderly to increase adherence to physical activity; iv) without controlling the intensity; using a longitudinal and randomized design, comparison of a combat sport modality with another type of physical exercise program more traditional, and measuring the external (e.g., via accelerometry/ physiological) and internal loads (e.g., via subjective perception of effort/ sense) of the sessions. Thus, the objective of this clinical trial was to analyze the effects of MT compared to FT on cardiac autonomic modulation and hemodynamic parameters in older adults. The hypothesis was that the groups would have similar results throughout the program, with improvements in cardiovascular health parameters for both groups, indicating that MT could be as good as FT on the variables studied; Therefore, it is an alternative in exercise programs for this population.

Participants

Participants were recruited in the Presidente Prudente region through advertisements on social networks and local media, flyers in health centers, squares, churches or by appearing to seek care at the study locations. To be included, participants must: 1) Be between 60 and 89 years old; 2) Present a medical certificate confirming that the older adult was able to practice physical exercise. As exclusion criteria, participants could not: 1) Perform any other systematic exercise practice, in addition to regular physical activity, such as walking; 2) Having serious cardiovascular diseases such as myocardial infarction, or procedures such as saphenous bypass and pacemaker; 4) Use crutches, canes, walkers, wheelchairs or other similar utensils; 5) Present a self-report of mobility difficulties to carry out the intervention practice; 6) Present an error greater than 5% in the cardiac autonomic modulation tracing. Participants signed the informed consent form before data collection and the study was conducted in the second half of 2019.

After collecting data at the initial moment, the older adult who participated in this study were randomly distributed into one of two groups: practicing MT or FT. The randomization process was carried out by a researcher who was not part of this project (with the aim of blinding the sample allocation process) through a sequence of numbers generated on the website http://www.randomization.com , randomization was done by groups, and according to sex.

The sample allocation process was carried out secretly with opaque, sealed envelopes and following the numerical allocation sequence. In the first practice session, the envelope was opened in front of the older adult who participated in the project, informing them which group they would be included in. This entire process is in line with the recommendations of the Consolidated Standards of Reporting Trials (CONSORT) for randomized clinical trials [ 37 ].

The present study was approved by the FCT/UNESP Research Ethics Committee (CAAE: 91923318.7.0000.5402), and was registered and presented publicly on the clinical trials platform clinicaltrials.gov/ with protocol number NCT03919968 in 01/02/2019 with the name “Effect of Muay Thai vs. Circuit Training on the Cardiovascular Health of Elderly”, following the standards proposed by CONSORT.

Sample calculation

Due to the lack of research in the literature involving MT and cardiovascular parameters in older adults with the necessary information to carry out the sample calculation, we opted for studies that investigated Tai Chi Chuan. The sample calculation was carried out based on the study of Tsai et al. [ 38 ] which compared the blood pressure of elderly people practicing Tai Chi Chuan with a control group. The variable diastolic blood pressure was used with a difference of 8 mmHg to be detected and a standard deviation of 8 mmHg, a test power of 80% and a significance of 5%. Adding 20% of possible sample losses, the sample size was at least 36 older adults (18 older adults for each group).

Primary outcomes

Cardiac autonomic modulation.

To evaluate cardiac autonomic modulation, the older adults were instructed that at least 12 h before carrying out the experimental protocol they should not use alcoholic and/or stimulant drinks/foods, such as coffee, tea and chocolates, so that there would be no direct influence on cardiac autonomic behavior at the time of collection [ 39 ]. Cardiac autonomic modulation was assessed using heart rate variability. For this purpose, the heart rate was recorded beat by beat for 30 min using a heart rate monitor (Polar Electro Oy, Kempele, Finland—model V800) positioned in the distal third of the sternum. The intervals between heartbeats (RR intervals) obtained by the heart rate monitor with a sampling frequency of 1000 Hz53 were exported by the Polar Flow Web Service ( https://flow.polar.com/ ) and saved in text format for later analysis by Kubios HRV® software (Biosignal Analysis and Medical Image Group, Department of Physics, University of Kuopio, Finland).

For analysis, the RR interval time series was initially subjected to preprocessing to eliminate premature ectopic beats and artifacts, with moderate filtering and interval correction with the cubic spline interpolation method. After pre-processing, the data were analyzed and subsequently visually screened to verify the absence of artifacts or cardiac arrhythmias that could interfere with the analyses [ 40 ]. Only RR interval series with more than 95% sinus beat will be included in the study [ 41 ]. 1000 consecutive RR intervals were used for analysis, taken from the stretch of greatest signal stability.

For data analysis, the HRV software Kubios HRV 2.0 Analysis Software for Windows (Analysis Group, Department of Applied Physics, University of Kuopio, Finland) was used [ 42 ] and the mean heart rate (HR mean), the average RR intervals (RR mean) and the linear indices of the time and frequency domains were extracted. In the time domain, the following were calculated: standard deviation of the RR intervals (SDNN), the square root of the mean of the square of the differences between adjacent normal RR intervals (rMSSD) and the percentage of adjacent RR intervals with a difference in duration greater than 50 ms (pNN50). In the frequency domain, the indices of: high frequency (HF), low frequency (LF) and the LF/HF ratio were calculated. Quantitative analysis of the Poincaré plot was also performed, obtaining the indices: SD1 (standard deviation of instantaneous beat-to-beat variability), SD2 (long-term standard deviation of continuous RR intervals), and the SD1/ SD2. Furthermore, a qualitative analysis of the plot was carried out, through the analysis of the figures formed by the attractor, as proposed by Tulppo et al. [ 43 ].

Blood pressure and heart rate

Systolic blood pressure, diastolic blood pressure and heart rate were measured using an Omron automatic oscillometric device (Model HEM-742, Japan). For these measurements, the individuals remained lying down at rest for five minutes, with their legs uncrossed, and were asked not to talk during this time. These procedures are in line with the American Society of Cardiology [ 44 ]. These measurements were taken again 10 to 15 min after the initial measurement. For analysis, the average of these two measurements was used.

Secondary outcomes

Pulse pressure and double product.

Pulse pressure was calculated as the difference between mean systolic blood pressure and mean diastolic blood pressure [ 45 ]. The double product is an estimated measure of myocardial oxygen consumption. Its value was obtained by multiplying the average heart rate by the average systolic blood pressure [ 46 ].

Anthropometric measurements

Body mass was measured using a digital scale (Plenna, Brazil) and height using a fixed stadiometer (Sanny, Brazil). Using these values, body mass index was calculated by dividing body mass by the square of height [ 47 ]. The older adults were evaluated barefoot and wearing light clothing.

Intervention

For intervention, participants were randomly divided into two groups: MT group and FT group. In both groups, the intervention lasted 12 weeks, carried out three times a week, on non-consecutive days, lasting approximately 60 min. The initial two weeks were dedicated to adaptation and familiarization, both with the exercise protocol and with the subjective perceived exertion scale, used to determine exercise intensity. The 60 min for both groups were divided into 10 min of general exercises, 40 min of specific exercises and 10 min of playful activities and simulations of what was learned during the session. Each day of the week focused on a specific part of the body. The first day of the week was focused on upper limb exercises, the second day on lower limbs and the last for the whole body. All activities respected the biological individuality of each older adult person and were always carried out in pairs with older adult of similar size and weight, in both groups. Furthermore, throughout the intervention period there was an increase in monthly overload, through an increase in the time spent performing the exercises, a reduction in active rest and passive rest, as well as an increase in the difficulty of the exercises (mainly in the case of MT) and increased weights (mainly in the case of FT). Furthermore, these variables were considered in the interventions [ 48 ]:

Organization of sessions (periodized): adaptation (2 weeks);

Types and order of exercises: first session of the week for upper limbs; second session for lower limbs and third session for full body. Size of the exercises was it broader first and more localized later;

Resistance options used in FT: stick, dumbells, elastic, medicine ball, string, body weight;

Types of contraction: all exercises were isotonic;

Contraction speed (cadence): it was moderate so that the participant could correctly execute the movement with the degree of intensity within the expected range;

Range of motion: was complete or adapted according to the participant’s joint conditions;

Rest between sets: 1 min to 30 s in MT and 1 min to 50 s in FT according to the training protocol in the supplementary figures;

Strategies for controlling intensity: use of the Borg scale, accelerometer and participants' self-report;

Volume: number of exercises, number of sets, and number of repetitions as described in training protocol in the supplementary figures.

Intensity was controlled using the subjective perception of exertion scale by Borg et al. [ 49 ] from six (minimum effort) to 20 (maximum effort) at the end of every training session, however, to standardize with the accelerometer, we only present one week (the three days of training) at the end of each month of training. The intensity was maintained between 12 and 13, which is classified as moderate. Furthermore, training intensity was measured using an Actigraph GT3X accelerometer (ActiGraph, LLC, Pensacola, FL, USA) for one week (the three days of training) at the end of each month of training, strengthening intensity control. The accelerometer data was filtered, digitized and adjusted to a frequency of 30Hz with an interval (epoch) of 60 s. The accelerometer was analyzed using ActiLife 6 software (ActiGraph, LLC, Pensacola, FL, USA).

Muay Thai training

The MT group, in the initial 10 min of general exercises, performed stretching and physical warm-ups specific to the MT modality. In turn, throughout the 40 min of specific exercises, seven exercises were given per session, adapted for the elderly, according to the part of the body in focus that day. On Monday, exercises were carried out with punches (jab, straight, cross, hook and elbows), dodges and defenses. On Wednesday there were kicks (circular thigh and frontal height), knees and defenses, both stationary and in movement. On Friday, exercises were performed combined with upper and lower limb movements in the same exercise. Protections such as shin guards, gloves, Thai pads, gauntlets, rib protectors and head protectors were always used, prioritizing the safety of the elderly when carrying out activities. In the first month, the seven exercises per day were performed for two minutes, with one older adults always receiving the blows, while the other struck. There was a recovery time of one minute when changing exercises, making it easy to change equipment (Supplementary Figure S1) and after that time acting as a partner, receiving the blows for two minutes. In the second month, there were two and a half minutes of execution, with 30 s of recovery, two and a half minutes acting as a partner and maintaining the seven exercises. In the last month, it was the same protocol as the second month, but with eight exercises and an increase in their difficulty and complexity. The final 10 min of playful activities and simulations of the exercises learned were through activities that included some developed skills, as well as fighting simulation, through the sparing technique (a person who is not an opponent, but who helps to improve the technique practitioner).

Functional training

The FT group, during the 10 min of general exercises, performed stretching and physical warm-ups. In turn, throughout the 40 min of specific exercises, 13 exercises were given per day adapted for the elderly, according to the part of the body in focus that day. On Monday they performed the following exercises: barbell curls with a stick, lateral raises with dumbbells, triceps curls on an elastic band, shoulder presses with dumbbells, front and diagonal raises with a stick, high rows with a stick, front raises with elbow flexion with a medicine ball on the chair, open push-up on the wall, barbell curl with dumbbells, shoulder coordination with medicine ball (raising the ball above the shoulder), crucifix with elastic, inverted curl with elastic and abdominal standing on the side with dumbbells. On Wednesday the exercises were: squats sitting on a chair, marching on the agility ladder, supporting sumo squats, climbing on the step, walking on the line, lateral leg raise supporting, seated knee extension with shin guards, trunk semi-flexion (good day) with a stick, unilateral hip extension, maintained single-leg support exercise (balance), plantar flexion sitting with weight on your lap, sit-ups while standing, raising the leg and coordinating with the arm stationary. And on Friday: marching on the line with holding objects, climbing the step with trunk rotation, getting into the hula hoop, squatting, picking up and lifting above the shoulder, squats on the chair with front pole raise, marching with high knees and arm coordination, touching the cones with the hand, sitting on the chair and going around the cone (agility), trunk rotation with a medicine ball (passing the ball to the partner), touching the feet with a medicine ball (tying shoelaces), arm coordination with standing running and abdominal with squats.

In the first month there were two series (the older adult performed the same exercise twice), of 40 s of execution for each exercise with 20 s of active rest between them (to change exercises) and one minute of passive rest between series (Supplementary Figure S2). In the first month there were two series (the older adult performed the same exercise twice), of 40 s of execution for each exercise with 20 s of active rest between them (to change exercises) and one minute of passive rest between series. In the second month, there were three series (the older adult performed the same exercise three times), of 50 s of execution for each exercise with 10 s of active rest between them (to change exercises) and 50 s of passive rest between series. In the last month, the same protocol as in the second month was maintained, however the exercises had an increase in difficulty, complexity, dual task insertion and those with dumbbells, elastic bands, shin guards, among others, increased weight/resistance. The older adults were in pairs at each station (due to the number of older adults, to be similar to the MT who was in pairs and maintain interaction), but they performed the same exercises together, often facilitating the execution, such as trunk rotation. with medicine ball (passing the ball to the partner). The final 10 min of playful activities and simulations of the exercises learned were through activities that included some developed skills, such as “Dead Alive” in the lower limbs room or “Your master ordered” making movements that worked the entire body.

Statistical analysis

The Kolmogorov–Smirnov test was performed to verify data normality. The descriptive analysis was presented as mean and standard deviation or frequency (%). To check possible differences in the randomized groups at the beginning of training, the t-test for independent samples and Chi-square for frequencies were performed. Mauchly's test of sphericity with Greenhouse–Geisser correction when necessary. Analysis of variance (ANOVA; groups: MT vs. FT; moments: session of four, eight and 12 weeks) with repeated measures on the second factor was used to analyze and compare the intensity of a training session for each muscle group (upper limbs, lower limbs and global) at the end of each month. Furthermore, to analyze and compare the effects of MT and FT training on the older adults, analysis of covariance (ANCOVA; groups: MT vs. FT; moments: pre and post 12 weeks of training) was performed with repeated measures on the second factor adjusted by body mass that showed a difference between the groups at the initial moment. When differences in ANCOVA or ANOVA were detected, correction was applied using the Bonferroni post-roc test. The 95% confidence interval of the difference (95%CIdif) was also calculated for variables in which there was a significant effect of at least one factor. The effect size was measured using partial eta squared (η 2 p ) in which 0.01 was considered small, 0.06 medium and 0.14 large. [ 50 ]. The Statistical Package for the Social Sciences (SPSS) software version 25.0 was used for the analyses, GraphPad Prism version 8.0 for creating the graphs, Microsoft Excel version 16.0 for creating the plot graph and the significance established was p -value < 0.05.

The initial sample consisted of 50 older adults (41 women and 9 men) aged 66.04 ± 5.32 years, who were randomly divided into two groups: MT ( n = 25) and FT ( n = 25). At the initial moment, the groups were compared and no differences were observed between them, which can be considered a homogeneous sample. Among the older adults in MT, 60% ( n = 15) had high blood pressure, 4% ( n = 1) heart disease, 28% ( n = 7) diabetes, none smoked, 32% ( n = 8) consumed alcohol, 16% ( n = 4) were single, 44% ( n = 11) married, 16% ( n = 4) divorced/separated, 24% ( n = 6) widowed, 64% ( n = 16) of white ethnicity, none black, 8% ( n = 2) oriental and 28% ( n = 7) mixed race. In turn, the older adults in the FT were 56% ( n = 14) hypertensive (chi-square comparing the groups P = 0.774), 16% ( n = 4) diabetic ( P = 0.306), 12% ( n = 3) had heart disease ( P = 0.297), 4% ( n = 1) smoked ( P = 0.312), 32% ( n = 8) consumed alcoholic beverages ( P = 1.000), 8% ( n = 2) were single ( P = 0.384), 64% ( n = 16) married ( P = 0.156), 8% ( n = 2) divorced/separated ( P = 0.384), 20% ( n = 5) widowed ( P = 0.733), 68% ( n = 17) white ( P = 0.765), 4% ( n = 1) black ( P = 0.312), 4% ( n = 1) oriental ( P = 0.552) and 24% ( n = 6) mixed race ( P = 0.747).

During the intervention period, seven older adults left the study due to illness, surgery, excessive heat and/or logistical problems and two were excluded from the analyses, one for not having complete assessments and the other due to an error in the cardiac autonomic modulation tracing greater than 5% (18% sample loss). Thus, the final sample was composed of 41 older adults (9 men and 33 women), 19 in MT (5 men) and 22 in FT (4 men) (Fig. 1 ).

Sample flowchart based on CONSORT

The MT and FT groups were compared at the initial moment, considering only those who completed the study and a difference was observed only in the body mass variable ( t = -2.138; F 40 = 2.814; P = 0.039) (Table 1 ).

Table 2 presents the effects of MT and FT on cardiac autonomic modulation indexes adjusted by body mass at the initial moment. In the mean HR, no group effect was shown (F 1,39 = 1.009; p = 0.321; η 2 p = 0.025, small), nor the interaction (F 1,39 = 2.583; p = 0.116; η 2 p = 0.128, medium), but there was an effect of the moment (F 1,39 = 5.701; p = 0.022; η 2 p = 0.062, medium). However, when Bonferroni correction was applied, this difference was not confirmed. No statistical differences were observed in the other indices of cardiac autonomic modulation.

The intensity of FT and MT was analyzed at the end of each month using the subjective perception of effort scale according to the days of the week and the muscle group in focus, Monday upper limbs, Wednesday lower limbs and Friday -day global exercises working the whole body are presented in Fig. 2 . It can be seen that the intensity of the groups was similar and that there was an increase and/or maintenance of training intensity each month. In the upper limbs there was no effect of group (F 1,10 = 1.179; p = 0.303; η 2 p = 0.105, medium), nor the interaction (F 2,20 = 1.255; p = 0.307; η 2 p = 0.112, medium), but there was an effect of the moment (F 2,20 = 8.521; p = 0.002; η 2 p = 0.460, large). The CT showed an increase in intensity due to the subjective perception of effort in the upper limbs compared to the moment four weeks with 12 weeks ( P = 0.030; 95%CI dif = -4.1; -0.2). Globally there was no group effect (F 1,9 = 0.609; p = 0.455; η 2 p = 0.063, medium), nor the interaction (F 2,18 = 0.562; p = 0.580; η 2 p = 0.059, small), but there was an effect of the moment (F 2,18 = 3.608; p = 0.048; η 2 p = 0.286, large), however, it was not confirmed in the Bonferroni correction. There was also reinforcement of intensity control using the accelerometer and it can be observed that throughout the intervention, in both groups, the time spent in light physical activity decreased, moderate physical activity increased and, at times, vigorous and very intense physical activity vigorous (Supplementary Figure S3).

Description and comparison of the intensity of Functional Training and Muay Thai measured by subjective perception of effort according to the days of the week. Note: MT = Muay Thai; FT = functional training; SPE = subjective perception of effort; * indicates difference between moments

In the visual analysis of the Poincaré plot, although both groups showed a little more dispersion after 12 weeks of training, it was observed that the amplitude of the dispersion of the points in the RR intervals was similar in the FT and MT groups (Fig. 3 ).

Poincaré plot graph observed in the Muay Thai training group and Functional Training group. Note: Pre = initial moment; Post = moment after 12 weeks of intervention

The cardiovascular effects after 12 weeks of MT training intervention compared to FT were analyzed in the variables of systolic and diastolic blood pressure, pulse pressure, heart rate and double product. For diastolic blood pressure, there was no group effect (F 1,39 = 1.876; P = 0.179; η 2 p = 0.046, small), but there was an effect of the moment (F 1,39 = 8.206; P = 0.007; η 2 p = 0.174, large), as well as interaction effect between group and moment (F 1,39 = 7.950; P = 0.008; η 2 p = 0.169, large). Specifically, the MT group at the post-training moment had lower diastolic blood pressure ( P = 0.010; 95%CI dif = -13.3; -1.89) in relation to the FT group. Furthermore, the MT group showed a decrease in diastolic blood pressure during training ( P = 0.002; 95%CI dif = -10.3; -2.6). The double product showed no group effect (F 1,39 = 3.959; p = 0.054; η 2 p = 0.092, medium) and interaction (F 1,39 = 2.620; p = 0.114; η 2 p = 0.063, medium), but it had a moment effect (F 1,39 = 6.230; p = 0.017; η 2 p = 0.138, medium) Therefore, the MT group at the post-training moment presented a lower double product ( P = 0.029; 95%CI dif = -2647.0; -151.7) in relation to the FT group. No statistical differences were evident for the other hemodynamic parameters (Fig. 4 ).

Effect of Functional Training compared to Muay Thai training on systolic, diastolic and pulse blood pressure, resting heart rate and double product in older adults. Note: F = strength; η 2 p = partial eta squared; mmHg = millimeters of mercury; bpm = beats per minute; Pre = initial moment; Post = moment after 12 weeks of intervention; * indicates difference between moments; # indicates difference between groups

Among the main findings of the present study, a significant reduction in diastolic blood pressure was observed in the MT group and lower values post-training compared to FT. Furthermore, the double product of the MT post-intervention moment was smaller than that of the FT. An increase in training intensity was also found over the 12 weeks in FT, with no difference between the groups.

The World Health Organization has highlighted the importance of increasing intensities in weekly physical activity [ 51 ]. Previous studies have shown that moderate to vigorous physical intensity is linked to greater cardiac autonomic modulation [ 52 , 53 , 54 ]. Oliveira-Dantas et al. [ 55 ] in a study with elderly hypertensive women reported that the practice of exercise through resistance training, compared to control, after 10 weeks, contributed to increases in cardiac autonomic modulation. Another study with adult sedentary workers also showed improvements in cardiac autonomic modulation after 12 weeks of FT [ 28 ].

Despite presenting themselves as similar, previous studies with older adults [ 56 , 57 , 58 , 59 ] present an adaptation of dance movements that are used in traditional Muay Thai rituals, called Wai Kru, some present them as Thai boxing or Thai dance, but they are not fighting movements, they are dance. These showed benefits in balance and functional fitness parameters [ 56 , 57 , 58 , 59 ]. Considering our knowledge, only one study analyzed the effects of the Muay Thai combat sport modality with older adults and after 8 weeks of training it promoted benefits on balance and flexibility [ 60 ].

The present study did not find differences for other indexes indicative of autonomic modulation, which does not corroborate previous studies [ 32 , 61 ]. It is noteworthy that our study compared two different types of training, while the comparative group in other studies was the control, in which participants did not practice any type of physical exercise [ 32 , 61 ], which certainly contributed to the observed discrepancies. Furthermore, in addition to the difference in intervention time, the study of Rezende Barbosa et al. [ 62 ] was carried out only with postmenopausal women and, combined with FT, included aerobic exercises in the intervention, which may also have contributed to the difference in results in relation to those of the present study. Still, a determining factor in promoting benefits on cardiac autonomic modulation is training intensity [ 26 , 63 ]. Therefore, aging can reduce the performance of practitioners, which is why controlling the intensity of training is essential. As we can see, in general, the older adults did not reach high intensity in training, which may also have influenced our results.

Another important result of the present study is the maintenance of the Poincaré plot indices in the FT and MT groups. The SD1 index and the SD1/SD2 ratio have parasympathetic autonomic influence, while SD2 represents global variability [ 64 , 65 , 66 ]. Unlike the present study, Rezende Barbosa et al. [ 32 ], after 18 weeks of FT, they observed an increase in SD1, SD2 and SD1/SD2 ratios, with the increase in SD1 being significant when compared to the group. Perhaps, it is possible can attribute this lack of positive results to the time of intervention, since for autonomic modulation it may have been an insufficient time, given that the cardiovascular condition of elderly people is more compromised than younger individuals and, therefore, of slower progression and response. However, the analysis of the Poincaré plot in the present study allowed us to observe the maintenance of cardiac autonomic modulation in the MT and FT groups. This is an important result of reducing the harmful effects of aging on cardiac autonomic modulation.

In young populations, the practice of MT has already demonstrated benefits on cardiovascular parameters [ 20 , 21 , 22 ], however, there is no study on the practice of MT and cardiovascular parameters in older adults. The intermittent characteristic of MT may explain the significant reduction observed in diastolic blood pressure in present study. Despite different populations, middle-aged hypertensive adults, previous studies with 12-week intermittent exercise interventions improved myocardial function and reduced systolic and diastolic blood pressure [ 67 ]. The present study corroborates previous studies in adults with hypertension, in which the practice of Tai Chi resulted in a reduction in blood pressure compared to brisk walking, after three months of intervention [ 68 ]. Additionally, a meta-analysis indicated that Tai Chi significantly reduced systolic and diastolic blood pressure compared to the control group in adults with essential hypertension [ 69 ]. However, Tai Chi Chuan is considered a soft modality, with lighter movements, close to Yoga, so they are different modalities. Another aspect is that approximately 60% of the volunteers in the present study had high blood pressure, but their values were controlled and this may be one of the reasons why there was no reduction in SBP, only in diastolic blood pressure, since it is more difficult to reduce blood pressure values of normotensive individuals. Finally, because they are considered hard modalities (FT and MT), with movements of moderate to vigorous intensity, the older adults who signed up to participate in the interventions may have better cardiovascular health, whereas those with greater heart problems may be afraid of the characteristic of the modality, unlike if they were modalities like Tai Chi and Yoga, for example. However, it is a characteristic of the sample that could not be controlled, and could be considered a sample selection bias.

In the literature, the effects of FT on blood pressure are still controversial. Rezende Barbosa et al. [ 70 ] in a 12-week FT study with healthy women, no significant differences were observed in systolic and diastolic blood pressure. Nonetheless Choi et al. [ 33 ], after 12 weeks of FT, they observed a reduction in systolic and diastolic blood pressure in healthy older adults, while the control group increased these variables. It is important to highlight that in the aging process, the maintenance of cardiovascular parameters or a slight improvement are already considered beneficial effects, since in the older adults the tendency is to increase blood pressure due to changes resulting from the decrease in arterial compliance [ 71 ]. As mentioned previously, most previous studies compared the intervention with a sedentary control group (or one that did not receive any intervention), which increases the tendency for positive results in the intervention group (result bias), while ours compared two modalities in that (at least hypothetically) both would bring benefit, although they are different, both modalities showed similar results in this population.

Pulse pressure is a viable tool for measuring vascular aging and a good marker of cardiovascular risk in the elderly, as the higher the pulse pressure values, the greater the cardiovascular risk [ 72 ]. Furthermore, pulse pressure is an independent indicator of arterial stiffness and is more associated with cardiovascular events than systolic or diastolic blood pressure alone [ 73 ]. Previous studies such as Choi et al. [ 33 ] showed a reduction in pulse pressure after 12 weeks of FT when compared to the control group, which was not observed in the present study. This result may be due to the groups having similar results in this variable, not implying a difference between them. However, the absence of differences between the groups does not mean that both groups improved as expected, as the comparative analyzes did not show a significant effect of time (even if there was no group effect). Therefore, in some aspects it did not improve significantly pre vs. post (regardless of the intervention, which is different from expectations).

In our study, the double product was also analyzed, which is an index that indicates myocardial oxygen consumption or cardiac workload demand [ 74 , 75 ]. The double product of the MT post-intervention moment was smaller than the FT. The decrease in MT double product may originate from the reduction in myocardial oxygen demand and improvement in myocardial performance [ 74 , 75 ]. Therefore, practicing MT can improve the effectiveness of cardiac function with less energy use. Additionally, the effect of nitric oxide on reducing systolic blood pressure and improving heart rate function may also have contributed to this finding. In general, MT showed a tendency to improve the hemodynamic parameters of practitioners. This may have occurred due to the characteristic of the modality in which there are peaks of higher intensities within the session. Consequently, the use of pulsometers to control intensity during the session is suggested in future studies.

Combat sports modalities have specificities such as physical confrontation, frequent intervals and plyometrics, which limit the ways of monitoring the intensity of this type of training [ 36 ]. The present study advances in using accelerometry to complement the subjective perception of effort, since ratings of perceived effort are one of the most used and valid ways of monitoring and/or controlling the intensity of combat sports training [ 36 , 76 ]. However, it does not always have a high relationship with the physiological changes produced during physical exercise [ 77 ]. Studies have shown significant correlations between subjective perception of exertion, blood lactate and heart rate [ 36 ]. Even though these studies did not use an accelerometer, this equipment can provide physical activity intensity values (light, moderate, vigorous and very vigorous), with greater accuracy and precision than self-report measures or pedometers, for example [ 78 ].

Despite the relevance of the study, it is necessary to mention some limitations such as the lack of control of habitual physical activity and food intake, which may influence the variables analyzed. Another point is that it was not possible to blind the evaluators and volunteers, as this is a study that uses physical exercise programs as an intervention. Finally, the lack of use of pulsometers to control intensity during training sessions what could have caused older adults have not reached and maintained high intensity in training. As strengths, the study design stands out, which was a randomized clinical trial using two types of training, and intensity monitoring using the subjective perception of exertion scale and accelerometer. As practical applications of this study, healthcare professionals often provide recommendations for patients to exercise regularly to promote a healthy lifestyle and reduce the risk of chronic diseases. In this sense, MT can be recommended by health professionals for the older adults due to its ease of application, as long as it is adapted, as it is a dynamic physical activity and can be worked on in groups.

Thus, it is possible to conclude that 12 weeks of MT practice was able to reduce diastolic blood pressure post-training in older adults. Furthermore, MT showed lower values of diastolic blood pressure and double product in the post-training period of older adults compared to FT. An increase in training intensity was also found over the 12 weeks in FT, with no difference between the groups. However, it is recommended that future randomized clinical trials use and compare these training models, including a control group, to confirm these promising results.

Availability of data and materials

The data that supports the findings of this study are available from Bruna T. C. Saraiva (corresponding author) but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission from Bruna T. C. Saraiva.

Gibbons CH. Basics of autonomic nervous system function. Handb Clin Neurol. 2019;160:407–18.

Article PubMed Google Scholar

Catai AM, Pastre CM, de Godoy MF, da Silva E, de Takahashi ACM, Vanderlei LCM. Heart rate variability: are you using it properly? Standardisation checklist of procedures. Braz J Phys Ther. 2020;24:91–102.

Hämmerle P, Eick C, Blum S, Schlageter V, Bauer A, Rizas KD, et al. Heart rate variability triangular index as a predictor of cardiovascular mortality in patients with atrial fibrillation. J Am Heart Assoc. 2020;9:e016075.

Article PubMed PubMed Central Google Scholar

Koopman JJE, van Bodegom D, Maan AC, Li Z, Ziem JB, Westendorp RGJ, et al. Heart rate variability, but not heart rate, is associated with handgrip strength and mortality in older Africans at very low cardiovascular risk: A population-based study. Int J Cardiol. 2015;187:559–61.

Kohler IV, Sudharsanan N, Bandawe C, Kohler H-P. Aging and hypertension among the global poor—Panel data evidence from Malawi. PLOS Global Public Health. 2022;2:e0000600.

Tan JPH, Beilharz JE, Vollmer-Conna U, Cvejic E. Heart rate variability as a marker of healthy ageing. Int J Cardiol. 2019;275:101–3.

Djärv T, Wikman A, Lagergren P. Number and burden of cardiovascular diseases in relation to health-related quality of life in a cross-sectional population-based cohort study. BMJ Open. 2012;2:e001554.

Peacock E, Joyce C, Craig LS, Lenane Z, Holt EW, Muntner P, et al. Low medication adherence is associated with decline in health-related quality of life: results of a longitudinal analysis among older women and men with hypertension. J Hypertens. 2021;39:153–61.

Article CAS PubMed Google Scholar

Kanbara K, Morita Y, Hasuo H, Abe T. The association between heart rate variability and quality of life in patients with functional somatic syndrome and healthy controls. Appl Psychophysiol Biofeedback. 2021;46:279–85.

MacDonald HV, Johnson BT, Huedo-Medina TB, Livingston J, Forsyth KC, Kraemer WJ, et al. Dynamic resistance training as stand-alone antihypertensive lifestyle therapy: a meta-analysis. J Am Heart Assoc. 2016;5:e003231.

Nogueira IC, de Santos ZMSA, Mont’ Alverne DGB, Martins ABT, de Magalhães CBA. Efeitos do exercício físico no controle da hipertensão arterial em idosos: uma revisão sistemática. Revista Brasileira de Geriatria e Gerontologia. 2012;15:587–601.

Article Google Scholar

Park S, Han K, Lee S, Kim Y, Lee Y, Kang MW, et al. Cardiovascular or mortality risk of controlled hypertension and importance of physical activity. Heart. 2021;107:1472–9.

Raffin J, Barthélémy J-C, Dupré C, Pichot V, Berger M, Féasson L, et al. Exercise frequency determines heart rate variability gains in older people: a meta-analysis and meta-regression. Sports Med. 2019;49:719–29.

Ravier G, Dugué B, Grappe F, Rouillon J-D. Maximal accumulated oxygen deficit and blood responses of ammonia, lactate and pH after anaerobic test: a comparison between International and National Elite Karate Athletes. Int J Sports Med. 2006;27:810–7.

Franchini E, Cormack S, Takito MY. Effects of high-intensity interval training on olympic combat sports athletes’ performance and physiological adaptation: a systematic review. J Strength Cond Res. 2019;33:242–52.

Valdés-Badilla P, Herrera-Valenzuela T, Ramirez-Campillo R, Aedo-Muñoz E, Báez-San Martín E, Ojeda-Aravena A, et al. Effects of olympic combat sports on older adults’ health status: a systematic review. Int J Environ Res Public Health. 2021;18:7381.

Crisafulli A, Vitelli S, Cappai I, Milia R, Tocco F, Melis F, et al. Physiological responses and energy cost during a simulation of a Muay Thai boxing match. Appl Physiol Nutr Metab. 2009;34:143–50.

Saraiva BTC, Scarabottolo CC, Christofaro DGD, Silva GCR, Freitas Junior IF, Vanderlei LCM, et al. Effects of 16 weeks of Muay Thai training on the body composition of overweight/obese adolescents Ido movement for culture. J Martial Arts Anthropol. 2021;21:35–44.

Google Scholar

Saraiva BTC, Ritti-Dias RM, Scarabottolo CC, da Silva ALF, Tebar WR, Christofaro DGD. Effects of nine months practice of martial arts on aerobic fitness in children and adolescentes. Sci Sports. 2023;38:394–400.

Saraiva BTC, do Prado WL, Vanderlei LCM, Milanez VF, de Damato TMM, dos Santos AB, et al. Acute effects of Muay Thai on blood pressure and heart rate in adolescents with overweight/obesity. Obesities. 2022;2:94–102.

Saraiva BTC, Ritti-Dias RM, Farah BQ, Suetake VYB, Diniz TA, Costa Júnior P, et al. Cardiovascular effects of 16 weeks of martial arts training in adolescents. Revista Brasileira de Medicina do Esporte. 2018;24:212–5.

Saraiva BTC, Franchini E, Vanderlei LCM, Milanez VF, Tebar WR, Beretta VS, et al. Effects of 16-week Muay Thai practice on cardiovascular parameters in children and adolescents with overweight/obesity. Sport Sci Health. 2024;20:647–57.

Rapkiewicz JA, Nunes JP, Mayhew JL, Ribeiro AS, Nabuco HC, Fávero MT, et al. Effects of Muay Thai training frequency on body composition and physical fitness in healthy untrained women. J Sports Med Phys Fitness. 2018;58:1808.

Centers for Disease Control and Prevention (CDC). Adult participation in aerobic and muscle-strengthening physical activities–United States, 2011. MMWR Morb Mortal Wkly Rep. 2013;62:326–30.

de Abreu RM, Rehder-Santos P, Simões RP, Catai AM. Can high-intensity interval training change cardiac autonomic control? A systematic review. Braz J Phys Ther. 2019;23:279–89.

Granero-Gallegos A, González-Quílez A, Plews D, Carrasco-Poyatos M. HRV-based training for improving VO2max in endurance athletes. A systematic review with meta-analysis. Int J Environ Res Public Health. 2020;17:7999.

Article CAS PubMed PubMed Central Google Scholar

Venturelli M, Cè E, Limonta E, Schena F, Caimi B, Carugo S, et al. Effects of endurance, circuit, and relaxing training on cardiovascular risk factors in hypertensive elderly patients. Age (Omaha). 2015;37:101.

Oliveira-Junior SA, Boullosa D, Mendonça MLM, Vieira LFC, Mattos WW, Amaral BOC, et al. Effects of circuit weight-interval training on physical fitness, cardiac autonomic control, and quality of life in sedentary workers. Int J Environ Res Public Health. 2021;18:4606.

Marcos-Pardo PJ, Orquin-Castrillón FJ, Gea-García GM, Menayo-Antúnez R, González-Gálvez N, de Vale RGS, et al. Effects of a moderate-to-high intensity resistance circuit training on fat mass, functional capacity, muscular strength, and quality of life in elderly: a randomized controlled trial. Sci Rep. 2019;9:7830.

Heinrich KM, Crawford DA, Langford CR, Kehler A, Andrews V. High-intensity functional training shows promise for improving physical functioning and activity in community-dwelling older adults: a pilot study. J Geriatr Phys Ther. 2021;44:9–17.

De Matos DG, Mazini Filho ML, Moreira OC, De Oliveira CE, De Oliveira Venturini GR, Da Silva-Grigoletto ME, et al. Effects of eight weeks of functional training in the functional autonomy of elderly women: a pilot study. J Sports Med Phys Fitness. 2017;57:272.

Rezende Barbosa MP, Vanderlei LC, Neves LM, Takahashi C, Torquato PR, Silva AK, et al. Functional training in postmenopause: Cardiac autonomic modulation and cardiorespiratory parameters, a randomized trial. Geriatr Gerontol Int. 2019;19:823–8.

Choi H-M, Hurr C, Kim S. Effects of elastic band exercise on functional fitness and blood pressure response in the healthy elderly. Int J Environ Res Public Health. 2020;17:7144.

Origua Rios S, Marks J, Estevan I, Barnett LM. Health benefits of hard martial arts in adults: a systematic review. J Sports Sci. 2018;36:1614–22.

Zou L, Sasaki J, Wei G-X, Huang T, Yeung A, Neto O, et al. Effects of mind-body exercises (Tai Chi/Yoga) on heart rate variability parameters and perceived stress: a systematic review with meta-analysis of randomized controlled trials. J Clin Med. 2018;7:404.

Slimani M, Davis P, Franchini E, Moalla W. Rating of perceived exertion for quantification of training and combat loads during combat sport-specific activities: a short review. J Strength Cond Res. 2017;31:2889–902.

Schulz KF, Altman DG, Moher D. CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010;340:c332–c332.

Tsai J-C, Wang W-H, Chan P, Lin L-J, Wang C-H, Tomlinson B, et al. The beneficial effects of Tai Chi Chuan on blood pressure and lipid profile and anxiety status in a randomized controlled trial. J Altern Complement Med. 2003;9:747–54.

Ramos E, Vanderlei L, Ramos D, Teixeira L, Pitta F, Veloso M. Influence of pursed-lip breathing on heart rate variability and cardiorespiratory parameters in subjects with chronic obstructive pulmonary disease (COPD). Braz J Phys Ther. 2009;13:288–93.

Quintana DS, Heathers JAJ. Considerations in the assessment of heart rate variability in biobehavioral research. Front Psychol. 2014;5:805.

Radespiel-Tröger M, Rauh R, Mahlke C, Gottschalk T, Mück-Weymann M. Agreement of two different methods for measurement of heart rate variability. Clin Auton Res. 2003;13:99–102.

Niskanen J-P, Tarvainen MP, Ranta-aho PO, Karjalainen PA. Software for advanced HRV analysis. Comput Methods Programs Biomed. 2004;76:73–81.

Tulppo MP, Mäkikallio TH, Seppänen T, Laukkanen RT, Huikuri HV. Vagal modulation of heart rate during exercise: effects of age and physical fitness. Am J Physiol-Heart Circul Physiol. 1998;274:H424–9.

Article CAS Google Scholar

Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves J, Hill MN, et al. Recommendations for blood pressure measurement in humans and experimental animals. Circulation. 2005;111:697–716.

Darne B, Girerd X, Safar M, Cambien F, Guize L. Pulsatile versus steady component of blood pressure: a cross-sectional analysis and a prospective analysis on cardiovascular mortality. Hypertension. 1989;13:392–400.

Rafie AHS, Sungar GW, Dewey FE, Hadley D, Myers J, Froelicher VF. Prognostic value of double product reserve. Eur J Cardiovasc Prev Rehabil. 2008;15:541–7.

Freitas Júnior IF. Padronização de medidas antropométricas e avaliação da composição corporal. São Paulo: Selo Literário 20 anos da Regulamentação da Profissão de Educação Física; 2018.

Bavaresco Gambassi B, Lopes dos Santos MD, de Furtado Almeida FJ. Basic guide for the application of the main variables of resistance training in elderly. Aging Clin Exp Res. 2019;31:1019–20.

Borg G, Hassmén P, Lagerström M. Perceived exertion related to heart rate and blood lactate during arm and leg exercise. Eur J Appl Physiol Occup Physiol. 1987;56:679–85.

Maher JM, Markey JC, Ebert-May D. The other half of the story: effect size analysis in quantitative research. CBE-Life Sci Educ. 2013;12:345–51.

Bull FC, Al-Ansari SS, Biddle S, Borodulin K, Buman MP, Cardon G, et al. World Health Organization 2020 guidelines on physical activity and sedentary behaviour. Br J Sports Med. 2020;54:1451–62.

Gulli G, Cevese A, Cappelletto P, Gasparini G, Schena F. Moderate aerobic training improves autonomic cardiovascular control in older women. Clin Auton Res. 2003;13:196–202.

Rodrigues JAL, Ferrari GD, Trapé ÁA, de Moraes VN, Gonçalves TCP, Tavares SS, et al. β2 adrenergic interaction and cardiac autonomic function: effects of aerobic training in overweight/obese individuals. Eur J Appl Physiol. 2020;120:613–24.

Christofaro DGD, Tebar WR, Vanderlei LCM, Fernandes RA, Mota J, Mielke GI, et al. Association of cardiac autonomic modulation with different intensities of physical activity in a small Brazilian inner city: a gender analysis. Eur J Sport Sci. 2023;23:649–55.

Oliveira-Dantas FF, do Brasileiro-Santos MS, Thomas SG, Silva AS, Silva DC, Browne RAV, et al. Short-term resistance training improves cardiac autonomic modulation and blood pressure in hypertensive older women: a randomized controlled trial. J Strength Cond Res. 2020;34:37–45.

Areeudomwong P, Saysalum S, Phuttanurattana N, Sripoom P, Buttagat V, Keawduangdee P. Balance and functional fitness benefits of a Thai boxing dance program among community-dwelling older adults at risk of falling: a randomized controlled study. Arch Gerontol Geriatr. 2019;83:231–8.

Noopud P, Suputtitada A, Khongprasert S, Kanungsukkasem V. Effects of Thai traditional dance on balance performance in daily life among older women. Aging Clin Exp Res. 2019;31:961–7.

Kaewjoho C, Mato L, Thaweewannakij T, Nakmareong S, Phadungkit S, Gaogasigam C, et al. Thai dance exercises benefited functional mobility and fall rates among community-dwelling older individuals. Hong Kong Physiother J. 2020;40:19–27.

Janyacharoen T, Srisamai T, Sawanyawisuth K. An ancient boxing exercise improves physical functions, balance, and quality of life in healthy elderly persons. Evid-Based Complement Altern Med. 2018;2018:1–4.

Sukmee K, Khongprasert S, Laohapakdee R. The effect of Muay Thai exercise on balance and flexibility in Thai elderly. J Sports Sci Health. 2020;21:432–45.

Yeh GY, Wayne PM, Phillips RS. T’ai Chi Exercise in Patients with Chronic Heart Failure. In: Tai Chi Chuan. Basel: KARGER; 2008. p. 195–208.

Chapter Google Scholar

da de Rezende Barbosa MPC, Oliveira VC, da Silva AKF, Pérez-Riera AR, Vanderlei LC. Effectiveness of functional training on cardiorespiratory parameters: a systematic review and meta-analysis of randomized controlled trials. Clin Physiol Funct Imaging. 2018;38:539–46.

Carrasco-Poyatos M, González-Quílez A, Altini M, Granero-Gallegos A. Heart rate variability-guided training in professional runners: effects on performance and vagal modulation. Physiol Behav. 2022;244:113654.

da Silva S, Guida HL, dos SantosAntônio AM, Vanderlei L, Ferreira LL, de Abreu L, et al. Auditory stimulation with music influences the geometric indices of heart rate variability in men. Int Arch Med. 2014;7:27.

Santana MD, de Souza AC, de Abreu L, Valenti VE. Association between oral variables and heart rate variability. Int Arch Med. 2013;6:49.

Vanderlei LCM, Pastre CM, Hoshi RA, de Carvalho TD, de Godoy MF. Noções básicas de variabilidade da frequência cardíaca e sua aplicabilidade clínica. Rev Bras Cir Cardiovasc. 2009;24:205–17.

Molmen-Hansen HE, Stolen T, Tjonna AE, Aamot IL, Ekeberg IS, Tyldum GA, et al. Aerobic interval training reduces blood pressure and improves myocardial function in hypertensive patients. Eur J Prev Cardiol. 2012;19:151–60.

Chan AWK, Chair SY, Lee DTF, Leung DYP, Sit JWH, Cheng HY, et al. Tai Chi exercise is more effective than brisk walking in reducing cardiovascular disease risk factors among adults with hypertension: a randomised controlled trial. Int J Nurs Stud. 2018;88:44–52.

Liang H, Luo S, Chen X, Lu Y, Liu Z, Wei L. Effects of Tai Chi exercise on cardiovascular disease risk factors and quality of life in adults with essential hypertension: a meta-analysis. Heart Lung. 2020;49:353–63.

da de Rezende Barbosa MPC, Netto Júnior J, Cassemiro BM, de Souza NM, Bernardo AFB, da Silva AKF, et al. Impact of functional training on cardiac autonomic modulation, cardiopulmonary parameters and quality of life in healthy women. Clin Physiol Funct Imaging. 2016;36:318–25.

La SC. hipertensión arterial en el anciano. Hipertens Riesgo Vasc. 2017;34:26–9.

Vaccarino V, Holford TR, Krumholz HM. Pulse pressure and risk for myocardial infarction and heart failure in the elderly. J Am Coll Cardiol. 2000;36:130–8.

O’Rourke M, Frohlich ED. Pulse Pressure. Hypertension. 1999;34:372–4.

Fornitano LD, de Godoy MF. Duplo produto elevado como preditor de ausência de coronariopatia obstrutiva de grau importante em pacientes com teste ergométrico positivo. Arq Bras Cardiol. 2006;86:138.

Gobel FL, Norstrom LA, Nelson RR, Jorgensen CR, Wang Y. The rate-pressure product as an index of myocardial oxygen consumption during exercise in patients with angina pectoris. Circulation. 1978;57:549–56.

Kirk C, Langan-Evans C, Clark DR, Morton JP. Quantification of training load distribution in mixed martial arts athletes: a lack of periodisation and load management. PLoS One. 2021;16:e0251266.

Chen MJ, Fan X, Moe ST. Criterion-related validity of the Borg ratings of perceived exertion scale in healthy individuals: a meta-analysis. J Sports Sci. 2002;20:873–99.

Bailey DP, Fairclough SJ, Savory LA, Denton SJ, Pang D, Deane CS, et al. Accelerometry-assessed sedentary behaviour and physical activity levels during the segmented school day in 10–14-year-old children: the HAPPY study. Eur J Pediatr. 2012;171:1805–13.

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Acknowledgements

Thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 for financing part of the study.

This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.

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Alex S. Ribeiro

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Saraiva, B.T.C., Franchini, E., Ribeiro, A.S. et al. Effects of 12 weeks of functional training vs. Muay Thai on cardiac autonomic modulation and hemodynamic parameters in older adults: a randomized clinical trial. BMC Cardiovasc Disord 24 , 433 (2024). https://doi.org/10.1186/s12872-024-04096-3

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DOI : https://doi.org/10.1186/s12872-024-04096-3

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Analysis of total flavonoid variation and other functional substances in rils of tartary buckwheat, with near-infrared model construction for rapid non-destructive detection.

1. Introduction

2. materials and methods, 2.1. experimental materials and spectra acquisition, 2.1.1. experimental materials, 2.1.2. spectra acquisition, 2.2. determination of the contents of total flavonoid, ve, and gaba, 2.2.1. determination of total flavonoids of tartary buckwheat, 2.2.2. determination of ve in tartary buckwheat, 2.2.3. determination of gaba content in tartary buckwheat, 2.3. data processing and model evaluation, 2.3.1. data processing, 2.3.2. model evaluation, 3.1. determination of functional components of tartary buckwheat and analysis of variation, 3.2. construction of the near infrared model, 3.2.1. near infrared spectrum of tartary buckwheat, 3.2.2. partitioning of the sample set, 3.3. creation of total flavonoid prediction models, 3.4. effects of different pretreatment methods on ve modeling, 3.5. effects of different pretreatment methods on the modeling of gaba, 4. discussion, 4.1. sample diversity, 4.2. pre-processing of the spectrum, 4.3. sample splitting, 4.4. extraction of the characteristic spectrum, 4.5. modeling of whole grains, 4.6. potential limitations, 5. conclusions, author contributions, data availability statement, acknowledgments, conflicts of interest.

Luo, S.; Huang, J.M.; Yi, Y.; Zhang, Z.Y. Development status, problems, advantages and countermeasures of guizhou buckwheat industry. Tillage Cultiv. 2017 , 6 , 49–53+68. [ Google Scholar ]
Qu, Z.H.; Bai, J.; Liu, R.M.; Wang, D.Q.; Zuo, W.B. Effects of variety and environment on flavonoid content in F. esculentum Moench. J. Shanxi Agric. Sci. 2023 , 51 , 1404–1409. [ Google Scholar ]
Zhu, L.W.; Zhou, Y.; Cai, F.; Deng, J.; Huang, J.; Zhang, X.N.; Zhang, J.G.; Chen, Q.F. Quantitative analysis of perennial buckwheat leaves protein and GABA using near infrared spectroscopy. Spectrosc. Spectr. Anal. 2020 , 40 , 2421–2426. [ Google Scholar ]
Zhang, Z.L.; Zhou, M.L.; Tang, Y.; Li, F.L.; Tang, Y.X.; Shao, J.R.; Xue, W.T.; Wu, Y.M. Bioactive compounds in functional buckwheat food. Food Res. Int. 2012 , 49 , 389–395. [ Google Scholar ] [ CrossRef ]
Chang, Z.H.; NiMa, Y.Z.; Huang, H.J.; Laba, Z.X.; Gao, X.L.; Tian, P.J.; Yin, Z.J. Exploring the mechanism of Tartary Fagopyrum tataricum on pancreatic cancer based on data mining. Tibet J. Agric. Sci. 2023 , 45 , 29–34. [ Google Scholar ]
Wang, Y.F.; Chen, X.Y.; Han, S.Y.; Zhu, L.S.; Liu, S.M.; Lv, H.; Chu, J.X. Experimental study on total flavones from buckwheat leaf in fighting pain and in flammation. Acad. J. Shanghai Univ. Tradit. Chin. Med. 2004 , 11 , 54–55. [ Google Scholar ]
Zhu, L.S.; Ma, X.C.; Han, S.Y.; Liu, S.M.; Lv, H. Effects of total flavones of buckwheat leaf on blood lipid and lipid peroxides. Chin. J. Clin. Rehabil. 2004 , 24 , 5178–5179. [ Google Scholar ]
Ke, J.; Ran, B.; Sun, P.; Cheng, Y.; Chen, Q.; Li, H. An evaluation of the absolute content of flavonoids and the identification of their relationship with the flavonoid biosynthesis genes in Tartary buckwheat seeds. Agronomy 2023 , 13 , 3006. [ Google Scholar ] [ CrossRef ]
Tan, P.Y.; Guo, W.B. Research progress of Tartary buckwheat flavonoids on human body’s physiological function and mechanism. Med. Recapitul. 2018 , 24 , 1627–1632. [ Google Scholar ]
Qiu, J.; Liu, Y.P.; Yue, Y.F.; Qin, Y.C.; Li, Z.G. Dietary Tartary buckwheat intake attenuates insulin resistance and improves lipid profiles in patients with type 2 diabetes: A randomized controlled trial. Nutr. Res. 2016 , 36 , 1392–1401. [ Google Scholar ] [ CrossRef ]
Lei, L.T.; Zhou, Y.C.; Pu, X.Y. Preparation of flavonoid compounds from Tartary buckwheat and study on their antioxidative and hypoglycemic effects. Chin. Food Ind. 2023 , 02 , 102–104. [ Google Scholar ]
Qin, P.Y.; Wang, Q.A.; Shan, F.; Hou, Z.H.; Ren, G.X. Nutritional composition and flavonoids content of flour from different buckwheat cultivars. Int. J. Food Sci. Tech. 2010 , 45 , 951–958. [ Google Scholar ] [ CrossRef ]
Yao, P.; Huang, Y.; Dong, Q.; Wan, M.; Wang, A.; Chen, Y.; Li, C.; Wu, Q.; Chen, H.; Zhao, H. FtMYB6, a light-induced SG7 R2R3-MYB transcription factor, promotes flavonol biosynthesis in Tartary buckwheat ( Fagopyrum tataricum ). J. Agric. Food Chem. 2020 , 68 , 13685–13696. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Duan, C.Y.; Xiong, X.; Jing, M.Y.; Gao, Y.; Hou, F.F.; Xing, G.M.; Li, S. Genetic analysis and QTL localization of vitamin E content in Hemerocallis citrina Baroni. J. Hebei Agric. Univ. 2023 , 46 , 38–45. [ Google Scholar ]
Ma, J.T.; Qiu, D.Y.; Pang, Y.Z.; Gao, H.W.; Wang, X.M.; Qin, Y.C. Diverse roles of tocopherols in response to abiotic and biotic stresses and strategies for genetic biofortification in plants. Mol. Breed. 2020 , 40 , 18. [ Google Scholar ] [ CrossRef ]
Sozen, E.; Demirel, T.; Ozer, N.K. Vitamin E: Regulatory role in the cardiovascular system. IUBMB Life 2019 , 71 , 507–515. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Ekeuku, S.O.; Etim, E.P.; Pang, K.L.; Chin, K.Y.; Mai, C.W. Vitamin E in the management of pancreatic cancer: A scoping review. World J. Gastrointest. Oncol. 2023 , 15 , 943–958. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Wu, Z.; Wang, N.S.; Li, Y.H. Correlation of serum glutamate and gamma-aminobutyric acid levels with clinical symptoms in chronic schizophrenia patients and their diagnostic value for cognitive impairment. Int. J. Lab. Med. 2024 , 45 , 95–98+103. [ Google Scholar ]
Yan, R. The levels and clinical significance of serum glutamate and T-aminobutyric acid in patients with depression. J. Int. Psychl. 2022 , 49 , 609–611. [ Google Scholar ]
Tong, X.M.; Chai, C.X.; Wang, Y.Q. Effect of germination on grain quality of Tartary buckwheat and optimization of technology. Food Mach. 2021 , 4 , 176–183. [ Google Scholar ]
Xi, Z.Y. Study on Non-Destructive Detection Method of Buckwheat Based on Near-Infrared Spectroscopy Technology. Master’s Thesis, Kunming University of Science and Technology, Kunming, China, 2013. [ Google Scholar ]
Xu, B.C.; Ding, X.L. The quantitative methods of flavonoids in buckwheat ( Fagopyrum tataricum ). J. Food Sci. Biotechnol. 2003 , 02 , 98–101. [ Google Scholar ]
Tang, C.Y.; Wang, T.; Tu, J.; Liu, G.H.; Li, P.; Zhao, J. Comparison of colorimetry and HPLC for determination of γ-aminobutyric acid in mulberry leaf tea. Food Sci. 2018 , 39 , 256–260. [ Google Scholar ]
Zheng, Z.X. Application of near-infrared spectroscopy analysis technology in the feed processing industry. Fujian Agric. Mach. 2019 , 1 , 24–27. [ Google Scholar ]
Yang, N.; Ren, G.X. Application of near-infrared reflectance spectroscopy to the evaluation of rutin and d-chiro-inositol contents in Tartary buckwheat. J. Agric. Food Chem. 2008 , 761–764. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Li, W.L.; Han, H.F.; Cheng, Z.W.; Zhang, Y.; Liu, S.Y.; Qu, H.B. A feasibility research on the monitoring of traditional Chinese medicine production process using NIR-based multivariate process trajectories. Sens. Actuators B Chem. 2016 , 231 , 313–323. [ Google Scholar ] [ CrossRef ]
Platov, Y.T.; Metlenkin, D.A.; Platova, R.A.; Rassulov, V.A.; Vereshchagin, A.I.; Marin, V.A. Buckwheat identification by combined UV-VIS-NIR spectroscopy and multivariate analysis. J. Appl. Spectrosc. 2021 , 88 , 723–730. [ Google Scholar ] [ CrossRef ]
Zhu, L.W.; Yan, J.X.; Huang, J.; Shi, T.X.; Cai, F.; Li, H.Y.; Chen, Q.F.; Chen, Q.J. Rapid determination of amino acids in golden Tartary buckwheat based on near infrared spectroscopy and artificial neural network. Spectrosc. Spectr. Anal. 2022 , 42 , 49–55. [ Google Scholar ]
Zhang, J.; Guo, J.; Zhang, M.L.; Zhang, X.; E, J.J. Establishment of rapid detection model of buckwheat nutritional components based on near infrared spectroscopy. J. Chin. Cereals Oils Assoc. 2020 , 35 , 151–158. [ Google Scholar ]
Zhang, B.J.; Chen, S.S.; Li, K.Y.; Li, L.H.; Xu, R.H.; An, C.; Xiong, F.M.; Zhang, Y.; Dong, L.L.; Ren, M.J. Construction and application of detection model for amylose and amylopectin content in sorghum grains based on near infrared spectroscopy. Sci. Agric. Sin. 2022 , 55 , 26–35. [ Google Scholar ]
Wang, Y.Y.; Dai, Y.J.; Wang, Y.Y.; Yang, J.L.; Xiang, D.H.; Yang, Y.Q.; Zeng, S.W. Research on non-destructive detection of protein and fat content in Torreya based on near-infrared spectroscopy technology. Sci. Technol. Food Ind. 2024 , 45 , 1–8. [ Google Scholar ]
Wang, C.W. The Vegetable Quality Evaluation of Perennial Buckwheat. Master’s Thesis, Guizhou Normal University, Guiyang, China, 2015. [ Google Scholar ]
Chen, M.M.; Qiu, Y.C.; Song, Y.; Yang, S.Q.; Zuo, F.; Qian, L.L. Study on origin tracing of mung bean based on near-infrared spectrum. J. Heilongjiang Bayi Agric. Univ. 2024 , 36 , 49–54. [ Google Scholar ]
Yan, H.; Neves, M.D.G.; Wise, B.M.; Moraes, I.A.; Barbin, D.F.; Siesler, H.W. The application of handheld near-infrared spectroscopy and raman spectroscopic imaging for the identification and quality control of food products. Molecules 2023 , 28 , 7891. [ Google Scholar ] [ CrossRef ]
Lv, D.; Li, R.Y.; Zheng, R.; Zheng, J.Q.; Meng, Z.Y.; Shi, T.X.; Chen, Q.F. Variation analysis of flavonoids content in seeds and seed traits of tartary buckwheat germplasn resources. Mol. Plant Breed. 2020 , 18 , 4762–4774. [ Google Scholar ]
Ren, C.Z.; Shan, F.; Wang, M.; Li, Y.L. Review on nutrition and functionality and food product development of buckwheat. Chin. J. Grain Oil Sci. 2022 , 37 , 261–269. [ Google Scholar ]
Galvao, R.K.H.; Araujo, M.C.U.; Jose, G.E.; Pontes, M.J.C.; Silva, E.C.; Saldanha, T.C.B. A method for calibration and validation subset partitioning. Talanta 2005 , 67 , 736–740. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Rabatel, G.; Marini, F.; Walczak, B.; Roger, J. VSN: Variable sorting for normalization. J. Chemom. 2019 , 34 , e3164. [ Google Scholar ] [ CrossRef ]
Chu, X.L.; Li, Y.H. Practical Handbook of Near Infrared Spectroscopy , 1st ed.; Chemical Industry Press: Beijing, China, 2023; pp. 139–140. [ Google Scholar ]
Barnes, R.J.; Dhanoa, M.S.; Lister, S.J. Correction to the description of standard normal variate (SNV) and detrend (DT) transformations in practical spectroscopy with applications in food and beverage analysis—2nd Edition. J. Near Infrared Spectrosc. 1993 , 1 , 185–186. [ Google Scholar ] [ CrossRef ]
Sun, J.H.; Zhang, W.; Shi, J.Q.; Li, Y.K. Selection and application of spectral data preprocessing strategy. Acta Metrol. Sin. 2023 , 44 , 1284–1292. [ Google Scholar ]
Wang, L.Q.; Yao, J.; Wang, R.Y.; Chen, Y.H.; Luo, S.N.; Wang, W.N.; Zhang, Y.R. Research on detection of soybean meal quality by nir based on PLS-GRNN. Spectrosc. Spect. Anal. 2022 , 42 , 1433–1438. [ Google Scholar ]
Khamsopha, D.; Woranitta, S.; Teerachaichayut, S. Utilizing near infrared hyperspectral imaging for quantitatively predicting adulteration in tapioca starch. Food Control 2021 , 123 , 107781. [ Google Scholar ] [ CrossRef ]
Li, S.P.; Wang, J.Y.; Wang, L. Construction and application of amylose content calibration model based on near-infrared spectroscopy. Grain Oil Sci. Technol. 2023 , 36 , 139–143. [ Google Scholar ]
Chai, Y.H.; Yu, Y.; Zhu, H.; Li, Z.M.; Dong, H.; Yang, H.S. Identification of common buckwheat ( Fagopyrum esculentum Moench) adulterated in Tartary buckwheat ( Fagopyrum tataricum (L.) Gaertn) flour based on near-infrared spectroscopy and chemometrics. Curr. Res. Food Sci. 2023 , 7 , 100573. [ Google Scholar ] [ CrossRef ]
Cui, C.; Liu, C.L.; Sun, X.R.; Wu, J.Z. Peanut frostbite detection method based on near infrared hyperspectral imaging technology. Food Ind. Technol. 2024 , 45 , 226–233. [ Google Scholar ]
Centner, V.; Massart, D.L.; Denoord, O.E.; Dejong, S.; Vandeginste, B.M.; Sterna, C. Elimination of uninformative variables for multivariate calibration. Anal. Chem. 1996 , 68 , 3851–3858. [ Google Scholar ] [ CrossRef ]
Li, B.; Su, C.T.; Yin, H.; Liu, Y.D. Hyperspectral imaging technology combined with machine learning for detection of moldy rice. Spectrosc. Spectr. Anal. 2023 , 43 , 2391–2396. [ Google Scholar ]
Chen, P.; Dai, J.W.; Li, J.Y.; Xu, Y.P.; Liu, D.; Chu, X.L. Progress of chemometric methods in near- infrared spectroscopy. Chem. Reag. 2023 , 45 , 105–112. [ Google Scholar ]
Li, Z.; Hong, M.Y.; Cui, S.L.; Chen, M.; Liu, X.K.; Chen, H.Y.; Liu, L.F. Rapid detection method of flavonoid content in peanut seed based on near infrared technology. Spectrosc. Spectr. Anal. 2024 , 44 , 1112–1116. [ Google Scholar ]
Hashemi, S.M.; Perry, G.; Rajcan, I.; Eskandari, M. SoyMAGIC: An unprecedented platform for genetic studies and breeding activities in soybean. Front Plant Sci. 2022 , 13 , 945471. [ Google Scholar ] [ CrossRef ]
Zhang, H.J.; Wu, J.H.; Luo, Y.; Li, L.J.; Yang, H.; Yu, X.Q.; Wang, X.S.; Chen, L.; Mei, H.W. Comparison of near infrared spectroscopy models for determining protein and amylose contents between calibration samples of recombinant inbred lines and conventional varieties of rice. Sci. Agric. Sin. 2007 , 6 , 941–948. [ Google Scholar ] [ CrossRef ]
Jasinski, S.; Lécureuil, A.; Durandet, M.; Bernard-Moulin, P.; Guerche, P. Arabidopsis seed content QTL mapping using high-throughput phenotyping: The assets of near infrared spectroscopy. Front. Plant Sci. 2016 , 7 , 1682. [ Google Scholar ] [ CrossRef ]
Illarionova, S.; Shadrin, D.; Trekin, A.; Ignatiev, V.; Oseledets, I. Generation of the nir spectral band for satellite images with convolutional neural networks. Sensors 2021 , 21 , 5646. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Moses, W.J.; Gitelson, A.A.; Berdnikov, S.; Povazhnyy, V. Satellite estimation of chlorophyll-α concentration using the red and NIR bands of MERIS—The Azovsea case study. IEEE Geosci. Remote Sens. Lett. 2009 , 6 , 845–849. [ Google Scholar ] [ CrossRef ]
Holman, F.H.; Riche, A.B.; Michalski, A.; Castle, M.; Wooster, M.J.; Hawkesford, M.J. High throughput field phenotyping of wheat plant height and growth rate in field plot trials using UAV based remote sensing. Remote Sens. 2016 , 8 , 1031. [ Google Scholar ] [ CrossRef ]

Click here to enlarge figure

Functional Component	Number of Samples (NS)	Range	Mean Value (MV)	Skewness	Kurtosis	Standard Deviation (SD)	Coefficient of Variation (CV)
Total flavonoid	175	1.20–3.37	2.42	−0.462	0.276	0.36	15.06
VE	173	1.82–5.26	3.33	0.061	0.502	0.55	16.53
GABA	173	0.37–2.50	1.34	0.054	−0.818	0.49	36.93

Functional Component	NS	Sample Size	Range	MV	SD
Total flavonoid	training set	140	1.20–3.37	2.44	0.37
Total flavonoid	test set	35	1.71–2.90	2.39	0.35
VE	training set	138	1.82–5.26	3.37	0.54
VE	test set	35	1.94–4.30	3.26	0.54
GABA	training set	138	0.37–2.50	1.31	0.51
GABA	test set	35	0.53–2.27	1.39	0.43

Pretreatment Method	Rc	Rp	RMSECV	RMSEP	RPD
Normalization	0.9586	0.7725	0.1062	0.2245	1.5719
Normalization + MSC	0.9621	0.8324	0.1018	0.1959	1.8013
Normalization + SNV	0.9491	0.7303	0.1175	0.2509	1.4063
Normalization + First derivative	0.9956	0.9419	0.0350	0.1178	2.9944
Normalization + Second derivative	0.9954	0.9389	0.0356	0.1217	2.8988
Normalization + SG	0.9534	0.7483	0.1126	0.2401	1.4692

Pretreatment Method	Rc	Rp	RMSECV	RMSEP	RPD
Normalization	0.9856	0.7748	0.0915	0.3483	1.5562
Normalization + MSC	0.9861	0.8483	0.0900	1.5780	0.3435
Normalization + SNV	0.9870	0.8586	0.0869	0.2794	1.9399
Normalization + First derivative	0.9957	0.9427	0.0504	0.1848	2.9330
Normalization + Second derivative	0.9980	0.9329	0.0343	0.1974	2.7459
Normalization + SG	0.9807	0.8633	0.1057	0.3008	1.8021

Pretreatment Method	Rc	Rp	RMSECV	RMSEP	RPD
Normalization	0.9936	0.8903	0.0579	0.2007	2.1786
Normalization + MSC	0.9929	0.9161	0.0611	0.5031	0.8692
Normalization + SNV	0.9941	0.9322	0.0553	0.1599	2.7352
Normalization + First derivative	0.9973	0.9032	0.0378	0.2028	2.1560
Normalization + Second derivative	0.0.9989	0.8950	0.0245	0.1944	2.2494
Normalization + SG	0.9943	0.9067	0.0547	0.1793	2.4385

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Zhu, L.; Du, Q.; Shi, T.; Huang, J.; Deng, J.; Li, H.; Cai, F.; Chen, Q. Analysis of Total Flavonoid Variation and Other Functional Substances in RILs of Tartary Buckwheat, with Near-Infrared Model Construction for Rapid Non-Destructive Detection. Agronomy 2024 , 14 , 1826. https://doi.org/10.3390/agronomy14081826

Zhu L, Du Q, Shi T, Huang J, Deng J, Li H, Cai F, Chen Q. Analysis of Total Flavonoid Variation and Other Functional Substances in RILs of Tartary Buckwheat, with Near-Infrared Model Construction for Rapid Non-Destructive Detection. Agronomy . 2024; 14(8):1826. https://doi.org/10.3390/agronomy14081826

Zhu, Liwei, Qianxi Du, Taoxiong Shi, Juan Huang, Jiao Deng, Hongyou Li, Fang Cai, and Qingfu Chen. 2024. "Analysis of Total Flavonoid Variation and Other Functional Substances in RILs of Tartary Buckwheat, with Near-Infrared Model Construction for Rapid Non-Destructive Detection" Agronomy 14, no. 8: 1826. https://doi.org/10.3390/agronomy14081826

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Chapter 2
The five-step model of functional behaviour analysis (FBA).
Functional Behaviour Assessment (FBA)
Functional Behavior Assessment
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