Electroretinography in adults with high-functioning autism spectrum disorder

Abstract

The electroretinogram (ERG) allows the investigation of retinal signaling pathways and has increasingly been applied in individuals with mental disorders in search for potential biomarkers of neurodevelopmental disorders. Preceding ERG examinations in individuals with autism spectrum disorders (ASD) showed inconsistent results, which might be due to the small number of participants, heterogeneity of the ASD population, differences in age ranges, and stimulation methods. The aim of this study was to investigate functional retinal responses in adults with ASD by means of the light-adapted (photopic) ERG. Light-adapted ERG measurements were obtained with the RETeval ® system applying three different stimulation protocols. In the final analysis, the ERG parameters a-wave, b-wave, the photopic negative response (PhNR), the photopic hill parameters as well as additional amplitude ratios were compared between 32 adults with high-functioning ASD and 31 non-autistic controls. Both groups were matched with regard to sex and age. No significant functional retinal differences in amplitude or peak time of the a- or b-wave, PhNR, the photopic hill parameters or the ERG-amplitude ratios could be detected in individuals with ASD compared to non-autistic participants. The absence of electrophysiological functional retinal alterations in ASD, suggests that changes in visual perception, such as increased attention to detail or visual hypersensitivity in ASD, are not due to impairments at early levels of retinal signal processing.

Full text

RESEARCH ARTICLE
Electroretinography in adults with high-functioning autism
spectrum disorder
Evelyn B. N. Friedel
1,2,3
| Mirjam Schäfer
1
| Dominique Endres
1
| Simon Maier
1
|
Kimon Runge
1
| Michael Bach
2
| Sven P. Heinrich
2
| Dieter Ebert
1
|
Katharina Domschke
1,4
| Ludger Tebartz van Elst
1
| Kathrin Nickel
1
1
Department of Psychiatry and Psychotherapy,
Medical Center –University of Freiburg,
Faculty of Medicine, University of Freiburg,
Freiburg, Germany
2
Eye Center, Medical Center –University of
Freiburg, Faculty of Medicine, University of
Freiburg, Freiburg, Germany
3
Faculty of Biology, University of Freiburg,
Freiburg, Germany
4
Center for Basics in Neuromodulation, Faculty
of Medicine, University of Freiburg, Freiburg,
Germany
Correspondence
PD Dr. Kathrin Nickel, Hauptstraße 5, 79104
Freiburg, Germany.
Email: kathrin.nickel@uniklinik-freiburg.de
Funding information
The study was funded by the “German Research
Foundation”(DFG ID: HE 3504/11-1jTE
280/24-1).
Abstract
The electroretinogram (ERG) allows the investigation of retinal signaling path-
ways and has increasingly been applied in individuals with mental disorders in
search for potential biomarkers of neurodevelopmental disorders. Preceding ERG
examinations in individuals with autism spectrum disorders (ASD) showed incon-
sistent results, which might be due to the small number of participants, heteroge-
neity of the ASD population, differences in age ranges, and stimulation methods.
The aim of this study was to investigate functional retinal responses in adults with
ASD by means of the light-adapted (photopic) ERG. Light-adapted ERG mea-
surements were obtained with the RETeval
®
system applying three different stim-
ulation protocols. In the final analysis, the ERG parameters a-wave, b-wave, the
photopic negative response (PhNR), the photopic hill parameters as well as addi-
tional amplitude ratios were compared between 32 adults with high-functioning
ASD and 31 non-autistic controls. Both groups were matched with regard to sex
and age. No significant functional retinal differences in amplitude or peak time of
the a- or b-wave, PhNR, the photopic hill parameters or the ERG-amplitude
ratios could be detected in individuals with ASD compared to non-autistic partici-
pants. The absence of electrophysiological functional retinal alterations in ASD,
suggests that changes in visual perception, such as increased attention to detail or
visual hypersensitivity in ASD, are not due to impairments at early levels of reti-
nal signal processing.
Lay Summary
The electroretinogram (ERG), an ophthalmologic method to assess the integrity
of retinal functioning, was investigated in individuals with autism spectrum disor-
der compared to non-autistic controls. Individuals with autism spectrum disorder
showed normal ERG parameters when compared to non-autistic controls. These
results suggest that changes in the visual perception or visual hypersensitivity in
autism spectrum disorder are not due to dysfunctions at early levels of retinal sig-
nal processing.
KEYWORDS
ASD, autism spectrum disorder, a-wave, b-wave, electroretinogram, ERG, PhNR, photopic negative
response
Evelyn B. N. Friedel and Mirjam Schäfer are co-first authors. Ludger Tebartz van Elst and Kathrin Nickel are co-senior authors.
Received: 25 July 2022 Accepted: 19 September 2022
DOI: 10.1002/aur.2823
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Autism Research published by International Society for Autism Research and Wiley Periodicals LLC.
Autism Research. 2022;1–12. wileyonlinelibrary.com/journal/aur 1

INTRODUCTION
Despite decades of psychiatric research on autism spec-
trum disorders (ASD), the exact etiology of ASD remains
unclear in most cases (Lord et al., 2018). A multifactorial
genesis with a strong genetic component and a contribu-
tion of environmental factors is discussed
(Currenti, 2010). In addition to key ASD symptoms, such
as impairments in social communication and interaction
as well as restricted behaviors or interests (American Psy-
chiatric Association, 2013), alterations in the processing
of sensory information as an important concomitant have
increasingly gained the attention of autism research. In
the Diagnostic and Statistical Manual of Mental Disor-
ders (DSM-5), the hyper- and hyporeactivity to sensory
information or special interest in sensory aspects of the
environment were considered for the first time (American
Psychiatric Association, 2013). With regard to visual sen-
sory symptoms, a hypersensitivity in terms of focusing on
tiny pieces and details, disliking bright lights or reduced
eye contact as well as a hyposensitivity with an attraction
to light and a fascination for reflections have been
described (Simmons et al., 2009).
Therefore, various studies have attempted to identify
associations between perceptual difficulties and impair-
ments in the underlying visual processing in individuals
with ASD (Little, 2018).
In this context, the electroretinogram (ERG), an oph-
thalmological method for the assessment of the functional
integrity of the retina (Robson et al., 2018,2022)waspre-
viously applied in ASD. The ERG represents the electrical
response of the retina following photopic stimulation
(Creel, 2015). Depending on the adaptational state of the
retina (light (LA)- or dark-adapted (DA)) and the selected
flash parameters for stimulation, different ERG compo-
nents can be distinguished and evaluated (Frishman
et al., 2018; Robson et al., 2022). The a- and b-wave as
well as the photopic negative response (PhNR) represent
the most common and important ERG components
(Figure 1). In the light-adapted ERG (LA-ERG), the a-
wave is composed of cone photoreceptor activity in the
outer retina and contributions of cone OFF-bipolar cells
in the inner retina, while the b-wave is the combined cone
ON- and OFF-bipolar cell response and reflects the inner
retina integrity (Creel, 2015; Robson et al., 2018,2022).
The PhNR following the b-wave arises in the innermost
retina, and is primarily related to the function of the reti-
nal ganglion cells (Frishman et al., 2018;Wuetal.,2016).
Observations of preceding studies applying ERG in
individuals with ASD are inconsistent (Table 1). This
may be due to small sample sizes, differences in the inves-
tigated age groups, heterogeneity of the ASD populations
(e.g., primary and secondary forms), and the different
ERG methods applied, such as DA- or LA-ERG
examinations.
Two earlier studies assessing both children and adults
with ASD pointed toward a smaller DA-ERG b-wave
amplitude in 48% (Ritvo et al., 1988) and 45% (Creel
et al., 1989) of the evaluated individuals with an ASD.
Additionally, an increased peak time for the DA-ERG b-
wave was reported in ASD, while no alterations of the a-
wave were detected (Constable et al., 2016; Creel
et al., 1989).
Furthermore, Constable et al. (2016) observed a
decreased b-wave amplitude in the LA-ERG of a small
sample (N=14) of children and adults with high-
functioning ASD following a stimulation with the stan-
dard ISCEV flash (0.5 log phot cds/m
2
; Robson
et al., 2022). Moreover, they detected smaller ON
responses using a prolonged 120 ms flash stimulus as well
as differences in the shape of the LA oscillatory poten-
tials (OPs) in younger individuals with ASD, while no
group differences were reported in the DA-15-Hz- or LA-
30-Hz-flicker phase and amplitude responses (Constable
et al., 2016).
In a subsequent LA-ERG study, the same authors
confirmed smaller b-wave amplitudes, but only at higher
flash strengths (Constable et al., 2020). They additionally
found attenuated a-wave amplitudes at higher flash
strengths, as well as increased b-wave peak times in chil-
dren and adolescents with ASD (Constable et al., 2020).
Another investigation (Constable et al., 2022) analyzed
LA-ERG responses in ASD with a discrete wavelet trans-
form approach and not only confirmed decreased b-wave
amplitudes at high flash strengths but also found a
reduced energy of the OPs in individuals with ASD,
which might contribute to the observed b-wave alter-
ations. The OPs mainly originating in the amacrine cells
are reported to be sensitive to disruption of dopamine- or
GABA-modulated neuronal pathways (Constable
et al., 2022; Wachtmeister, 1998). A recent study
described a promising machine learning model for ASD
detection based on ERG data using spectral and time-
FIGURE 1 Exemplary representation of an ERG curve in response
to a stimulation with the (C) PhNR-flash. The a- and b-wave as well as
the photopic negative response (PhNR) at minimum and at 72 ms are
depicted. A-wave and PhNR amplitudes were measured from the pre-
stimulus baseline to the respective troughs. The amplitude of the b-wave
was defined from the a-wave minimum to the subsequent maximum
deflection. PhNR, photopic negative response.
2FRIEDEL ET AL.

TABLE 1 Previous ERG studies in individuals with ASD
Study N subjects
Age in years mean SD
(range)
Sex: male/female,
ethnicity IQ mean SD (range) Psychiatric medication
Diagnosis and psychometric
instruments Method Results (HC vs. ASD/ADHD)
(Ritvo et al., 1988) 27 autistic individuals (4–60) 22 m/5 f <50: 3 50–69: 6 >70: 18 4 medicated (1 phenobarbital,
2 thioridazine, 1 anesthesia)
Clinical assessment, DSM-III
299.0
fERG: (1) scotopic dim blue (2)
scotopic red (3) scotopic bright
white (4) red 30-Hz flicker (5)
photopic bright-white
DA-ERG -b-wave amplitude #
in 13 (48%) autistic individuals
20 HC 21.9 12.8 (10–54) 15 m/5 f normal
(Creel et al., 1989) 22 HFA (7–60) 19 m/3 f n.a. 2 thioridazine Clinical assessment fERG: (1) scotopic dim blue (2)
scotopic red (3) scotopic bright
white (4) red 30-Hz flicker (5)
photopic bright-white
DA-ERG -b-wave amplitude #
in 10 (45%) HFA -b-wave
implicit times "in 5 (23%)
HFA
50 HC similar range 43 m/7 f n.a.
(Tebartz van Elst
et al., 2015)
33 ASD 39.5 1.9 22 m/11 f n.a. 10 with antidepressant
medication (i.e., SSRI, SNRI,
SNRI with mirtazapine)
Asperger’s syndrome (ICD-10:
F84.5), ASD (DSM-IV: 299.80)
Clinical assessment, AQ, EQ,
BVAQ, ADI-R, ADOS
PERG No differences in visual acuity,
PERG contrast gain,
background noise
33 HC 34.4 2.1 21 m/12 f n.a.
(Constable
et al., 2016)
11 HFA 37.2 13.2 (13.8–57.6) 10 m/1 f 116 10 n.a. Clinical assessment, DSM-IV-
TR, ADOS (10/11), AQ
fERG: DA-ERG 15-Hz flicker
-flash series (Naka-Rushton
curves) LA-ERG 30-Hz flicker
120 ms flash (ON–OFF) -flash
series (photopic hill)
DA-ERG -b-wave time to peak
"-No alteration in a- or b-
wave amplitude or a-wave
peak time LA-ERG -b-wave
amplitude #-No alteration in
a-or b-wave peak time, or
PhNR -No alteration in
photopic hill parameters -OP
alterations
15 HC (DA-ERG) 36.9 13.2 (12–58) 11 m/4 f n.a.
14 HC (LA-ERG) 35.3 12.9 (14–58) 11 m/3 f
(Constable
et al., 2020)
90 ASD (177 eyes) 13.0 4.2 (6–25.8) 130 m/47 f (eyes)
143 caucasian
98.9 16.5 12 dopamine antagonists, 7
SSRI, 6 melatonin
DSM-IV/5, ADOS, ADOS-2 LA-fERG with RETeval
®
-ISCEV nine-step flash series
LA-ERG -a- and b-wave
amplitude #at high flash
strengths -b-wave peak time "
-b:a ratio #-Altered photopic
hill parameters
87 HC (174 eyes) 13.8 4.8 (5.4–26.6) 82 m/92 f (eyes)
117 caucasian
n.a. 1 SSRI
(Constable
et al., 2021)
55 ASD 13.6 4.7 (5.4–26.7) 41 m/14 f 99 19 (60–136) 9 CNS-acting medication (1
tegretol, 8 medications that
targeted dopamine and seotonin
levels)
DSM-IV/5, ADOS, ADOS-2,
3Di
LA-fERG with RETeval
®
-ISCEV nine-step flash series
LA-ERG -b-wave amplitude#
-PhNR and PhNR at 72 ms
unaltered
87 HC 14.0 4.8 (5.4–27.3) 43 m/44 f n.a.
(Lee et al., 2022) 57 ASD 13.7 4.8 (6–27) 43 m/13 f 47 caucasian,
3 asian,
1 afro-caribbean,
1 latino
99.6 18.9 4 SSRI, 2 methylphenidate, 1
dopamine antagonist, 1
antiepileptic medication
DSM-IV/5, ICD-10, ADOS,
ADOS-2, 3Di
LA-fERG with RETeval
®
-ISCEV nine-step flash series
LA-ERG (ASD) -b-wave
amplitude #LA-ERG
(ADHD) -b-wave amplitude "
-faster b-wave peak time
-PhNR amplitude at 72 ms "
15 ADHD 15.3 3.5 (8–20) 8 m/7 f 11 caucasian,
2 asian, 1
afro-caribbean, 1 latino
92.9 14.2 7 methylphenidate
59 HC 13.3 4.6 (5–25) 31 m/28 f 35 caucasian,
18 asian, 6 mixed
n.a.
(Continues)
FRIEDEL ET AL.3

domain features which reportedly achieves a classifica-
tion accuracy of 86% and sensitivity of 98%
(Mohammad-Manjur et al., 2022).
Furthermore, differences in the parameters describing
the photopic hill of the b-wave amplitudes in response to
a flash series have been reported in individuals with ASD
(Constable et al., 2020). The photopic hill describes the
phenomenon that as the brightness of the stimuli
increases, the amplitude of the photopic b-wave
increases, reaches a brief saturation, and then declines
again to a non-zero plateau (Hamilton et al., 2007;
McCulloch et al., 2019).
However, the investigation of the PhNR points
toward normal ganglion cell function in individuals with
ASD (Constable et al., 2021). Correspondingly, a pattern
electroretinogram (PERG) study investigating 33 adults
with high-functioning autism compared to 33 healthy
controls (HC) detected no alterations in the PERG con-
trast gain, background noise or visual acuity (Tebartz
van Elst et al., 2015).
Aims of the study
We aimed at investigating ERG parameters including the
a-wave, the b-wave, the PhNR and the photopic hill
parameters applying several ERG stimulus protocols in
adults with ASD compared to HC. Based on the previous
LA-ERG investigations, we expected a reduced ampli-
tude of the b-wave and alterations in photopic hill
parameters in individuals with ASD.
METHODS
Participants
All examinations were performed at the Department of
Psychiatry and Psychotherapy of the University Medical
Center Freiburg. The local Ethics Committee approved
the study (Approval ID: 314/18). All participants gave
written informed consent.
Individuals with ASD met diagnostic criteria of Asper-
ger’s syndrome according to the criteria of the International
Classification of Diseases (ICD-10) (F84.5) and ASD
according to the Diagnostic and Statistical Manual of Men-
tal Disorders (DSM-5) (299.00). In addition to the diagnosis
established by an experienced senior psychiatrist, autistic
symptoms were assessed with the following questionnaires:
the Autism Spectrum Quotient (AQ) (Baron-Cohen
et al., 2001), the Empathy Quotient (EQ) (Baron-Cohen &
Wheelwright, 2004), and the Social Responsiveness Scale
2 (SRS-2) (Constantino, 2013). To investigate a homoge-
neous study sample, individuals with secondary syndromal
genetic forms of autism were not included.
HC were matched with regard to sex and age to the
ASD group and filled in the above questionnaires to rule
TABLE 1 (Continued)
Study N subjects
Age in years mean SD
(range)
Sex: male/female,
ethnicity IQ mean SD (range) Psychiatric medication
Diagnosis and psychometric
instruments Method Results (HC vs. ASD/ADHD)
(Constable
et al., 2022)
55 ASD 14.2 4.9 (6–27.3) 40 m/15 f 101 20 7 methylphenidate, 1
antiepileptic medication
DSM-IV-TR/DSM-5, ADOS-2,
3Di
LA-fERG with RETeval
®
-Five
flash series -DWT-analysis
LA-ERG (ASD) -b-wave
amplitude #at high flash
strengths -OP energy (op80 and
op160 coefficients) #LA-ERG
(ADHD) -b-wave amplitude "
at high flash strengths -b20 and
b40 coefficients "
15 ADHD 15.8 3.2 (8.4–21.8) 8 m/7 f 88 10 1 methylphenidate
156 HC 13.2 5.0 (3.1–26.7) 50 m/112 f n.a.
Abbreviations: ADHD, attention-deficit/hyperactivity disorder; ADI-R, Autism Diagnostic Interview –revised (Lord et al., 1994); ADOS, Autism Diagnostic Observation Schedule (Lord et al., 2000); ASD, autism spectrum disorder; AQ, Autism Spectrum Quotient
(Baron-Cohen et al., 2001); BVAQ, Bermond-Vorst Alexithymia Questionnaire; CNS, central nervous system; DA, dark-adapted; DSM, Diagnostic and Statistical Manual; 3Di, Developmental, dimensional and diagnostic interview (Skuse et al., 2004); DWT, discrete
wavelet transform; EQ, Empathy Quotient (Baron-Cohen et al., 2004); f, female; fERG, flash electroretinogram; HC, healthy controls; HFA, high-functioning autism; Hz, Hertz; ICD, International Classification of Diseases; IQ, intelligence quotient; ISCEV,
International Society for Clinical Electrophysiology of Vision; LA, light-adapted; m, male; N, number; n.a., not available; OP, oscillatory potential; PERG, pattern electroretinogram; PhNR, photopic negative response; SD, standard deviation; SEM, standard error of
the mean; SNRI, Serotonin norepinephrine reuptake inhibitor; SSRI, Selective serotonin reuptake inhibitor.
4FRIEDEL ET AL.

out the presence of an ASD. The following instruments
were collected for both study groups: the Structured Clin-
ical Interview for DSM (SCID-I and -II; [Wittchen
et al., 1997]) and the Symptom Checklist (SCL-90-R;
[Derogatis & Savitz, 1999]) to rule out other psychiatric
diseases, the Beck Depression Inventory (BDI-II; [Beck
et al., 1961; Hautzinger et al., 2006]) to assess depressive
symptoms in the ASD and to exclude them in the HC
group as well as the Wender-Utah Rating Scale (WURS-
k; [Retz-Junginger et al., 2002]) to evaluate the presence
of symptoms of an attention-deficit/hyperactivity disor-
der (ADHD) in childhood. The crystallized intelligence
was estimated with the Multiple Choice Vocabulary Test
(MWT-B; [Lehrl et al., 1995]).
Exclusion criteria were the presence of any psychiatric
disease for the HC group and psychotic symptoms, bipo-
lar disorder or substance abuse for the ASD group.
Depression and ADHD are frequent comorbidities of
ASD. Therefore, they were not defined as exclusion cri-
teria for the ASD group. An age < 18 years or > 65 -
years, ophthalmological diseases (except for correctable
refraction errors), myopia exceeding 6 dpt, or hyper-
opia above 6 dpt, a decimal visual acuity below 0.7 in the
Freiburg Visual Acuity and Contrast Test (FrACT;
[Bach, 2007]), somatic diseases such as diabetes mellitus
or arterial hypertension, neurological diseases, for exam-
ple seizures, were further defined as exclusion criteria for
both groups.
The individuals with ASD and HC already partici-
pated in an optical coherence tomography (OCT) study
of our research group (Friedel et al., 2022, unpublished).
ERG recordings and data extraction
LA-ERG measurements of both eyes were performed
monocularly with the RETeval
®
system from LKC Tech-
nologies, Inc. (2016) and the corresponding software (ver-
sion 2.10.2). Sensor Strip skin electrodes positioned
about 2 mm under the lower eyelid were applied for sig-
nal recording.
As the amplitude of the ERG waveform can be
affected by the placement of the skin electrode (Hobby
et al., 2018), we measured the vertical distance between
the upper edge of the Sensor Strip electrode and the lower
eye lid using ImageJ (version: 1.53 k) and the pictures of
the device intern camera which were taken during record-
ing and extracted with the RETeval
®
RFF Extractor
®
software.
Three different stimulation protocols (for details, see
Supplemental Table 1) were used in Troland mode, where
flash strengths compensate pupil size, eliminating the
need for mydriasis.
(A) ISCEV flash: Sixty flashes according to the rec-
ommendations of the International Society for Clinical
Electrophysiology of Vision (ISCEV) standard were pre-
sented. Luminance of the white flash was 85 Tdson
848 Td white background light. Stimulation frequency
was 1.96 Hz (Robson et al., 2022).
(B) Flash series: A series of white flashes with the lumi-
nance levels: 12, 22, 36, 71, 85, 113, 178, 251, 356, and
446 Tds were randomly presented on white background
illumination of 1130 Td. Sixty flashes were presented per
luminance level with a stimulation frequency of 1.96 Hz.
This random flash series was adapted from the previously
described stimulation procedure applied by Constable
et al. (2020) in children with ASD.
(C) PhNR: In accordance with the ISCEV extended
protocol for PhNR stimulation (Frishman et al., 2018),
200 red (621 nm) flashes with a luminance of 38 Tdswere
presented on rod-saturating blue (470 nm) background light
of 380 Td. Stimulation frequency was 3.43 Hz.
Measurements were conducted under normal room
lighting conditions. Participants were instructed to fixate
the red LED within the device. For all protocols LA-
ERG recordings were automatically averaged in
stimulus-synchronized manner.
ERG data were exported with the RETeval
®
RFF
Extractor
®
software provided by LKC Technologies Inc.
(version 2.9.3.0). Peak amplitudes in μV and corresponding
peak times in ms were extracted for the a-wave, b-wave and
the PhNR. A-wave amplitude (first max. negative deflec-
tion) and the PhNR (max. negative deflection between
30and100ms)weremeasuredinrelationtothebaseline,
b-wave amplitude (max. positive deflection) was measured
from the a-wave trough. Additionally, the amplitude of the
PhNR at 72 ms (as suggested by Preiser et al., 2013)aswell
as the corresponding P-Ratio (b-wave amplitude/PhNR
amplitude at 72 ms) was extracted. Further, the signal-to-
noise ratio (SNR) for b-wave amplitudes and the W-Ratio
for the PhNR at minimum ((b-wave amplitudePhNR
amplitude)/(b-wave amplitudea-wave amplitude);
Mortlock et al., 2010) were exported.
Previous investigations detected lower ERG ampli-
tudes in people with dark-pigmented irises compared to
blue-eyed subjects having light-pigmented irises
(Al Abdlseaed et al., 2010). Therefore, the iris color index
of all participants was extracted with the RETeval
®
RFF
Extractor
®
software and compared between HC and indi-
viduals with ASD. The ratio between the pupil (25th per-
centile of gray values across the horizontal diameter of
the pupil excluding saturated values) and the iris gray
values (25th percentile of the gray values within two
1 mm horizontal lines, aligned at the left and right pupils’
edges) corresponds to the iris color index and is based on
the device intern examination videos.
Before data export, all recordings were individually
checked for artifacts, baseline drifts and correct peak detection.
Data handling and statistical analysis
Further calculations and statistical analyses were con-
ducted with “R”(R Core Team, 2021) in RStudio
FRIEDEL ET AL.5

(RStudio Team, 2022). The “tidyverse”package
(Wickham et al., 2019) was used for data wrangling, the
“ggplot”(Kassambara, 2020) and “cowplot”
(Wilke, 2020) packages for graphical representations.
The ratio between the b-wave amplitude and the a-
wave amplitude (b/a-ratio) was calculated for all test pro-
tocols and luminance levels. Subsequently data of both
eyes were averaged per participant.
Photopic hill parameter estimation
For the b-wave amplitudes of the “(B) flash series”,the
luminance response function also called “photopic hill”
was modeled for every participant according to the pro-
cedure described by Hamilton et al. (2007), McCulloch
et al. (2019) and Constable et al. (2020).
Vb¼GI
μ

ln μ
I
ðÞ
B2
2
6
43
7
5þVmax I
Iþσð1Þ
(1) V
b
refers to the b-wave amplitude (μV), Ito the flash
luminance level (cds/m
2
), Gis the maximal peak ampli-
tude of the Gaussian fit (μV), μthe corresponding lumi-
nance level for G(cds/m
2
), Bindicates the width of the
Gaussian curve and can be approximated by 1.0, V
max
refers to the maximal saturated amplitude (μV), while σ
describes the corresponding luminance level needed for
half maximal saturation (cds/m
2
). The first part of the
photopic hill equation corresponds to a logistic growth
function which represents the OFF pathway, the second
term describes a Gaussian curve fit indicating ON path-
way functioning.
The above-described model formula was implemented
within a nonlinear regression function (function
“nlsLM”; package “minpack.lm”[Elzhov et al., 2022]) to
estimate the parameters G,μ,V
max
, and σfor all
participants.
For better inter-study comparisons, Troland-based
flash luminance levels were recalculated to cds/m
2
assuming a 6 mm pupil diameter (LKC Technologies,
Inc., 2016) and log transformed for graphical
representation.
Group comparisons
Psychometric and demographic data as well as the iris
color index and the vertical electrode position were com-
pared with non-parametric Wilcoxon-tests and Fisher-
exact-tests (package “stats,”R Core Team, 2021).
To avoid influences of outliers, ERG data were sum-
marized by the median and the corresponding 95%
bootstrapped-confidence interval and group differences
were estimated by fitting a robust regression model
(function “lmrob”; package “robustbase”[Maechler
et al., 2021]). MM-type estimators (Koller &
Stahel, 2011) were used in the model to robustly predict
effects of group affiliation (β
group
) on the given ERG
measure (Y), while controlling for age and sex as con-
founding variables. Values for participant’s ages were
centered by subtracting the overall mean age. Male HC
(with the lowest ages) served as reference (β
0
). The regres-
sion coefficients indicate the changes in the measured var-
iable (Y) when one of the predictor variables changes, for
example, difference from HC (reference =β
0
) to ASD
(β
group
coefficient), while keeping all other predictors
unchanged. The regression formula was
Y¼β0þβage age þβsex sex þβgroup group:ð2Þ
The significance level was defined as α=0.05 and
family-wise adjusted for the different ERG measures
evaluated: amplitudes, peak times, ratios and photopic
hill parameters. The Benjamini and Hochberg (1995) pro-
cedure was used for the adjustment.
The WURS-k (Retz-Junginger et al., 2002) and BDI-
II (Beck et al., 1996; Hautzinger et al., 2006) scores were
not added to the model, because of the strong correlation
with the group variable. Therefore, subsequent analyses
were conducted on subsamples of individuals with ASD.
We considered only individuals with ASD (1) without
elevated ADHD-related symptoms in childhood
(WURS-k < 30), (2) without increased depressiveness
(BDI-II < 14) and (3) without psychiatric medication
intake. The ERG peak amplitudes and peak times of
those subsamples were separately compared to the HC
group.
Since Lee et al. (2022) reported elevated ERG ampli-
tudes in individuals with ADHD, we additionally com-
pared the ERG peak amplitudes of ASD individuals with
elevated ADHD symptoms in childhood (ASD+ADHD;
WURS-k ≥30) to the ASD individuals without elevated
ADHD symptoms in childhood (ASD-ADHD; WURS-
k < 30) and compared both subgroups separately against
the HC group.
RESULTS
Participants
Thirty-nine individuals with an ASD and 36 HC were ini-
tially investigated. Seven individuals with ASD and five
HC had to be excluded due to elevated artifacts or base-
line drifts in the ERG recordings, which prevents proper
peak detection in the ERG measurements. The final sam-
ple for the analysis consisted of 32 individuals with ASD
as well as 31 HC. Participants also took part in an optical
coherence tomography (OCT) study of our research
group (Friedel et al., 2022, unpublished). While groups
6FRIEDEL ET AL.

did not differ in age, sex nor IQ (Lehrl et al., 1995), the
ASD group showed elevated autistic symptoms according
to the AQ (Baron-Cohen et al., 2001) and SRS-2 ques-
tionnaires (Constantino, 2013) as well as decreased EQ
(Baron-Cohen et al., 2004) scores. Compared to the HC,
the ASD group had elevated depressive symptom scores
assessed with the BDI-II (Beck et al., 1961; Hautzinger
et al., 2006) and increased symptom scores of an ADHD
in childhood according to the WURS-k (Retz-Junginger
et al., 2002) (Table 2). Twenty individuals with an ASD
received psychiatric medication. Fourteen took antide-
pressants (six SNRI, three SSRI, two bupropion, four tri-
cyclic and three tetracyclic antidepressants, one
agomelatine) and three methylphenidates. As ASD is
often associated with a stimulus overload due to the
increased attention to detail or sleeping problems, seven
participants took atypical neuroleptics. Additionally, one
individual with ASD received pregabalin and two mood
stabilizers (one lamotrigine, one lithium).
ERG results
No significant differences were detected with regard to
the placement of skin electrodes between individuals with
ASD and HC (right eye (p=0.06): HC mean (SD): 1.05
(0.56) mm (range: 0.21–2.56 mm) and individuals with
ASD mean (SD): 0.87 (0.65) mm (range: 0.28–3.31 mm);
left eye (p=0.61): HC mean (SD): 1.12 (0.54) mm
(range: 0.31–2.64 mm) and individuals with ASD mean
(SD): 1.07 (0.55) mm (range: 0.39–2.37 mm). Likewise,
the iris color index between HC and autistic individuals
was not significantly different between both groups
(HC mean (SD): 1.2 (0.06), range: 1.07–1.29; individuals
with ASD mean (SD): 1.19 (0.07), range: 1.09–
1.39; p=0.25).
ERG amplitudes: The robust regression models
showed no significant differences in ERG peak ampli-
tudes between individuals with ASD and HC regardless
of the stimulation protocol ((A) ISCEV flash (a-wave:
p=0.84; b-wave: p=0.18), (B) flash series (see p-values
in Figure 2and Supplemental Table 2), (C) PhNR (nei-
ther for the a- (p=0.49) or b-wave (p=0.49) nor for the
PhNR amplitudes (at minimum: p=0.44 or at 72 ms:
p=0.42) (Figure 2and Supplemental Table 2).
Further, no significant differences of the photopic hill
parameters G(p=0.21), V
max
(p=0.43), μ(p=0.11) or
σ(p=0.29) which were derived from the (B) flash series
could be detected between the ASD and HC group
(Supplemental Table 3).
ERG peak times: Similarly, in none of the three stimu-
lation protocols ((A) ISCEV flash (a-wave: p=0.11; b-
wave: p=0.16), (B) flash series (see p-values in Supple-
mental Table 2), (C) PhNR (a-wave: p=0.10; b-wave:
p=0.35; PhNR: p=0.16)), significant differences in the
ERG peak times of the a-, b-wave or the PhNR at mini-
mum were detected when comparing the ASD and HC
group with robust regression models (Supplemental
Table 2).
ERG ratios: Likewise, the additionally extracted and
calculated ratios for the ERG peak amplitudes showed
no significant group differences in the robust regression
models. Neither the b-wave signal-to-noise ratio (b-
SNR), nor the ratio between the b- and a-wave ampli-
tudes (b/a) differed significantly between individuals with
ASD and HC in any of the three stimulation protocols
((A) ISCEV flash (b-SNR: p=0.36; b/a: p=0.67),
(B) flash series (see p-values in Supplemental Table 3),
(C) PhNR (b-SNR: p=0.10; b/a: p=0.26))
(Supplemental Table 3). Further, no group differences
were detected for either PhNR ratio, neither the P-Ratio
(b-wave amplitude/PhNR amplitude at 72 ms [Preiser
et al., 2013]; p=0.74) nor the W-Ratio ((b-wave amplitu-
dePhNR amplitude)/(b-wave amplitudea-wave ampli-
tude) (Mortlock et al., 2010); p=0.91) (Supplemental
Table 3).
Subgroup analyses: The subsequently performed ana-
lyses considering (1) individuals without elevated
TABLE 2 Demographic and psychometric data
Parameter HC (N=31) ASD (N=32) p-value*
Sex (male/female) Count 19/12 19/13 > 0.99
Age in years Mean (SD) 35 (11) 34 (11) 0.89
IQ (MWT-B) 107 (11) 111 (16); n.a. =1 0.74
SRS-2 30 (12) 101 (24); n.a. =2 < 0.001
AQ 12 (5) 33 (7); n.a. =1 < 0.001
EQ 49 (11) 24 (12) < 0.001
BDI-II 3 (3) 18 (13) < 0.001
WURS-k 12 (9) 27 (15) < 0.001
Psychiatric medication (yes/no) Count 0/31 20/12
Abbreviations: AQ, Autism Spectrum Quotient (Baron-Cohen et al., 2001); BDI-II, Beck Depression Inventory II (Beck et al., 1996; Hautzi nger et al., 2006); EQ,
Empathy Quotient (Baron-Cohen et al., 2004); IQ, Intelligence Quotient; MWT-B, Multiple Choice Vocabulary Test (Lehrl et al., 1995); n.a., not available; SRS-2, Social
Responsiveness Scale 2 (Constantino, 2013); WURS-k, Wender Utah Rating Scale (Retz-Junginger et al., 2002). Group comparisons based on *Fishers-exact- and
Wilcoxon-tests.
FRIEDEL ET AL.7

ADHD-related symptoms in childhood (WURS-k < 30)
(N=19), (2) without increased depressiveness (BDI-
II < 14) (N=17), and (3) without psychiatric medication
(N=12) completely resemble the results of the overall
comparisons between the ASD and HC groups. In none
of the three stimulation protocols, significant differences
were detected regarding the ERG peak amplitudes (a-, b-
wave, PhNR and PhNR at 72 ms), peak times (a-, b-
wave or the PhNR) and the ratios for the ERG peak
amplitudes (b-wave SNR, b/a-ratio, P- and W-Ratio)
when comparing the three different subsamples of indi-
viduals with ASD separately to the HC group. Supple-
mental Table 4 shows the results of the subgroup
analyses.
Because Lee et al. (2022) found that individuals with
ASD and ADHD show opposite ERG b-wave amplitude
characteristics compared to HC with an elevated b-wave
amplitude in ADHD and a decreased b-wave in ASD, we
compared the a- and b-wave peak amplitudes as well as
the PhNR amplitudes between HC, ASD individuals
with symptoms of an ADHD in childhood
(ASD+ADHD; N=13) and those without ADHD
symptoms in childhood (ASD-ADHD; N=19). We
detected no significant differences between the sub-
groups, however, ASD individuals with comorbid
ADHD symptoms in childhood (ASD+ADHD) showed
slightly higher b-wave amplitudes compared to HC and
ASD-ADHD (Supplemental Figure 1).
DISCUSSION
In the present study, 32 individuals with ASD and 31 HC
were examined with LA-ERG applying three different
stimulation protocols: the ISCEV flash, a flash series for
the additional evaluation of the photopic hill parameters
FIGURE 2 ERG peak amplitudes for both groups and all three stimulation protocols: ISCEV-flash (a), flash series (b) and PhNR-flash (c). The
median and 95% bootstrapped-confidence interval (notches in boxplots and error bars in the line luminance response function) as well as the p-values
for the group coefficients of the robust regression models are depicted. ASD, autism spectrum disorder; HC, healthy controls; ISCEV, International
Society for Clinical Electrophysiology of Vision; PhNR, photopic negative response.
8FRIEDEL ET AL.

(G,μ,V
max
, and σ) as well as a PhNR stimulation. Nei-
ther the amplitudes nor the peak times of the a- or b-
wave, or the PhNR were altered in ASD compared to
HC. Likewise, ERG amplitude ratios and photopic hill
parameters showed no differences in individuals with
ASD compared to HC.
So far, there are only a few LA-ERG studies examin-
ing individuals with ASD and the results to date regard-
ing functional retinal alterations in autism are
inconsistent. Constable et al. (2016) observed attenuated
b-wave amplitudes in the LA-ERG of high-functioning
autistic adolescents and adults. In a subsequent examina-
tion in children and young adults, Constable et al. (2020)
confirmed the b-wave amplitude attenuation, but only in
response to high flash strengths. They additionally found
a significant reduction in the a-wave amplitude, a pro-
longed b-wave peak time, a decreased b/a-ratio as well as
alterations in the parameters V
max
and Gof the lumi-
nance response model (photopic hill) in children and
young adults with ASD (Constable et al., 2020). A recent
study by Lee et al. (2022) confirmed those b-wave alter-
ations (attenuated amplitude and prolonged peak time)
in children and young adults with ASD, while no a-wave
alterations were detected.
Although the stimulation protocol and the device
used for ERG recordings are comparable between the
current investigation and the studies conducted by Con-
stable et al. (2020) and Lee et al. (2022), our results of
unaltered retinal functioning in ASD are not in line with
the previous findings of attenuated a- and b-waves in
individuals with ASD (Constable et al., 2020; Lee
et al., 2022). Not even a hint on reduced ERG was seen;
rather, amplitudes were minutely larger in ASD.
The deviating results may be due to differences in the
investigated age groups, IQ ranges or sex ratios. While Con-
stable et al. (2020,2022)andLeeetal.(2022) focused on
younger autistic individuals between 6–27 years of age, we
focused on adults (mean age: 34 years). Although the for-
mer studies (Constable et al., 2020,2022; Lee et al., 2022)
excluded autistic participants with an IQ score below 65 or
with Fragile-X or Rett syndrome, their autistic groups
might have covered a wider scope of different forms of
ASD compared to our study sample, which encompassed a
homogeneous sample of patients with Asperger’ssyndrome
with a higher average IQ (mean IQ =107 (11) compared to
mean IQ =98.9 (16.5) in Constable et al.’s(
2020)sample
andmeanIQ=99.6 (18.9) in Lee et al.’s(
2022)sample).
This prompts the question whether changes in the develop-
mental trajectories of individuals with autism or differences
in the investigated ASD subtypes might have led to the
diverging results. Furthermore, differences in the statistical
handling of datasets may have contributed to differing
results. While we averaged the eyes, Constable et al. (2020)
included the eye with the highest b-wave amplitude.
The earliest ERG studies in individuals with ASD
(Creel et al., 1989; Ritvo et al., 1988) demonstrated a
reduced b-wave using DA-ERG, which is not directly
comparable to our LA-ERG results as different retinal
signaling pathways are stimulated with a dark adapted
retina.
Observations from animal models support the hypothe-
sis of differing results based on the studied subtypes of
ASD, especially with regard to the inclusion of syndromal
forms and alterations in adaptational retinal states.
Perche et al. (2018) investigated Fmr1
/y
mice, a model
for Fragile-X syndrome, with the DA-ERG and found
alterations in both, the a- and b-wave amplitudes. Likewise,
mice lacking the Engrailed 2 gene, which is associated with
ASD and cognitive deficits (Brielmaier et al., 2012), show
alterations in the DA-ERG a- and b-wave amplitudes
(Zhang et al., 2019), while the LA-ERG of those mice
resembles normal components (Zhang et al., 2019). BTBR
mice on the other hand, which are a model for idiopathic
ASD, have normal b-wave amplitudes in both, the DA-
and LA-ERG but attenuated DA- and LA-ERG a-wave
amplitudes (Cheng et al., 2020).
In contrast to Constable et al. (2016), we found no alter-
ation in the b-wave amplitude in ASD. In addition to differ-
ences in the investigated age groups (high-functioning
adolescents and adults in Constable et al. (2016) and adults
in our study), the different results may also be due to deviat-
ing sex ratios of the examined groups (90.9% male in Con-
stable et al. (2016) and 59.4% male in our study).
Moreover, Constable et al. (2016) used corneal DTL elec-
trodes, whereas we applied skin electrodes for recording.
Nevertheless, our observations of unaltered PhNR
amplitudes in ASD are in line with the results described
by Constable et al. (2021) and Lee et al. (2022) and sup-
port the assumption of normal ganglion cell responses in
individuals with ASD, which was previously reported
(Tebartz van Elst et al., 2015). Tebartz van Elst et al.
(2015) investigated 33 high-functioning patients with
ASD and 33 HC with the Pattern-ERG (PERG), which
is mainly driven by retinal ganglion cells, and found no
alterations in the PERG of individuals with ASD.
Despite the absence of any functional retinal alter-
ations in adults with ASD, longitudinal studies across dif-
ferent age groups and ASD subtypes, for example with
and without comorbid ADHD, are required to improve
our understanding of the earlier ERG findings in individ-
uals with ASD.
Limitations
As a cross-sectional study, no firm conclusions can be
drawn about possible longitudinal alterations in retinal
functioning or age-dependent effects. Even if the match-
ing procedure respected age and sex, the groups differed
with regard to comorbidities and the intake of psychiatric
medication. In the subsequent analysis, we took these
possible confounders into account and found no func-
tional retinal alterations in individuals with ASD without
comorbid symptoms or the intake of medication.
FRIEDEL ET AL.9

However, the sample size of these subgroups was small,
which may have limited their predictive power. We used
skin electrodes to improve compliance. However, previ-
ous studies showed that skin electrodes may lead to lower
amplitudes compared to corneal electrodes (Mortlock
et al., 2010). As we focused on individuals with high-
functioning ASD, our results are not applicable to sec-
ondary syndromal forms of ASD. In addition, we com-
pared ASD individuals with and without comorbid
ADHD symptoms in childhood, but we did not assess
current ADHD symptoms, which would be desirable for
future studies.
SUMMARY
We found that retinal cell function in terms of amplitude
and peak time of the a-wave, b-wave or PhNR, the phot-
opic hill parameters as well as the amplitude ratios are
not significantly altered in adults with ASD compared to
age- and sex-matched HC.
AUTHOR CONTRIBUTIONS
Kathrin Nickel, Evelyn B. N. Friedel and Mirjam Schä-
fer wrote the paper. Evelyn B. N. Friedel performed the
data and statistical analysis in cooperation with Mirjam
Schäfer and Kathrin Nickel. Evelyn B. N. Friedel created
graphical representations. Kathrin Nickel, Ludger
Tebartz van Elst, Evelyn B. N. Friedel and Dominique
Endres organized the study and created the study design.
Evelyn B. N. Friedel, Sven P. Heinrich and Michael Bach
performed the technical set-up and implemented the
ERG stimulation procedures. Kathrin Nickel, Domi-
nique Endres, Dieter Ebert and Kimon Runge recruited
the individuals with ASD and established the diagnosis.
Mirjam Schäfer performed the measurements. Ludger
Tebartz van Elst, Katharina Domschke, Simon Maier,
Dominique Endres, Dieter Ebe, Kimon Runge, Michael
Bach and Sven P. Heinrich revised the manuscript criti-
cally focusing on clinical and statistical aspects. All
authors were critically involved in the theoretical discus-
sion and composition of the manuscript. All authors read
and approved the final version of the manuscript.
ACKNOWLEDGMENTS
Open access funding was enabled and organized by pro-
ject DEAL.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request.
ETHICS STATEMENT
The Ethics Committee of the University Medical Center
Freiburg (Approval ID: 314/18) approved the study. All
participants gave written informed consent to participate.
ORCID
Evelyn B. N. Friedel https://orcid.org/0000-0001-9862-
9656
Kimon Runge https://orcid.org/0000-0002-0263-4360
Michael Bach https://orcid.org/0000-0003-2028-535X
Kathrin Nickel https://orcid.org/0000-0001-9863-317X
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article.
How to cite this article: Friedel, E. B. N., Schäfer,
M., Endres, D., Maier, S., Runge, K., Bach, M.,
Heinrich, S. P., Ebert, D., Domschke, K., Tebartz
van Elst, L., & Nickel, K. (2022).
Electroretinography in adults with
high-functioning autism spectrum disorder. Autism
Research,1–12. https://doi.org/10.1002/aur.2823
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