speech impediment in japanese

Learning japanese with a lisp (speech impediment)

Because of my lisp, I don’t feel like I sound anywhere close to what I hear in the lesson/review.

If there anyone else on here who has a lisp, or any other learner that can give me any tips that helped them. I would be very grateful, thank you.

Output is overrated. Forget about it for now. Focus on understanding. Chances are that your pronounciation would be shit even if you didn’t have a lisp since you haven’t had enough input yet.

In general - my first note is that even without any kind of speech sound error/distortion, you’re having to train your brain to use new and different sound patterns/sequences - it’s going to feel unnatural and hard to say. Expect to need to say things a bit slower and work up to faster. I often feel ridiculously slow. If you want it to be easier to actually get the sounds out, you will eventually need to practice doing it, just in sheer terms of the motor skill of producing the sound sequences, separate from the language skill of knowing what words to say (if your native language has particularly similar phonology to Japanese, this may be less of an issue).

The skill of producing a sentence accurately is more motorically demanding than a single word. I often find it helpful if I’m having trouble producing a particular sentence to work backwards (or from whichever part I’m having most trouble with) - starting with trying to produce the tricky part until it’s fairly fluent, then adding on a few more words until I can get the whole sentence. This came up a lot for me with some grammar points (なければなりません comes to mind as being quite annoying when I tried to say it in sentences)

Specific tips for pronunciation correction would depend on the type of lisp (frontal or lateral), and the sounds it affects. Generally - for s,z sounds (the usual lisp culprits), you want the air to flow out the middle of your tongue (the sides of your tongue kind of brace against the inside of your teeth and your tongue forms a bit of a groove) and not escape from the sides, and your tongue should be behind your teeth. Sorry if that’s super obvious - I figured it was worth mentioning.

If I understand correctly, you have difficulty with “s” sounds, or something along those lines, right? For instance, perhaps trying to make an “s” sound produces a “th” sound, right?

I wouldn’t worry that much about it. Japanese people have serious difficulties hearing the difference between “s” and “th” sounds in English, which leads me to believe that if you spoke “s” sounds in Japanese with “th” sounds it actually wouldn’t impact things that much, because it “all sounds roughly the same” to them. It will sound obviously off to you because you speak a language where hearing the difference between “s” and “th” is important, but it’s similar to “r” and “l”. They don’t distinguish those sounds in Japanese, so basically anything in the “r” to “l” range sounds almost the same to them.

That’s just my hunch from watching them listen to English though.

Thank you everyone for your replies to my post, it means a lot to me.

Thanks @Kanamana66 , to be honest your reply made me laugh I feel better now

Disclaimer: I am one very new beginner giving advice to another new beginner! But I work in an alternative school for kids with learning and communication disabilities, so I have many students with speech difficulties. I think shadowing exercises would be very helpful for you. Luckily, many learners are interested in pitch accent and pronunciation so you can find these on YouTube. I believe there is a way so slow down the video to get it to the sped that will work best for you. Focus on accuracy, even if that means going at snail pace.

If you want feedback for pronunciation, the HelloTalk app has a feature where you can post recorded speech and ask for corrections.

Thought I have to say it, but if there are specific sounds that vex you no matter what, it might behoove you to get an SLP…I don’t know what the cost or difficulties are for doing this asan adult, but speech is so nuanced…

As an SLP - depends where you are, in Ontario, typically in the range of $120-$140/session.

A lot of SLPs say they only take kids, but that usually means ‘I don’t do neuro stuff like strokes’ - if you explain that the concern is speech sounds/articulation, they will quite likely take you.

Depending on the type of lisp, you often see faster progress with adults because there’s motivation, and they understand better what you’re trying to tell them to change and usually will follow through on practicing better. That said, the habit has been there a lot longer, so it definitely requires some conscious thought to make changes.

If it’s something you want to change, definitely reach out and look for someone to help you with it!

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Navigating Speech Impediment While Speaking Japanese

• January 26, 2023

(Please feel free to reply to this whether or not you have a speech impediment – while help from other speakers with speech impediments would be amazing, I’m just looking for any speakers’ input on if I am understandable)

One of the effects of my communication impediment prevents me from pronouncing です at the end of sentences without difficulty. Rather than “De-Su” the intended pronunciation, I can only pronounce “De-Thh”. I have been navigating this by only saying “De” at the end of sentences to indicate です. I can pronounce ですか “De-Tska”. Attempting any other pronunciation is laborious.

Will Japanese speakers understand me if I always pronounce です as “De”? Does anyone have any additional suggestions or advice for how I can navigate?

• Japanese Language
• Learn Japanese

If you look at the functions of で, they heavily suggest another chunk of phrase to follow right behind – this is highly confusing if you were to replace ですwith で. Something that vaguely sounds like ですか, I can autocorrect as long as that is the path of least resistance. If I hear で though, interpreting that to です isn’t going to happen.

Is it not possible to say “でthか”? That would be a close enough approximation. Using “ts” would make it sound like でつか which is still an intermediate step that requires another autocorrect. “th” has no Japanese counterpart, as such it’s probably easier to interpret directly to *a slightly mispronounced ですか, but definitely passable*.

I feel like when there is a problem of articulatory precision in a speech pathology, reformulating the sentence could be the way to go sometimes, or code switching.

For です code switching doesn’t help but perhaps it worth considering omitting when possible, or using less/more formal alternatives.

If you have similar articulatory problems with words, trying to find katakana/English equivalent helps.

Part of the learning process for everyone (and especially if you have speech impediment) is figuring out what linguistic devices are the most natural for you to express yourself. Basically, in your case it will be a list of words to avoid.

If your speech is particularly severe, consider using text-to-speech but I think close colleagues will be able to adapt, and customer language questions are probably constrained enough to allow for high pronunciation variation/noise.

I feel like the su is soft enough anyway that で+th would be fine. My Japanese friend legit talks like that anyway. All of the s sounds are super soft and sound closer to th. It’s not a hard s like it is in English. I think because it’s over annunciated in anime people get confused. I literally had someone compliment me for how relaxed my tongue was because the number one mistake foreigners make in pronunciation is the way they say their rs and over annunciation. As long as you try your best people will do their best to fill in what they don’t understand. Our brains do that automatically anyways. Fr xmpl yu cn prbbly rd ths. Don’t let yourself get stuck on one sound, because Japanese people are honestly happy to hear foreigners speak Japanese in general, regardless of how good they are.

[this]( https://youtu.be/9TzXD8dt7ks ) is an example (when he says です). I was trying to link you to one of my friends streams but ig he took them down bc he hasn’t streamed for a year…

Japanese doesn’t even have a “th” sound in its set of possible phonemes. Foreign words with “th” are usually transcribed as s, j or z in katakana (マーガレット・サッチャー, ジ・エンド、ザ・ワールド, etc）. Even if you say “th” a Japanese person will likely just hear it as an allophone of “s”, like how we English speakers hear the “tt” in “latter” or “butter” as a “t” even though the actual phoneme is practically the same as the らりるれろ “r” sound in Japanese. They’re less likely to notice an inappropriate “th” than English speakers are. You’ll be understood just fine even if it isn’t perfect.

I just break it down to syllables, practice slowly and then speed it up.

My biggest issue with my speech impediment is when there are sh and s sounds close together. So words like さしみ are challenging because my mouth wants to pronounce it like しゃしみ.

So by breaking it down and practicing pronunciation of each syllable and then putting it all together helps a lot.

If you can afford it, go to a speech pathologist. They can suggest exercises to help with the sounds that you have issues with.

At the risk of stating the obvious:

Mispronunciation is almost always gonna be better than complete omission.

There are Japanese people with lisps and other speech impediments, too, and they get understood (well, to varying degrees I suppose, as in any language).

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Speech impediment - slurred speech

Is there a name for a speech impediment like this where, for lack of a better description, it sounds like you're talking after you've bitten your tongue?

「いだだきまあず」　→　いただきます

「ごぢぞうざまでぢだあ」　→　ごちそうさまでした

Here's the video I took the screenshots from, but the audio doesn't completely match the words on the screen. (Also, I couldn't figure out how to link between a particular start and end time on the video.)

• spoken-language
• terminology

• Could you add an audio clip? There are several possibilities such as how hearing impaired people or how stroke victims speak. Or just quick, informal native speech. –  user3169 Commented Mar 7, 2017 at 4:05

There is a word 鼻声【はなごえ】 , which is understood by all native speakers. Exaggerated examples would be:

がぜびぎまじだぁ （風邪引きましたぁ） あ゛あ゛あ゛ 、ごめんなざい～ （ごめんなさい～）

But this usually refers to a temporary symptom of a normal human being.

For a yokai like this, I would use 鼻の詰まったような声, 鼻にかかったような声, くぐもった 声, こもった 声 or 濁った声. You can use 常に濁音で喋る although this doesn't look technical at all.

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How can I learn a language if I have a speech impediment?

• by 50 LANGUAGES Team

Language Learning with Speech Challenges

Learning a new language while having a speech impediment may seem daunting. It‘s essential to approach the process with patience and determination. Despite the challenge, many individuals with speech impediments have successfully acquired multiple languages.

Consider employing a language tutor who understands speech impediments. They can provide personalized strategies and techniques, catered to your unique needs. They‘re trained to provide feedback that respects your individual speech patterns.

Utilizing technology can aid in your language learning journey. Many applications now offer visual aids, interactive games, and written translations that can help improve language comprehension. This reduces reliance on speech for learning, allowing for a more inclusive experience.

Practice and repetition are key in language learning. Despite your impediment, speaking the language as much as possible will help. This can lead to improvements not just in language comprehension, but also in your overall speech abilities.

Written language exercises are valuable. They help develop a deep understanding of the language‘s structure and grammar. This focus on the written aspect can make communication easier, as you can write down your thoughts when speaking becomes difficult.

Using sign language can also be beneficial, especially if your speech impediment is severe. Sign language allows you to express yourself without speech. Many languages have corresponding sign language versions, providing another avenue for language acquisition.

Audio-visual materials like movies or music can enhance your listening comprehension skills. While this doesn‘t rely on your speech, it‘s a critical part of understanding and learning a language. Over time, this exposure can help develop your ability to communicate effectively.

Embrace the journey with positivity and resilience. You‘re bound to face challenges, but these hurdles are part of the learning process. Be patient with yourself, and take pride in every bit of progress you make. Remember, the objective is effective communication, not perfect pronunciation.

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Overcoming Speech Impediment: Symptoms to Treatment

There are many causes and solutions for impaired speech

• Types and Symptoms
• Speech Therapy
• Building Confidence

Speech impediments are conditions that can cause a variety of symptoms, such as an inability to understand language or speak with a stable sense of tone, speed, or fluidity. There are many different types of speech impediments, and they can begin during childhood or develop during adulthood.

Common causes include physical trauma, neurological disorders, or anxiety. If you or your child is experiencing signs of a speech impediment, you need to know that these conditions can be diagnosed and treated with professional speech therapy.

This article will discuss what you can do if you are concerned about a speech impediment and what you can expect during your diagnostic process and therapy.

FG Trade / Getty Images

Types and Symptoms of Speech Impediment

People can have speech problems due to developmental conditions that begin to show symptoms during early childhood or as a result of conditions that may occur during adulthood. 

The main classifications of speech impairment are aphasia (difficulty understanding or producing the correct words or phrases) or dysarthria (difficulty enunciating words).

Often, speech problems can be part of neurological or neurodevelopmental disorders that also cause other symptoms, such as multiple sclerosis (MS) or autism spectrum disorder .

There are several different symptoms of speech impediments, and you may experience one or more.

Can Symptoms Worsen?

Most speech disorders cause persistent symptoms and can temporarily get worse when you are tired, anxious, or sick.

Symptoms of dysarthria can include:

• Slurred speech
• Slow speech
• Choppy speech
• Hesitant speech
• Inability to control the volume of your speech
• Shaking or tremulous speech pattern
• Inability to pronounce certain sounds

Symptoms of aphasia may involve:

• Speech apraxia (difficulty coordinating speech)
• Difficulty understanding the meaning of what other people are saying
• Inability to use the correct words
• Inability to repeat words or phases
• Speech that has an irregular rhythm

You can have one or more of these speech patterns as part of your speech impediment, and their combination and frequency will help determine the type and cause of your speech problem.

Causes of Speech Impediment

The conditions that cause speech impediments can include developmental problems that are present from birth, neurological diseases such as Parkinson’s disease , or sudden neurological events, such as a stroke .

Some people can also experience temporary speech impairment due to anxiety, intoxication, medication side effects, postictal state (the time immediately after a seizure), or a change of consciousness.

Speech Impairment in Children

Children can have speech disorders associated with neurodevelopmental problems, which can interfere with speech development. Some childhood neurological or neurodevelopmental disorders may cause a regression (backsliding) of speech skills.

Common causes of childhood speech impediments include:

• Autism spectrum disorder : A neurodevelopmental disorder that affects social and interactive development
• Cerebral palsy :  A congenital (from birth) disorder that affects learning and control of physical movement
• Hearing loss : Can affect the way children hear and imitate speech
• Rett syndrome : A genetic neurodevelopmental condition that causes regression of physical and social skills beginning during the early school-age years.
• Adrenoleukodystrophy : A genetic disorder that causes a decline in motor and cognitive skills beginning during early childhood
• Childhood metabolic disorders : A group of conditions that affects the way children break down nutrients, often resulting in toxic damage to organs
• Brain tumor : A growth that may damage areas of the brain, including those that control speech or language
• Encephalitis : Brain inflammation or infection that may affect the way regions in the brain function
• Hydrocephalus : Excess fluid within the skull, which may develop after brain surgery and can cause brain damage

Do Childhood Speech Disorders Persist?

Speech disorders during childhood can have persistent effects throughout life. Therapy can often help improve speech skills.

Speech Impairment in Adulthood

Adult speech disorders develop due to conditions that damage the speech areas of the brain.

Common causes of adult speech impairment include:

• Head trauma 
• Nerve injury
• Throat tumor
• Stroke 
• Parkinson’s disease 
• Essential tremor
• Brain tumor
• Brain infection

Additionally, people may develop changes in speech with advancing age, even without a specific neurological cause. This can happen due to presbyphonia , which is a change in the volume and control of speech due to declining hormone levels and reduced elasticity and movement of the vocal cords.

Do Speech Disorders Resolve on Their Own?

Children and adults who have persistent speech disorders are unlikely to experience spontaneous improvement without therapy and should seek professional attention.

Steps to Treating Speech Impediment 

If you or your child has a speech impediment, your healthcare providers will work to diagnose the type of speech impediment as well as the underlying condition that caused it. Defining the cause and type of speech impediment will help determine your prognosis and treatment plan.

Sometimes the cause is known before symptoms begin, as is the case with trauma or MS. Impaired speech may first be a symptom of a condition, such as a stroke that causes aphasia as the primary symptom.

The diagnosis will include a comprehensive medical history, physical examination, and a thorough evaluation of speech and language. Diagnostic testing is directed by the medical history and clinical evaluation.

Diagnostic testing may include:

• Brain imaging , such as brain computerized tomography (CT) or magnetic residence imaging (MRI), if there’s concern about a disease process in the brain
• Swallowing evaluation if there’s concern about dysfunction of the muscles in the throat
• Electromyography (EMG) and nerve conduction studies (aka nerve conduction velocity, or NCV) if there’s concern about nerve and muscle damage
• Blood tests, which can help in diagnosing inflammatory disorders or infections

Your diagnostic tests will help pinpoint the cause of your speech problem. Your treatment will include specific therapy to help improve your speech, as well as medication or other interventions to treat the underlying disorder.

For example, if you are diagnosed with MS, you would likely receive disease-modifying therapy to help prevent MS progression. And if you are diagnosed with a brain tumor, you may need surgery, chemotherapy, or radiation to treat the tumor.

Therapy to Address Speech Impediment

Therapy for speech impairment is interactive and directed by a specialist who is experienced in treating speech problems . Sometimes, children receive speech therapy as part of a specialized learning program at school.

The duration and frequency of your speech therapy program depend on the underlying cause of your impediment, your improvement, and approval from your health insurance.

If you or your child has a serious speech problem, you may qualify for speech therapy. Working with your therapist can help you build confidence, particularly as you begin to see improvement.

Exercises during speech therapy may include:

• Pronouncing individual sounds, such as la la la or da da da
• Practicing pronunciation of words that you have trouble pronouncing
• Adjusting the rate or volume of your speech
• Mouth exercises
• Practicing language skills by naming objects or repeating what the therapist is saying

These therapies are meant to help achieve more fluent and understandable speech as well as an increased comfort level with speech and language.

Building Confidence With Speech Problems 

Some types of speech impairment might not qualify for therapy. If you have speech difficulties due to anxiety or a social phobia or if you don’t have access to therapy, you might benefit from activities that can help you practice your speech. 

You might consider one or more of the following for you or your child:

• Joining a local theater group
• Volunteering in a school or community activity that involves interaction with the public
• Signing up for a class that requires a significant amount of class participation
• Joining a support group for people who have problems with speech

Activities that you do on your own to improve your confidence with speaking can be most beneficial when you are in a non-judgmental and safe space.

Many different types of speech problems can affect children and adults. Some of these are congenital (present from birth), while others are acquired due to health conditions, medication side effects, substances, or mood and anxiety disorders. Because there are so many different types of speech problems, seeking a medical diagnosis so you can get the right therapy for your specific disorder is crucial.

Centers for Disease Control and Prevention. Language and speech disorders in children .

Han C, Tang J, Tang B, et al. The effectiveness and safety of noninvasive brain stimulation technology combined with speech training on aphasia after stroke: a systematic review and meta-analysis . Medicine (Baltimore). 2024;103(2):e36880. doi:10.1097/MD.0000000000036880

National Institute on Deafness and Other Communication Disorders. Quick statistics about voice, speech, language .

Mackey J, McCulloch H, Scheiner G, et al. Speech pathologists' perspectives on the use of augmentative and alternative communication devices with people with acquired brain injury and reflections from lived experience . Brain Impair. 2023;24(2):168-184. doi:10.1017/BrImp.2023.9

Allison KM, Doherty KM. Relation of speech-language profile and communication modality to participation of children with cerebral palsy . Am J Speech Lang Pathol . 2024:1-11. doi:10.1044/2023_AJSLP-23-00267

Saccente-Kennedy B, Gillies F, Desjardins M, et al. A systematic review of speech-language pathology interventions for presbyphonia using the rehabilitation treatment specification system . J Voice. 2024:S0892-1997(23)00396-X. doi:10.1016/j.jvoice.2023.12.010

By Heidi Moawad, MD Dr. Moawad is a neurologist and expert in brain health. She regularly writes and edits health content for medical books and publications.

CogniFit Blog: Brain Health News

Brain Training, Mental Health, and Wellness

Rhotacism: A complete guide to this speech impediment

Remember when you were a child and spoke by making your “R’s” sound like “W’s” and everything thought it was cute? That’s known as rhotacism and some people live with it even as adults. What is rhotacism, what is it like in other languages, and what are its symptoms? What does it look like as a speech impediment and what are some examples? What are its causes? How does it affect the brain ? Is it curable and how can it be fixed? This article will answer all your doubts about rhotacism. 

What is rhotacism?

Rhotacism is a speech impediment that is defined by the lack of ability, or difficulty in, pronouncing the sound R . Some speech pathologists, those who work with speech impediments may call this impediment de-rhotacization because the sounds don’t become rhotic, rather they lose their rhotic quality. It could also be called a residual R error.

It’s not such an uncommon phenomenon and actually also happens with the letter L , a phenomenon known as lambdacism . Sometimes people mistake these speech impediments for a lisp, of which they are not. Within the 2000-2001 school year, more than 700,000 students within the American public school system were categorized as having either a language impediment or a speech impediment. Ironically, all three speech impediments contain the troubled letter within them.

The word rhotacism comes from the New Latin rhotacism meaning peculiar or excessive use of [r]. The Latin word came from Ancient Greek word rhōtakismós which means to incorrectly use “rho” which is the equivalent of the Greek R. For language nerds, here’s a really great explanation of how the word came into being.

How does rhotacism work in different languages?

Rhotacism is, in theory , more common among people whose native language has a trilled R. For example, in Spanish the “rr” is a trilled R. Other languages with a trilled R include Bulgarian, Hungarian, Arabic, Finnish, Romanian, Indonesian, Russian , Italian, and most Swedish speakers. Some people might mock Asians, specifically Chinese, for not being able to pronounce the English word “broccoli” correctly- rather pronouncing it “browccoli”. This isn’t due to a rhotacism, however. It’s actually due to the fact that Mandarin (Chinese) words can have an “r” sound in the beginning of a word, but not in the middle or end of a word. This leads them to have issues in their phonotactics and creates an inability to pronounce the English “R” in the middle of words.

The leader of Hezbollah, Hasan Nasrallah, is a Lebanese leader and is mocked for his rhotacism when he says, “ Amwīka ” and “ Iswā’īl ” for the Arabic Amrīka (America), and Isrā’īl (Israel). He is a native Arabic speaker- a language which has the trilled R. Notice how he puts a W sound in those two words where the R sound usually is.

Symptoms of rhotacism

• Some people try to hide their impediment by avoiding words with R ’s in them.
• An overall inability to say R sounds
• Using trilled R’s or guttural R’s (such as the French R) when trying to pronounce the regular English R.

Rhotacism as a speech impediment

Using a strict classification, only about 5%-10% of the human population speaks in a completely normal way. Everyone else suffers from some type of speech disorder or another. For children of any language, the R sounds are usually the hardest to master and often end up being the last ones a child learns. That’s why baby talk if you think about it, doesn’t really use explicit or strong R sounds. In English, rhotacism often comes off as a W sound which is why “Roger Rabbit” sounds like “Woger Wabbit”. R is often more difficult because a child has to learn the different combination of the /r/ sounds, not just the letter itself, unlike other letters. For example, when it comes before and after vowel sounds. The combination of a vowel with the /r/ sound is called a phenome and in English, there are eight combinations of these:

–        The prevocalic R , such as “rain”

–        The RL , such as “girl”

–        The IRE, such as “tire”

–        The AR, such as “car”

–        The EAR , “such as “beer”

–        The OR , such as “seashore”

–        The ER , such as “butter”

–        The AIR , such as “software”

A speech impediment is a speech disorder , not a language disorder . Speech disorders are problems in being able to produce the sounds of speech whereas language disorders are problems with understanding and/or being able to use words. Language disorders, unlike speech disorders, have nothing to do with speech production.

Often what happens is that the person speaking isn’t tensing their tongue enough, or not moving their tongue correctly (up and backward depending on the dialect) which makes the W or “uh” sound come out. It may also be that the person is moving their lips instead of their tongue.

Examples of rhotacism

• Barry Kripke from the TV show The Big Bang Theory has both rhotacism and lambdacism- meaning he has issues pronouncing both his R ’s and his L ’s.
• The most famous of rhotacism would be Elmer Fudd from Looney Tunes . He pronounces the word “rabbit” [ˈɹ̠ʷæbɪ̈t] as “wabbit” [ˈwæbɪ̈t]
• In Monty Python’s Life of Brian , the 1979 film’s character Pilate suffers from rhotacism. In the film, people mock him for his inability to be understood easily.

Here’s a video with a woman who suffers from rhotacism. She explains how difficult it can be to have the speech impediment.

Causes of rhotacism

For many people, the causes of rhotacism are relatively unknown-, especially in adults. However, scientists theorize that the biggest cause is that the person grew up in an environment where they heard R ’s in a weird way, the shape of their mouths are different than normal, or their tongues and lips never learned how to produce the letter. In children, this could happen because the parents or adults around think the way the child talks (using baby talk) is cute and the child never actually learns how to produce it.

For one internet forum user, it has to do with how they learned the language , “I speak various languages, I pronounce the “R” normal in Dutch, French, and Spanish, but I have a rhotacism when speaking English. It’s the way I learnt it.”

For other people, speech issues are a secondary condition to an already existing, serious condition. Physically, it would be a cleft lip or a cleft palate. Neurologically, it could be a condition such as cerebral palsy. It may also be a tongue tie . Almost everyone has a stretch of skin that runs along the bottom of their tongue. If that skin is too tight and reaches the tip of the tongue, it can make pronouncing (and learning how to pronounce) R ’s and L ’s difficult. If the tongue tie isn’t fixed early on, it can be incredibly difficult to fix and learn how to pronounce later.

How the brain affects rhotacism

The brain affects rhotacism only for those who suffer from it not due to a physical impediment (such as a cleft palate). For some, this could happen because the brain doesn’t have the phonemic awareness and never actually learned what the letter R is supposed to sound like. This is common with kids whose parents spoke to them in “baby talk” and encouraged the child’s baby talk, too. This kind of behavior only strengthens a child’s inner concept that / R / is pronounced like “w” or “uh”.

Another reason could be that the brain connections simply don’t allow the lips or mouth to move in the way they need to in order to pronounce the R . This inability has little to do with physical incapabilities and more to do with mental ones. Some people with rhotacism have an issue with their oral-motor skills which means that there isn’t sufficient communication in the parts of the brain responsible for speech production.

Treatment for rhotacism

Is rhotacism curable.

It can have negative social effects- especially among younger children, such as bullying, which lowers self-esteem and can have a lasting effect. However, if the impediment is caught early enough on and is treated rather quickly, there is a good overall prognosis meaning it’s curable.

        However, some people never end up being able to properly pronounce that R and they end up substituting other sounds, such as the velar approximant (like w sounds) , the uvular approximant (also known as the “French R ”), and the uvular trill ( like the trilled R in Spanish).

How to fix rhotacism

Rhotacism is fixed by speech therapy . Before anything else, there needs to be an assessment from a Speech Language Pathologist (SLP) who will help decide if the problem can be fixed. If a child is involved, the SLP would predict if the child can outgrow the problem or not. After the diagnosis, a speech therapist will work with the person who suffers from the speech impediment by possibly having weekly visits with some homework and practice instructions. Therapy happens in spouts- a period of a few weeks and a break. There is a follow-up to see if there has been an improvement in pronunciation. In the U.S., children who are in school and have a speech disorder are placed in a special education program. Most school districts provide these children with speech therapy during school hours.

Another option, often used alongside speech therapy, is using a speech therapy hand-held tool that helps isolate the sound being pronounced badly and gives an image of the proper tongue placement to enable better pronunciation.

One study tested a handheld tactical tool (known as Speech Buddies) and the traditional speech therapy methods. The study found that students who used the hand-held tool (alongside speech therapy) improved 33% faster than those who used only the traditional speech therapy methods.

Have you or someone you know ever struggled with rhotacism? Let us know what you think in the comments below!

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Types of Speech Impediments

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Articulation Errors

Ankyloglossia, treating speech disorders.

A speech impediment, also known as a speech disorder , is a condition that can affect a person’s ability to form sounds and words, making their speech difficult to understand.

Speech disorders generally become evident in early childhood, as children start speaking and learning language. While many children initially have trouble with certain sounds and words, most are able to speak easily by the time they are five years old. However, some speech disorders persist. Approximately 5% of children aged three to 17 in the United States experience speech disorders.

There are many different types of speech impediments, including:

• Articulation errors

This article explores the causes, symptoms, and treatment of the different types of speech disorders.

Speech impediments that break the flow of speech are known as disfluencies. Stuttering is the most common form of disfluency, however there are other types as well.

Symptoms and Characteristics of Disfluencies

These are some of the characteristics of disfluencies:

• Repeating certain phrases, words, or sounds after the age of 4 (For example: “O…orange,” “I like…like orange juice,” “I want…I want orange juice”)
• Adding in extra sounds or words into sentences (For example: “We…uh…went to buy…um…orange juice”)
• Elongating words (For example: Saying “orange joooose” instead of "orange juice")
• Replacing words (For example: “What…Where is the orange juice?”)
• Hesitating while speaking (For example: A long pause while thinking)
• Pausing mid-speech (For example: Stopping abruptly mid-speech, due to lack of airflow, causing no sounds to come out, leading to a tense pause)

In addition, someone with disfluencies may also experience the following symptoms while speaking:

• Vocal tension and strain
• Head jerking
• Eye blinking
• Lip trembling

Causes of Disfluencies

People with disfluencies tend to have neurological differences in areas of the brain that control language processing and coordinate speech, which may be caused by:

• Genetic factors
• Trauma or infection to the brain
• Environmental stressors that cause anxiety or emotional distress
• Neurodevelopmental conditions like attention-deficit hyperactivity disorder (ADHD)

Articulation disorders occur when a person has trouble placing their tongue in the correct position to form certain speech sounds. Lisping is the most common type of articulation disorder.

Symptoms and Characteristics of Articulation Errors

These are some of the characteristics of articulation disorders:

• Substituting one sound for another . People typically have trouble with ‘r’ and ‘l’ sounds. (For example: Being unable to say “rabbit” and saying “wabbit” instead)
• Lisping , which refers specifically to difficulty with ‘s’ and ‘z’ sounds. (For example: Saying “thugar” instead of “sugar” or producing a whistling sound while trying to pronounce these letters)
• Omitting sounds (For example: Saying “coo” instead of “school”)
• Adding sounds (For example: Saying “pinanio” instead of “piano”)
• Making other speech errors that can make it difficult to decipher what the person is saying. For instance, only family members may be able to understand what they’re trying to say.

Causes of Articulation Errors

Articulation errors may be caused by:

• Genetic factors, as it can run in families
• Hearing loss , as mishearing sounds can affect the person’s ability to reproduce the sound
• Changes in the bones or muscles that are needed for speech, including a cleft palate (a hole in the roof of the mouth) and tooth problems
• Damage to the nerves or parts of the brain that coordinate speech, caused by conditions such as cerebral palsy , for instance

Ankyloglossia, also known as tongue-tie, is a condition where the person’s tongue is attached to the bottom of their mouth. This can restrict the tongue’s movement and make it hard for the person to move their tongue.

Symptoms and Characteristics of Ankyloglossia

Ankyloglossia is characterized by difficulty pronouncing ‘d,’ ‘n,’ ‘s,’ ‘t,’ ‘th,’ and ‘z’ sounds that require the person’s tongue to touch the roof of their mouth or their upper teeth, as their tongue may not be able to reach there.

Apart from speech impediments, people with ankyloglossia may also experience other symptoms as a result of their tongue-tie. These symptoms include:

• Difficulty breastfeeding in newborns
• Trouble swallowing
• Limited ability to move the tongue from side to side or stick it out
• Difficulty with activities like playing wind instruments, licking ice cream, or kissing
• Mouth breathing

Causes of Ankyloglossia

Ankyloglossia is a congenital condition, which means it is present from birth. A tissue known as the lingual frenulum attaches the tongue to the base of the mouth. People with ankyloglossia have a shorter lingual frenulum, or it is attached further along their tongue than most people’s.

Dysarthria is a condition where people slur their words because they cannot control the muscles that are required for speech, due to brain, nerve, or organ damage.

Symptoms and Characteristics of Dysarthria

Dysarthria is characterized by:

• Slurred, choppy, or robotic speech
• Rapid, slow, or soft speech
• Breathy, hoarse, or nasal voice

Additionally, someone with dysarthria may also have other symptoms such as difficulty swallowing and inability to move their tongue, lips, or jaw easily.

Causes of Dysarthria

Dysarthria is caused by paralysis or weakness of the speech muscles. The causes of the weakness can vary depending on the type of dysarthria the person has:

• Central dysarthria is caused by brain damage. It may be the result of neuromuscular diseases, such as cerebral palsy, Huntington’s disease, multiple sclerosis, muscular dystrophy, Huntington’s disease, Parkinson’s disease, or Lou Gehrig’s disease. Central dysarthria may also be caused by injuries or illnesses that damage the brain, such as dementia, stroke, brain tumor, or traumatic brain injury .
• Peripheral dysarthria is caused by damage to the organs involved in speech. It may be caused by congenital structural problems, trauma to the mouth or face, or surgery to the tongue, mouth, head, neck, or voice box.

Apraxia, also known as dyspraxia, verbal apraxia, or apraxia of speech, is a neurological condition that can cause a person to have trouble moving the muscles they need to create sounds or words. The person’s brain knows what they want to say, but is unable to plan and sequence the words accordingly.

Symptoms and Characteristics of Apraxia

These are some of the characteristics of apraxia:

• Distorting sounds: The person may have trouble pronouncing certain sounds, particularly vowels, because they may be unable to move their tongue or jaw in the manner required to produce the right sound. Longer or more complex words may be especially harder to manage.
• Being inconsistent in their speech: For instance, the person may be able to pronounce a word correctly once, but may not be able to repeat it. Or, they may pronounce it correctly today and differently on another day.
• Grasping for words: The person may appear to be searching for the right word or sound, or attempt the pronunciation several times before getting it right.
• Making errors with the rhythm or tone of speech: The person may struggle with using tone and inflection to communicate meaning. For instance, they may not stress any of the words in a sentence, have trouble going from one syllable in a word to another, or pause at an inappropriate part of a sentence.

Causes of Apraxia

Apraxia occurs when nerve pathways in the brain are interrupted, which can make it difficult for the brain to send messages to the organs involved in speaking. The causes of these neurological disturbances can vary depending on the type of apraxia the person has:

• Childhood apraxia of speech (CAS): This condition is present from birth and is often hereditary. A person may be more likely to have it if a biological relative has a learning disability or communication disorder.
• Acquired apraxia of speech (AOS): This condition can occur in adults, due to brain damage as a result of a tumor, head injury , stroke, or other illness that affects the parts of the brain involved in speech.

If you have a speech impediment, or suspect your child might have one, it can be helpful to visit your healthcare provider. Your primary care physician can refer you to a speech-language pathologist, who can evaluate speech, diagnose speech disorders, and recommend treatment options.

The diagnostic process may involve a physical examination as well as psychological, neurological, or hearing tests, in order to confirm the diagnosis and rule out other causes.

Treatment for speech disorders often involves speech therapy, which can help you learn how to move your muscles and position your tongue correctly in order to create specific sounds. It can be quite effective in improving your speech.

Children often grow out of milder speech disorders; however, special education and speech therapy can help with more serious ones.

For ankyloglossia, or tongue-tie, a minor surgery known as a frenectomy can help detach the tongue from the bottom of the mouth.

A Word From Verywell

A speech impediment can make it difficult to pronounce certain sounds, speak clearly, or communicate fluently. 

Living with a speech disorder can be frustrating because people may cut you off while you’re speaking, try to finish your sentences, or treat you differently. It can be helpful to talk to your healthcare providers about how to cope with these situations.

You may also benefit from joining a support group, where you can connect with others living with speech disorders.

National Library of Medicine. Speech disorders . Medline Plus.

Centers for Disease Control and Prevention. Language and speech disorders .

Cincinnati Children's Hospital. Stuttering .

National Institute on Deafness and Other Communication Disorders. Quick statistics about voice, speech, and language .

Cleveland Clinic. Speech impediment .

Lee H, Sim H, Lee E, Choi D. Disfluency characteristics of children with attention-deficit/hyperactivity disorder symptoms . J Commun Disord . 2017;65:54-64. doi:10.1016/j.jcomdis.2016.12.001

Nemours Foundation. Speech problems .

Penn Medicine. Speech and language disorders .

Cleveland Clinic. Tongue-tie .

University of Rochester Medical Center. Ankyloglossia .

Cleveland Clinic. Dysarthria .

National Institute on Deafness and Other Communication Disorders. Apraxia of speech .

Cleveland Clinic. Childhood apraxia of speech .

Stanford Children’s Hospital. Speech sound disorders in children .

Abbastabar H, Alizadeh A, Darparesh M, Mohseni S, Roozbeh N. Spatial distribution and the prevalence of speech disorders in the provinces of Iran . J Med Life . 2015;8(Spec Iss 2):99-104.

By Sanjana Gupta Sanjana is a health writer and editor. Her work spans various health-related topics, including mental health, fitness, nutrition, and wellness.

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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  

Nature Communications volume  15 , Article number:  6367 ( 2024 ) Cite this article

Metrics details

• 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.

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.

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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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A neurological disorder stole her voice. Jennifer Wexton takes it back on the House floor.

After a rare neurological disorder robbed Rep. Jennifer Wexton of her ability to speak clearly, she has been given her voice back with the help of a powerful artificial intelligence program. (AP video Kaito Au/ production Mary Conlon).

Rep. Jennifer Wexton, D-Va., uses an AI program on her iPad at her home in Leesburg, Va., Friday, July 19, 2024. A rare neurological disease robbed Wexton of her ability to speak clearly. But with the help of a powerful artificial intelligence program, the Virginia Democrat will use a clone of her voice to deliver what is believed to be the first speech on the House floor ever given via a voice cloned by artificial intelligence, thrusting Wexton into a broader debate about over artificial intelligence. (AP Photo/John McDonnell)

• Copy Link copied

FILE - Democrat Jennifer Wexton speaks at her election night party after defeating Rep. Barbara Comstock, R-Va., Tuesday, Nov. 6, 2018, in Dulles, Va. A rare neurological disease robbed Wexton of her ability to speak clearly. But with the help of a powerful artificial intelligence program, the Virginia Democrat will use a clone of her voice to deliver what is believed to be the first speech on the House floor ever given via a voice cloned by artificial intelligence, thrusting Wexton into a broader debate about over artificial intelligence. (AP Photo/Alex Brandon, File)

In this image from video Rep. Jennifer Wexton, D-Va., uses an AI program on her iPad to speak in the chamber of the House of Representatives, Thursday, July 25, 2024 at the Capitol in Washington. A rare neurological disease robbed Wexton of her ability to speak clearly. But with the help of a powerful artificial intelligence program, the Virginia Democrat usee a clone of her voice to deliver what is believed to be the first speech on the House floor ever given via a voice cloned by artificial intelligence, thrusting Wexton into a broader debate about over artificial intelligence. (House Television via AP)

When Jennifer Wexton rose Thursday to speak on the House floor, something she has done countless times before, the congresswoman used a voice she thought was gone forever.

After a rare neurological disorder robbed her of her ability to speak clearly, Wexton has been given her voice back with the help of a powerful artificial intelligence program, allowing the Virginia Democrat to make a clone of her speaking voice using old recordings of speeches and appearances she made as a congresswoman. She used that program to deliver what is believed to be the first speech on the House floor ever given via a voice cloned by artificial intelligence.

“It was a special moment that I never imagined could happen. I cried happy tears when I first heard it,” Wexton told The Associated Press in the first interview she’s participated in since attaining her new voice.

Standing at a lectern on the floor, Wexton rose to commemorate Disability Pride Month, a time each July that aims to commemorate the Americans with Disabilities Act, the landmark 1990s civil rights law aimed at protecting Americans with disabilities. But her speech was also a symbol of her strength in the face of a debilitating disease.

“I used to be one of those people who hated the sound of my voice,” she remarked from the floor. “When my ads came on TV, I would cringe and change the channel. But you truly don’t know what you’ve got til it’s gone, because hearing the new AI of my old voice for the first time was music to my ears. It was the most beautiful thing I had ever heard.”

AP AUDIO: A neurological disorder stole her voice. Jennifer Wexton takes it back on the House floor.

AP correspondent Jackie Quinn reports on a U.S. congresswoman who has lost her voice to a disabling disease, but makes history with artificial intelligence in a House Floor speech.

Wexton’s voice now plays out of her iPad, propped up using a rainbow-colored floral case. During the interview at her dining room table in Leesburg, Virginia, the congresswoman typed out her thoughts, used a stylus to move the text around, hit play and then the AI program put that text into Wexton’s voice. It’s a lengthy process, so the AP provided Wexton with a few questions ahead of the interview to give the congresswoman time to type her answers.

Wexton was diagnosed with progressive supranuclear palsy in 2023, an aggressive neurological disorder that impacts many aspects of life, including speech. Sitting across from a credenza filled with photos marking the high points of her personal life - weddings, family trips, her children - the congresswoman called the diagnosis “cruel” for someone whose “entire professional life has been built around using my voice,” from Virginia prosecutor to state Senator to member of Congress.

“A politician who can’t do public speaking will be a former politician before too long. But this AI voice model has given me a new opportunity to have my voice heard and it reminds listeners that I am still me,” Wexton told the AP.

The congresswoman, whose runaway win in 2018 signaled the success Democrats would have that year, initially announced a Parkinson’s diagnosis in April 2023 , striking an upbeat tone by telling supporters they were “welcome to empathize” with her, but not to “feel sorry for me.” Her tone in September 2023 was vastly different : She described her PSP diagnosis as “Parkinson’s on steroids” and said she would not seek reelection in 2024.

“This new diagnosis is a tough one. There is no ‘getting better’ with PSP. I’ll continue treatment options to manage my symptoms, but they don’t work as well with my condition as they do for Parkinson’s,” she said at the time.

The diagnosis has changed Wexton’s personal and professional life. The congresswoman doesn’t look like she once did. Her posture slumped, her movements less precise, her natural voice muted - all impacts of the disease. As it became more difficult for Wexton to use her voice, she turned to a traditional text-to-speech app that many people with speech disorders often use. The voice sounded more like a robot than a human, but Wexton used it to conduct interviews and give speeches.

“This is not a situation I would have chosen to find myself in,” she said from the House floor. “I never thought that at my age and otherwise good health, something like PSP could, in the space of just over a year, rob me of my ability to speak, run or dance, and force me to stop doing the job that I love.”

ElevenLabs, a start-up with one of the most widely used AI-powered voice cloning models, saw Wexton speak using the older technology. They contacted her office several weeks ago and Wexton’s aides provided the company with several recordings, mostly speeches she had given as a member of Congress.

“Our technology gives individuals who have lost their voice the ability to speak as they once did, with the emotion and passion they feel, and we hoped to help the Congresswoman do just that,” said Dustin Blank, Head of Partnerships at the company.

Wexton told AP she first used the cloned voice to speak with President Joe Biden in the Oval Office earlier this month when he signed the National Plan to End Parkinson’s Act, a bill that Wexton called the “most consequential action we have taken in decades to combat Parkinson’s and related diseases, like my PSP.” A few days later, Wexton publicly debuted her cloned voice in a video, leading to an outpouring of support and thrusting the congresswoman into a debate over AI.

This is “not the way I thought I would be leaving Congress,” she said. “I didn’t anticipate being at the forefront of a debate over the future of AI.”

Using AI-powered cloning to give Wexton her voice back is one of the positive applications of this technology. However, voice cloning has also been used nefariously, like defrauding people and pushing fake political messaging. The most notable of these instances was when an AI-generated robocall impersonating President Joe Biden urged voters ahead of the New Hampshire primary not to vote. The call was quickly reported and resulted in serious consequences for those behind it, but the incident raised serious questions about the future of this technology and the companies behind it.

Wexton, whose district is home to scores of data centers that power AI, harbors those questions, too. After she debuted her voice clone, Wexton jokingly texted a few friends the same message: “AI isn’t entirely evil, just mostly.”

Hany Farid, a professor and digital forensics expert at the University of California, Berkeley, said Wexton’s example is the exception to the numerous nefarious uses for voice cloning technology.

“I found it really moving… and I am all for this application,” he said. “But I just want to emphasize, just because there are these really beautiful stories… doesn’t mean we should just ignore the pretty nasty things with these technologies.”

One way to ensure the technology is being used for good, said Farid, is “better checks and balances” to ensure “people aren’t doing nefarious things with your products.” That includes content credentials that say how the audio was developed, storage of all audio created using the technology and know-your-customer rules that require voice cloning companies to know who is using their technology.

Wexton agrees more guardrails are needed. Her team of advisers has taken precautions to make sure her likeness is protected, from limiting access to the voice to only three people and tightening security on the program.

“It is humanizing and it is empowering. It can also be dangerous,” she said. “I still believe that the dangerous potential of AI technology must be better understood and steps must be taken to prevent abuses of the technology like deepfakes from proliferating and part of that falls on lawmakers like us in Congress,” she later added.

In 2019, Wexton won bipartisan approval for an amendment directing the National Science Foundation to research public awareness around deepfake videos generated by AI.

Wexton also said the technology isn’t perfect. Because the audio used came from speeches and public events, it isn’t great for regular conversation, often making everything sound “like some big proclamation.” Her two college-aged sons, she said, don’t like it for that reason and, she quipped, she doesn’t use it to “ask my husband to please pass me the ketchup,” displaying a sense of humor that she is known for on Capitol Hill.

“At the end of the day, it will never be me. But it is more me than I ever could have hoped I could hear again and for that, I am so grateful and excited,” she said. “I plan to make the most of it.”

For doctors like Jori Fleisher, the Director of Rush CurePSP Center of Care , that sentiment is why this kind of technology could be life-altering for those diagnosed with the rare neurological disorder.

Too often PSP patients lose their voices and have to rely on traditional speech-to-text programs to communicate, Fleisher said. But those programs use robotic voices that often sound nothing like the patients. Fleisher notes that people with “neurological diseases are already stigmatized,” so speaking with a voice that sounds like a computer “perpetuates the stigma” and often leads them to withdraw from relationships and “worsens the social isolation that can be such a huge part of these conditions.”

“To know of and already deeply respect Representative Wexton and then hear her speak so beautifully in her own voice, using her own words through this technology, it is giving me goosebumps now,” she said, growing emotional. “It’s so empowering.”

The key, Fleisher added, is making this technology available to more people by encouraging patients in the early stages of PSP and other neurological disorders to “bank enough sounds from your own voice that it could be used later” and for insurance companies to cover this kind of treatment. Wexton said she tried to do this late last year through an Apple program, but her voice was already too impacted by the disease for their AI to use.

Wexton’s new voice particularly helps in more emotional moments when hearing sentiment in her speech is significantly more powerful than a more robotic sound. When asked how Barbara Comstock, the Republican congresswoman Wexton has grown close to since defeating her in 2018, had helped support her since Wexton revealed her diagnosis, the Democrat grew emotional and said, “She has been so gracious.”

“I was just thrilled for her,” Comstock said, recalling when she first heard Wexton’s AI voice. “Just great to hear she is getting her literal voice out there for others to see the power of the technology. … I am getting teary thinking about it again.”

After defeating Comstock in 2018, Wexton’s future in Virginia politics was bright, with many in the state speculating she could seek higher office. Her diagnosis has taken that future away — her political career will end next year — but it has given Wexton a new resolve.

“I want to be a voice, even an AI voice, for Americans facing accessibility challenges and other disabilities because too often people only see us for that disability,” Wexton said. “I hope that by continuing to do my job to the best of my ability, whether that means using a walker or a wheelchair to get to the House floor to vote or delivering my speeches through an AI-recreated version of my voice, that it can help show I am just as much me on the inside that I have always been.”

The Associated Press receives financial assistance from the Omidyar Network to support coverage of artificial intelligence and its impact on society. AP is solely responsible for all content. Find AP’s standards for working with philanthropies, a list of supporters and funded coverage areas at AP.org

10 Most Common Speech-Language Disorders & Impediments

As you get to know more about the field of speech-language pathology you’ll increasingly realize why SLPs are required to earn at least a master’s degree . This stuff is serious – and there’s nothing easy about it.

In 2016 the National Institute on Deafness and Other Communication Disorders reported that 7.7% of American children have been diagnosed with a speech or swallowing disorder. That comes out to nearly one in 12 children, and gets even bigger if you factor in adults.

Whether rooted in psycho-speech behavioral issues, muscular disorders, or brain damage, nearly all the diagnoses SLPs make fall within just 10 common categories…

Types of Speech Disorders & Impediments

Apraxia of speech (aos).

Apraxia of Speech (AOS) happens when the neural pathway between the brain and a person’s speech function (speech muscles) is lost or obscured. The person knows what they want to say – they can even write what they want to say on paper – however the brain is unable to send the correct messages so that speech muscles can articulate what they want to say, even though the speech muscles themselves work just fine. Many SLPs specialize in the treatment of Apraxia .

There are different levels of severity of AOS, ranging from mostly functional, to speech that is incoherent. And right now we know for certain it can be caused by brain damage, such as in an adult who has a stroke. This is called Acquired AOS.

However the scientific and medical community has been unable to detect brain damage – or even differences – in children who are born with this disorder, making the causes of Childhood AOS somewhat of a mystery. There is often a correlation present, with close family members suffering from learning or communication disorders, suggesting there may be a genetic link.

Mild cases might be harder to diagnose, especially in children where multiple unknown speech disorders may be present. Symptoms of mild forms of AOS are shared by a range of different speech disorders, and include mispronunciation of words and irregularities in tone, rhythm, or emphasis (prosody).

Stuttering – Stammering

Stuttering, also referred to as stammering, is so common that everyone knows what it sounds like and can easily recognize it. Everyone has probably had moments of stuttering at least once in their life. The National Institute on Deafness and Other Communication Disorders estimates that three million Americans stutter, and reports that of the up-to-10-percent of children who do stutter, three-quarters of them will outgrow it. It should not be confused with cluttering.

Most people don’t know that stuttering can also include non-verbal involuntary or semi-voluntary actions like blinking or abdominal tensing (tics). Speech language pathologists are trained to look for all the symptoms of stuttering , especially the non-verbal ones, and that is why an SLP is qualified to make a stuttering diagnosis.

The earliest this fluency disorder can become apparent is when a child is learning to talk. It may also surface later during childhood. Rarely if ever has it developed in adults, although many adults have kept a stutter from childhood.

Stuttering only becomes a problem when it has an impact on daily activities, or when it causes concern to parents or the child suffering from it. In some people, a stutter is triggered by certain events like talking on the phone. When people start to avoid specific activities so as not to trigger their stutter, this is a sure sign that the stutter has reached the level of a speech disorder.

The causes of stuttering are mostly a mystery. There is a correlation with family history indicating a genetic link. Another theory is that a stutter is a form of involuntary or semi-voluntary tic. Most studies of stuttering agree there are many factors involved.

Dysarthria is a symptom of nerve or muscle damage. It manifests itself as slurred speech, slowed speech, limited tongue, jaw, or lip movement, abnormal rhythm and pitch when speaking, changes in voice quality, difficulty articulating, labored speech, and other related symptoms.

It is caused by muscle damage, or nerve damage to the muscles involved in the process of speaking such as the diaphragm, lips, tongue, and vocal chords.

Because it is a symptom of nerve and/or muscle damage it can be caused by a wide range of phenomena that affect people of all ages. This can start during development in the womb or shortly after birth as a result of conditions like muscular dystrophy and cerebral palsy. In adults some of the most common causes of dysarthria are stroke, tumors, and MS.

A lay term, lisping can be recognized by anyone and is very common.

Speech language pathologists provide an extra level of expertise when treating patients with lisping disorders . They can make sure that a lisp is not being confused with another type of disorder such as apraxia, aphasia, impaired development of expressive language, or a speech impediment caused by hearing loss.

SLPs are also important in distinguishing between the five different types of lisps. Most laypersons can usually pick out the most common type, the interdental/dentalised lisp. This is when a speaker makes a “th” sound when trying to make the “s” sound. It is caused by the tongue reaching past or touching the front teeth.

Because lisps are functional speech disorders, SLPs can play a huge role in correcting these with results often being a complete elimination of the lisp. Treatment is particularly effective when implemented early, although adults can also benefit.

Experts recommend professional SLP intervention if a child has reached the age of four and still has an interdental/dentalised lisp. SLP intervention is recommended as soon as possible for all other types of lisps. Treatment includes pronunciation and annunciation coaching, re-teaching how a sound or word is supposed to be pronounced, practice in front of a mirror, and speech-muscle strengthening that can be as simple as drinking out of a straw.

Spasmodic Dysphonia

Spasmodic Dysphonia (SD) is a chronic long-term disorder that affects the voice. It is characterized by a spasming of the vocal chords when a person attempts to speak and results in a voice that can be described as shaky, hoarse, groaning, tight, or jittery. It can cause the emphasis of speech to vary considerably. Many SLPs specialize in the treatment of Spasmodic Dysphonia .

SLPs will most often encounter this disorder in adults, with the first symptoms usually occurring between the ages of 30 and 50. It can be caused by a range of things mostly related to aging, such as nervous system changes and muscle tone disorders.

It’s difficult to isolate vocal chord spasms as being responsible for a shaky or trembly voice, so diagnosing SD is a team effort for SLPs that also involves an ear, nose, and throat doctor (otolaryngologist) and a neurologist.

Have you ever heard people talking about how they are smart but also nervous in large groups of people, and then self-diagnose themselves as having Asperger’s? You might have heard a similar lay diagnosis for cluttering. This is an indication of how common this disorder is as well as how crucial SLPs are in making a proper cluttering diagnosis .

A fluency disorder, cluttering is characterized by a person’s speech being too rapid, too jerky, or both. To qualify as cluttering, the person’s speech must also have excessive amounts of “well,” “um,” “like,” “hmm,” or “so,” (speech disfluencies), an excessive exclusion or collapsing of syllables, or abnormal syllable stresses or rhythms.

The first symptoms of this disorder appear in childhood. Like other fluency disorders, SLPs can have a huge impact on improving or eliminating cluttering. Intervention is most effective early on in life, however adults can also benefit from working with an SLP.

Muteness – Selective Mutism

There are different kinds of mutism, and here we are talking about selective mutism. This used to be called elective mutism to emphasize its difference from disorders that caused mutism through damage to, or irregularities in, the speech process.

Selective mutism is when a person does not speak in some or most situations, however that person is physically capable of speaking. It most often occurs in children, and is commonly exemplified by a child speaking at home but not at school.

Selective mutism is related to psychology. It appears in children who are very shy, who have an anxiety disorder, or who are going through a period of social withdrawal or isolation. These psychological factors have their own origins and should be dealt with through counseling or another type of psychological intervention.

Diagnosing selective mutism involves a team of professionals including SLPs, pediatricians, psychologists, and psychiatrists. SLPs play an important role in this process because there are speech language disorders that can have the same effect as selective muteness – stuttering, aphasia, apraxia of speech, or dysarthria – and it’s important to eliminate these as possibilities.

And just because selective mutism is primarily a psychological phenomenon, that doesn’t mean SLPs can’t do anything. Quite the contrary.

The National Institute on Neurological Disorders and Stroke estimates that one million Americans have some form of aphasia.

Aphasia is a communication disorder caused by damage to the brain’s language capabilities. Aphasia differs from apraxia of speech and dysarthria in that it solely pertains to the brain’s speech and language center.

As such anyone can suffer from aphasia because brain damage can be caused by a number of factors. However SLPs are most likely to encounter aphasia in adults, especially those who have had a stroke. Other common causes of aphasia are brain tumors, traumatic brain injuries, and degenerative brain diseases.

In addition to neurologists, speech language pathologists have an important role in diagnosing aphasia. As an SLP you’ll assess factors such as a person’s reading and writing, functional communication, auditory comprehension, and verbal expression.

Speech Delay – Alalia

A speech delay, known to professionals as alalia, refers to the phenomenon when a child is not making normal attempts to verbally communicate. There can be a number of factors causing this to happen, and that’s why it’s critical for a speech language pathologist to be involved.

The are many potential reasons why a child would not be using age-appropriate communication. These can range anywhere from the child being a “late bloomer” – the child just takes a bit longer than average to speak – to the child having brain damage. It is the role of an SLP to go through a process of elimination, evaluating each possibility that could cause a speech delay, until an explanation is found.

Approaching a child with a speech delay starts by distinguishing among the two main categories an SLP will evaluate: speech and language.

Speech has a lot to do with the organs of speech – the tongue, mouth, and vocal chords – as well as the muscles and nerves that connect them with the brain. Disorders like apraxia of speech and dysarthria are two examples that affect the nerve connections and organs of speech. Other examples in this category could include a cleft palette or even hearing loss.

The other major category SLPs will evaluate is language. This relates more to the brain and can be affected by brain damage or developmental disorders like autism. There are many different types of brain damage that each manifest themselves differently, as well as developmental disorders, and the SLP will make evaluations for everything.

Issues Related to Autism

While the autism spectrum itself isn’t a speech disorder, it makes this list because the two go hand-in-hand more often than not.

The Centers for Disease Control and Prevention (CDC) reports that one out of every 68 children in our country have an autism spectrum disorder. And by definition, all children who have autism also have social communication problems.

Speech-language pathologists are often a critical voice on a team of professionals – also including pediatricians, occupational therapists, neurologists, developmental specialists, and physical therapists – who make an autism spectrum diagnosis .

In fact, the American Speech-Language Hearing Association reports that problems with communication are the first detectable signs of autism. That is why language disorders – specifically disordered verbal and nonverbal communication – are one of the primary diagnostic criteria for autism.

So what kinds of SLP disorders are you likely to encounter with someone on the autism spectrum?

A big one is apraxia of speech. A study that came out of Penn State in 2015 found that 64 percent of children who were diagnosed with autism also had childhood apraxia of speech.

This basic primer on the most common speech disorders offers little more than an interesting glimpse into the kind of issues that SLPs work with patients to resolve. But even knowing everything there is to know about communication science and speech disorders doesn’t tell the whole story of what this profession is all about. With every client in every therapy session, the goal is always to have the folks that come to you for help leave with a little more confidence than when they walked in the door that day. As a trusted SLP, you will build on those gains with every session, helping clients experience the joy and freedom that comes with the ability to express themselves freely. At the end of the day, this is what being an SLP is all about.

Ready to make a difference in speech pathology? Learn how to become a Speech-Language Pathologist today

• Emerson College - Master's in Speech-Language Pathology online - Prepare to become an SLP in as few as 20 months. No GRE required. Scholarships available.
• Arizona State University - Online - Online Bachelor of Science in Speech and Hearing Science - Designed to prepare graduates to work in behavioral health settings or transition to graduate programs in speech-language pathology and audiology.
• NYU Steinhardt - NYU Steinhardt's Master of Science in Communicative Sciences and Disorders online - ASHA-accredited. Bachelor's degree required. Graduate prepared to pursue licensure.
• Calvin University - Calvin University's Online Speech and Hearing Foundations Certificate - Helps You Gain a Strong Foundation for Your Speech-Language Pathology Career.

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How do you say this in Japanese? I have a speech impediment

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Speech impediment's equivalent phrase in Japanese is "言語障害." If you mean stuttering, the translation would be: 私は(少し)ドモリます。

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Does Anya have some sort of speech impediment in the original Japanese version?

The official Polish translation gave her a lisp so I've been wondering about that.

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