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Expert Review of Molecular Diagnostics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iero20
Salivary biomarkers associated with obstructive
sleep apnea: a systematic review
Sompop Bencharit , Robert G. Redenz , Erica R. Brody & Harmeet Chiang
To cite this article: Sompop Bencharit , Robert G. Redenz , Erica R. Brody & Harmeet Chiang
(2021): Salivary biomarkers associated with obstructive sleep apnea: a systematic review, Expert
Review of Molecular Diagnostics, DOI: 10.1080/14737159.2021.1873132
To link to this article: https://doi.org/10.1080/14737159.2021.1873132
Published online: 17 Jan 2021.
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REVIEW
Salivary biomarkers associated with obstructive sleep apnea: a systematic review
Sompop Bencharit
a,b
, Robert G. Redenz
c
, Erica R. Brody
d
and Harmeet Chiang
e
a
Department of General Practice and Department of Oral and Maxillofacial Surgery, School of Dentistry, Virginia Commonwealth University,
Richmond, Virginia, USA;
b
Department of Biomedical Engineering, College of Engineering, Virginia Commonwealth University, Richmond, Virginia,
USA;
c
School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA;
d
VCU Libraries | Tompkins-McCaw Library for the Health
Sciences, Virginia Commonwealth University, Richmond, Virginia, USA;
e
Department of General Practice, School of Dentistry, Virginia
Commonwealth University, Richmond, Virginia, USA
ABSTRACT
Introduction: This study aimed to define and characterize current literature describing salivary bio-
marker changes with the goal of improving diagnosis and treatment outcomes for sleep apnea.
Area Covered: A search of six databases yielded 401 peer-reviewed articles published through October 2019
corresponded to 221 unique references following deduplication. Twenty studies were selected. The sample
size ranged from 17 to 99. The samples were mostly whole saliva and selected glandular areas.
Expert Opinion: Most targeted studies focused on the level of salivary cortisol and ɑ-amylase. One
study used RNA transcriptome analysis of 96 genes. Only two explored novel targets using mass
spectrometry. ɑ-amylase, myeloperoxidase, and IL-6 were among those biomarkers found associated
with OSA. Cytokeratin, CystatinB, calgranulin A, and alpha-2-HS-glycoprotein are upregulated in OSA
patients based on non-targeting mass spectrometry. Salivary cortisol and ɑ-amylase and others
appeared to be associated with severity of OSA and OSA treatment. There were inconsistencies in
saliva collection and processing protocols. More studies are needed in exploring novel biomarkers to
examine if these biomarkers are capable of diagnosing and monitoring OSA through proteomics or
transcriptomics. Salivary biomarkers have a potential to be a noninvasive measure for the disease
diagnosis and treatment outcome monitoring for sleep apnea.
ARTICLE HISTORY
Received 23 November 2020
Accepted 5 January 2021
KEYWORDS
ɑ-amylase; biomarker;
diagnosis; monitoring; saliva;
salivary cortisol; sleep
apnea; systematic review
1. Introduction
Obstructive sleep apnea (OSA) is a sleep disorder character-
ized by breathing pauses during sleep or inadequate or
obstructive breathing also known as obstructive apneas or
hypopneas. This disruption of the respiratory airway can be
caused by repetitive collapsing of the upper airway during
sleep [1]. Patients with OSA are at risk of a variety of neurop-
sychiatric dysfunction, including lack of attention, cognition
problems, memory issues, irritability, depression, and sexual
dysfunction [2–8]. Patients with OSA also have higher risks of
cardiovascular and cerebrovascular diseases [8–11], pulmonary
hypertension and right heart failure [12,13], as well as meta-
bolic syndrome and type 2 diabetes [14,15]. Nonalcoholic fatty
liver disease [16] and gout [17] can also be linked to OSA. In
the western world, the prevalence of OSA is reported ~4% in
males and 2% in females [17,18] and appears to be associated
with an increased rate of obesity [19,20]. However, as many as
90% of persons with OSA may be undiagnosed, and therefore
unaware of having the condition [21–23]. A higher range of
~15-34% prevalence of OSA across different ethnics around
the world has been reported [24]. Undiagnosed OSA bears an
annual economic burden the U.S. estimated at over 149
USD billion annually, according to the American Academy of
Sleep Medicine (AASM) [25].
The diagnosis of OSA is based on polysomnography (PSG)- or
home sleep apnea testing (HSAT) and can be classified as mild,
moderate, and severe [26,27]. Overnight PSG is considered to be
the gold standard for diagnosing sleep-related breathing disor-
ders, which include obstructive sleep apnea (OSA), central sleep
apnea, and sleep-related hypoventilation/hypoxia. Despite the
proven effectiveness of overnight PSG in diagnosing sleep dis-
orders like OSA, this test is associated with excessively long
patient wait times and high costs. Consequently, development
of a cheaper, more effective alternative is necessary for quick and
effortless diagnosis of patients [28]. Identifying valid, convenient,
and inexpensive tools to identify those individuals at high risk for
OSA is of utmost importance. It is therefore essential to not only
raise the awareness of the condition, but also improve current
diagnostic modalities. The characteristics for an ideal biomarker
for OSA was described previously [29,30] as having highly sensi-
tive and specific to OSA, allowing diagnosis and monitoring of
disease severity and treatment effectiveness, being a surrogate of
pathogenetic pathway leading to measuring severity of OSA,
being simple and easy to measure, and finally allowing measure-
ments of multiple pathways associated with OSA.
A recent study suggests that there are a number of serum
biomarkers that might be useful in improving the diagnosis and
disease monitoring for OSA [31]. The serum biomarkers asso-
ciated with OSA include inflammatory markers (C-reactive
CONTACT Sompop Bencharit sbencharit@vcu.edu Department of General Practice, School of Dentistry, Virginia Commonwealth University, Richmond, VA
23298-0566
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
https://doi.org/10.1080/14737159.2021.1873132
© 2021 Informa UK Limited, trading as Taylor & Francis Group
protein (CRP), Tumor necrotic factor-α, interleukins: IL-8, IL-6),
cell adhesion molecules (fibrinogen), oxidative stress markers
(8-isoprostane, malondialdehyde,paraoxonase) and metabolic
markers for hemoglobin A1c, leptin, adiponectin, and resistin)
[31]. Two scoping reviews suggest that IL-6, IL-10, and CRP are
among the most reliable serum biomarkers [32]. Obtaining
serum biomarkers requires blood sampling and thus the appli-
cations of serum biomarkers can be limited.
The current gold standard for diagnosing and monitoring
disease severity and response to treatment for OSA is PSG.
However, the cost, time and requirement of experts led to
more research on biomarkers in serum. Still the serum-based
biomarkers require blood sampling and may not be suitable
for at-home monitoring or in specific populations such as
elderly or children. Therefore, there is a real need in finding
more noninvasive biomarkers that can easily be obtained and
can be developed as home monitoring biomarkers.
Saliva samples can be obtained noninvasively and are more
likely to be accepted by patients than plasma samples [33].
Saliva biomarkers have been shown to be associated with not
only oral but systemic diseases [34,35]. Salivary protein biomar-
kers are linked to metabolic syndrome and diabetes [35–38].
Saliva, therefore, has a potential to be a good source for devel-
oping a diagnostic tool for OSA. This systematic review aimed to
examine the current status of OSA associated salivary biomarker
studies and to define the salivary biomarkers discovered to date.
Based on the current biomarker studies, it also aimed to develop
recommendations for future studies.
2. Methods
2.1. Identification of relevant studies
A systematic review of studies in humans written in English
without date restriction that evaluated the potential relation-
ship between biological markers in saliva and diagnosis of
obstructive sleep apnea using PRISMA protocol [39].
2.1.1. Inclusion criteria
Peer-reviewed studies that involved the analysis of human
saliva obtained from a minimum of 10 patients diagnosed
with sleep apnea were selected for inclusion.
2.1.2. Exclusion criteria
Studies written in languages other than English were
excluded. Search strategies were created for each of the fol-
lowing bibliographic databases to obtain peer-reviewed arti-
cles and conference articles and published through
21 October 2019: MEDLINE (Ovid), Embase (OVID), Dentistry
and Oral Sciences Source (DOSS), Web of Science, CINAHL
Complete, and ProQuest Dissertations & Theses. Search
queries employed can be found in Appendix I. Additional
citations were obtained by reviewing the references cited in
the articles selected for inclusion in the review. All references
were managed by Microsoft Excel and Mendeley, a citation
management program. Duplicate references obtained from
the various databases searches were removed.
2.2. Study selection
A two-step process was employed to select articles for inclu-
sion in this review. Initially, two authors (S.B. and R.R.)
reviewed titles and abstracts of all articles obtained from the
electronic search. Articles meeting the inclusion criteria were
included in the full-text analysis. The final articles were
selected with agreement of two reviewers (S.B. and R.R.).
A third reviewer (H.C.) was invited to vote for article selection
when there was a disagreement.
2.3. Data collection
One author (R.R.) collected the following information from
each included article: sample size, study group composition,
sample type (e.g. saliva, serum, etc.), sample processing tech-
nique, biomarker detection technique and targeting, and bio-
markers identified. A second author (S.B.) verified the validity
of the charted data.
3. Results
A total of 401 articles were obtained from the electronic
literature search. After duplicates were removed, a total of
221 articles were reviewed for inclusion in the study. After
the abstracts were reviewed 176 articles were excluded. Thirty-
one articles were included in the full-text review. After the full-
text review, 11 articles were excluded. The final 20 articles
were included in the systematic review. Figure 1 shows the
database search process, excluded and included articles, and
reasons for the exclusion.
A variety of methods were used for subject selection across
the 20 studies (Table 1). Most studies conducted their own
PSG to confirm the OSAS diagnosis and even sub-divided their
OSAS patients into groups of mild, moderate, and severe
OSAS. Some studies did not have or did not specify if they
had control groups. The majority of studies used inclusion
Article Highlights
●Saliva sample collection for biomarker examination can be done
successfully during the PSG diagnostic process since the patient will
be in the control environment.
●Saliva sample treatment should be based on type of biomarker and
analysis. Protease or RNAase inhibitors may be needed to preserve
proteins and RNAs. Removing highly abundant proteins such as α-
amylase and immunoglobulins can improve the discovery of low
abundant protein biomarkers.
●Known salivary biomarkers such as cortisol and α-amylase can be
used to diagnose and monitor treatment outcomes for OSA. Using
night and morning ratios of these biomarkers seem to be an effective
way to reduce individual variations.
●There is a need to explore novel salivary biomarkers through gene
arrays, transcriptomics, and proteomics.
●There is also a need to link salivary biomarkers to serum biomarkers.
●Longitudinal studies monitoring the changes of salivary biomarkers
are needed to validate the benefit of salivary biomarkers.
2S. BENCHARIT ET AL.
criteria such as excessive daytime sleepiness, diagnosed or
suspected OSAS, and obesity, among others. Siber-
Hoogeboom et. al [40] used samples from rhonchopathy
patients. Zheng et. al (2014) divided their patients by presence
or absence of cardiovascular disease [41]. The details of
patient grouping for each study is shown in Table 1.
Information on the saliva sample collection and processing
was extracted from all included studies, displayed in Table 1.
Sixteen of the 20 articles used whole, unstimulated saliva for
their respective studies. Some studies came from the same
cohort. All three studies by Nizam et al [44–46] used the same
52 subjects. Two articles by Ghiciuc et. al [47,48] used the
same subject population. Among these, methods such as
a swab, Salivette (cotton roll), or expectorating were used to
obtain a saliva sample. The remaining studies used glandular,
unstimulated saliva from under the tongue, obtained using
a pipette [49], glandular, unstimulated from the palate and
uvula [8], and glandular, unstimulated from the palate and
uvula using Schirmer tear test strips [8,40]. Raff et. al [50] did
not specify how salivary samples were obtained.
Post-collection processing can also be seen in Table 1. Of
the 20 studies, 10 used methods of immediate centrifugation
of the salivary samples and then freezing until analysis. Six
studies froze the samples immediately, and upon analysis
centrifuged the saliva samples. Liu et al [50,51] Yan et al [52]
and Ghiciuc et al [48] did not specify whether the samples
were frozen. Traxdorf et. al [48,53] did not specify if the
samples were centrifuged. Raff et. al [50] did not specify how
salivary samples were processed. From the studies that speci-
fied freezing of the samples, eight total studies froze their
samples at −20°C and eight studies froze samples at −80°C.
Only one study [49,50] stated how long the samples were
frozen – no longer than 3 weeks.
Sample collection time depended on study design and
hypothesis. In general, studies that included PSG had samples
taken prior to and in the morning, after the PSG. Ten studies
specified sample collection was in the morning, anywhere
from 30 minutes to 2 hours after awakening, or a certain
window of time in the morning (for example 8:00–11:00).
Two studies had samples taken at night time. Four studies
had multiple different times per day where salivary samples
were taken, according to study design and hypothesis. Only
two studies did not specify when saliva samples were taken. In
general, most studies stated that they gave subjects instruc-
tions not to eat/drink or conduct oral hygiene practices within
30 minutes to 2 hours before sample collection.
Biomarker detection and analysis were different for each
study, depending on which biomarker they were investigating.
Eight studies used a type of enzyme-linked immunosorbent
assay (ELISA) to quantify their biomarkers in question. Three
studies used mass spectrometry for analysis but only two were
non-targeting. Three studies used immunoenzymatic kits for
Figure 1. A flow chart for study selection.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 3
Table 1. Sample characterization and processing.
Study
Sample
Size Grouping Sample type Collection process Sample processing
Akpinar et. al 2012 [42] 56 32 OSAS diagnosed patients and
age & gender matched
24 health subjects
Saliva – whole,
unstimulated
Collected in the morning
after sleep study. Rinse
mouth with water
Centrifuged and frozen until
analyzed
Celec et. al 2012 [70] 89 OSAS patients: before CPAP,
1 month and 6 months after
CPAP
Saliva – whole,
unstimulated
Collected between
8:00–10:00; 30 min after
toothbrushing
Frozen until analysis,
centrifuged
Ghiciuc et. al 2013 [47] 17 10 OSAS caucasian, male, obese,
before and 3 and 6 months
after CPAP; and 7 control –
male, obese
Saliva – whole,
unstimulated
(Salivette)
Avoid food, coffee, and
alcohol consumption,
teeth brushing, and any
physical
exercise during the
90 min after awakening,
Collected between
6:30–7:30, then 30, 60,
and 90 min thereafter
Centrifuged and frozen until
analyzed
Ghiciuc et. al 2016 [48] 17 OSAS patients and control –
male
Same population as
Ghiciuc et. al 2013
Saliva – whole,
unstimulated
(Salivette)
Same as Ghiciuc et. al 2013;
except collection time,
6:30–7:30, 12:00, and
19:00
Centrifuged and analyzed
Kawai et. al 2013 [49] 20 OSAS patients – male Saliva – glandular
(under tongue),
unstimulated
(Pipette)
not consume any food or
drink for 1 hour prior to
the
saliva collection.
Frozen until analysis,
centrifuged
Liu et. al 2013 [51] 45 30 OSAS patients and 15 control
Age 30–50
23 male 22 female
Airway surface liquid –
glandular (palate and
uvula), unstimulated
Collected at least 2 hours
after eating and
rinsing mouth with
water.
Protease inhibitor added to
sample, centrifuged
Nizam et. al 2016 [46] 52 17 Mild-moderate OSAS, 22
Severe OSAS, 13 control
(same population as Nizam
et al 2014)
Saliva – whole,
unstimulated
Collected samples in the
morning
Centrifuged and frozen at −80 C
until analyzed
Nizam et. al 2015 [45] 50 17 Mild-moderate 20 OSAS,
Severe OSAS, 13 control
(same population as Nizam
et al 2014)
Saliva – whole
unstimulated
Collected samples in the
morning
Centrifuged and frozen at −80 C
until analyzed
Nizam et. al 2014 [44] 52 17 Mild-moderate OSAS, 22
Severe OSAS, 13 control
Saliva – whole,
unstimulated
Collected samples in the
morning
Centrifuged and frozen at −80 C
until analyzed
Park et. al 2014 [71] 67 41 (Mild-moderate OSAS, severe
OSAS) and 26 control
Saliva – whole,
unstimulated
No big meal 60 min before
collection and not to eat
or drink dairy products
and sugar
foods on day collection.
Rinsed with water
10 minutes before
collection.
Collect at 22:00 and
07:00 (within 60 minutes
after waking up).
Refrigerated within
30 min of collection. Frozen
at or below −20°C within 4
h after collection until
analysis, centrifuged
Park et. al 2013 [66] 80 Pediatric subjects
21 Mild-moderate OSAS, 27
severe OSAS and 32 control
Saliva – whole,
unstimulated
At night before sleep study
and in the morning after
Frozen until analysis,
centrifuged
Patacchioli et. al 2014
[69]
34 Pediatric subjects
13 Mild OSAS, 14 Moderate-
severe OSAS and 7 control
Saliva – whole,
unstimulated (Swab
sampling)
8:30–9:00 (~1 h after
waking up), 12:00 (before
lunch), and 19:00 (before
discharge)
refrain from eating and
drinking > 30 min
Centrifuged and frozen at −20°
C until analyzed
Raff et. al 2011 [50] 18 18 OSAS patients randomized
blinded CPAP and placebo
Saliva – whole,
unstimulated
(Salivette)
Bedtime 23:00, and next
morning 7:00 and days 1,
7, and 14 of
Mucolytic, 2 weeks after
CPAP or placebo.
Not reported
Schmoller et. al 2009 [67] 38 Severe OSAS patients before and
after CPAP
Saliva – whole,
unstimulated
(Salivette)
6 times: evening before
bed,
morning after awakening
(supine), before and
1 hour after
breakfast, and before and
1 hour after lunch
Centrifuged and frozen at −80°
C until analyzed
(Continued )
4S. BENCHARIT ET AL.
cortisol. Two studies used spectrofluorometry for analysis.
Additional types of biomarker detection used were flow cyto-
metry, kinetic reaction assay and luminescence assay. These
are summarized in Table 2.
Of the 20 studies, 18 targeted for specific biomarkers, whereas
two had no specific targets. The most common biomarkers that
were targeted were cortisol, with six studies investigating its role
in OSAS. Two studies each targeted ɑ-amylase, myeloperoxidase,
and IL-6. Biomarkers that were tested for each study can be seen in
Table 2. The following studies tested novel biomarkers. Kawai et. al
[49] used mass spectrometry to target for phosphatidylcholine.
Zheng et. al [41] also used mass spectrometry to study alpha-2-HS-
glycoprotein and its role in OSAS patients. Nizam et. al [45,49]
largely targeted for a number of matrix metalloproteinases
(MMPs) using ELISA. Nizam et. al [44] targeted for interleukins
including IL-1α, IL-6, IL-21 and IL-33, with ELISA as well. Siber-
Hoogeboom et. al [40,44] investigated Trefoil factor family pep-
tides (TFF) 3 and 2, utilizing ELISA as well. Furthermore, Thimgan
et. al [54] used a low-density array to look at 96 genes and search
for an increase in transcripts for certain proteins.
4. Discussion
This systematic review is perhaps one of the first to specifically
examine salivary biomarkers and their relationship to OSA. The
studies reviewed provide preliminary evidence that saliva collec-
tion and salivary biomarkers give insight into OSA severity and the
effects of treatments such as continuous positive airway pressure
(CPAP). There are a few issues to consider. First, there was a lack of
consistency in saliva sample collection and treatment. Saliva secre-
tion as well as its composition changes throughout the day and
can be influenced by food, stress and other factors, e.g. exercise,
fluid uptake, medications [55–59]. The majority of included studies
exhibited attempts to control these factors by collecting samples
at the same time of the day, and prohibiting oral hygiene, drink-
ing, eating and taking medications prior to sample collection.
However, the details of the subjects’ treatment and saliva collec-
tion and storage are not clear in most of the studies. Some studies
reported no information about the time or collection process or
storage methods. While saliva samples are simple to collect, the
investigators often neglect important natures of saliva such as
diurnal nature of saliva secretion, individual variations (age, gen-
der, possibly ethnicity, and oral or systemic health), micro-
organism in saliva and oral cavity, oral hygiene (including hygiene
habit and other oral conditions), diet & medication, and physical
activities or exercises [34,55,56]. It is therefore important to stan-
dardize the salivary collection protocol including careful subject
selection, abstaining from oral hygiene protocol, time of collec-
tion, method of collection (ideally unstimulated whole saliva),
speedy processing (for instance using centrifugation at 4 C to
remove debris), adding biomarker stabilizers, and storing the
samples in limited time. More importantly, since saliva can have
a wide range of individual variations, it is important to have
a baseline sample to compare the changes of the biomarkers
Table 1. (Continued).
Study
Sample
Size Grouping Sample type Collection process Sample processing
Siber-Hoogeboom et. al
2017 [40]
99 TFF3 analysis, 57 samples: 9
mild, 7 moderate, 6 severe
OSA, 8 rhonchopathy and 7
control;
TFF2 analysis, 33 samples: 4
mild, 10 moderate, 6 severe
OSA, 4 rhonchopathy and 9
control
Saliva – glandular
(palate and uvula),
unstimulated
(Schirmer tear test
strips)
No report on time of
sample collection, or if
subjects did anything
before collection.
Collection strips were frozen at
−80°C until analysis, then
eluted with aqua dest and
centrifuged
Thimgan et. al 2015 [54] 40 14 OSAS patients and 18
suspected OSAS patients and
8 controls
Saliva – whole,
stimulated by
chewing (Salievette)
No prescription
medications, no caffeine,
and had not eaten 1 hour
prior to collection.
No report on time of
sample collection.
Frozen at −80°C until analysis
Tóthová et. al 2019 [43] 24 OSAS patients who never used
CPAP prior to the study
Saliva – whole,
unstimulated
Collected in the evening
and
morning for two
consecutive days.
without
CPAP, and with CPAP.
No eating or tooth
brushing 1 h prior to
collection.
Centrifuged 4°C and frozen at
−80°C until analyzed
Traxdorf et. al 2016 [53] 60 34 OSAS patients, 6
Ronchopathy, and 20 controls
Saliva – whole,
unstimulated
(Salivette)
Collected between
9:00–11:00
Frozen at −80°C until analyzed
Yan et. al 2019 [52] 58 Mild OSAS, moderate OSAS,
severe OSAS and control
Saliva – whole,
unstimulated
(Salivette)
7 am (30 min after wake up) Centrifuged at 4°C and analyzed
Zheng et. al 2014 [41] 23 Non-CVD and CVD patients with
OSAS
Saliva – whole,
stimulated
No drinking and eating for
previous evening, and no
oral
hygiene practices 2
h before saliva collection.
Centrifuged at 4°C, added
protease inhibitors, and
frozen at −80°C until
analyzed
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 5
Table 2. Changes in salivary biomarkers.
Study
Biomarker detection
technique Target or non-target (NT) Biomarkers
Akpinar et. al 2012 [42] Flow cytometry Targeted for salivary myeloperoxidase and
serum C-reactive protein
Salivary MPO (and serum CRP) were significantly
higher in OSA patients; and
Salivary MPO (and BMI) was associated with
Apnea-Hypopnea Index (AHI)
Celec et. al 2012 [43] Spectrofluorometry NT AGEs and fructosamine decreased with CPAP.
Ghiciuc et. al 2013 [47] Immunoenzymatic kits for
direct salivary assay of
cortisol
Targeted for cortisol OSAS patients had lower cortisol levels in the
morning than controls, however, the levels were
higher 3 and 6 months after CPAP.
(In control, salivary cortisol spiked after waking
up and leveled down. In OSAS the level did not
spike. The spike pattern restored after CPAP.)
Ghiciuc et. al 2016 [48] Immunoenzymatic kits for
direct salivary assay of
cortisol
Targeted for cortisol and testosterone OSAS patients had lower cortisol levels in the
morning and lower testosterone in evening
compared to control. Testosterone/Cortisol ratio
is higher for control in the morning and lower in
the evening than OSAS.
Kawai et. al 2013 [49] Liquid chromatography-mass
spectrometry
Targeted for phosphatidylcholine PC concentration negatively correlated with
hypopnea index.
Liu et. al 2013 [51] 2D Gel electrophoresis and
mass spectrometry
NT Upregulated in control:
Actin beta,
Igγ heavy chain,
α-amylase,
Collagen α1(V),
carbonic anhydrase VI
Upregulated in patients:
Cytokeratin,
CystatinB,
calgranulin A
Nizam et. al 2016 [46] Enzyme-linked
immunosorbent assay
(ELISA)
Targeted for Salivary IL-1b, IL-6, IL-22,
IL-33, and PTX3
Increased IL-6 in both OSAS groups. Increased apelin
in severe OSAS group.
Nizam et. al 2015 [45] Immunofluorometric assay,
enzyme-linked
immunosorbent assay,
western immunoblotting,
and gelatine zymography
Targeted for matrix metalloproteinase-2, −8,
−9, tissue inhibitor of matrix
metalloproteinase-1, myeloperoxidase,
neutrophil elastase (NE), neutrophil
gelatinase-associated lipocalin
Salivary NE and proMMP-2 levels were significantly
lower in the
OSAS groups than the control group. Serum
proMMP-9/
degree of MMP-9 activation in saliva were
significantly lower in the severe OSAS group than
the control group. Serum proMMP-2, NE and
salivary proMMP-9
and −2 negatively correlated with indicators of
OSAS severity.
Nizam et. al 2014 [44] Enzyme-linked
immunosorbent assay
(ELISA)
Targeted for IL-1β, IL-6, IL-21, IL-33, and
pentraxin-3
IL-6 and IL-33 were higher in both OSAS groups.
Park et. al 2014 [71] Kinetic reaction assay Targeted for α
α-amylase activities
Salivary α-amylase subtraction and ratio was higher
in severe OSAS than mild-moderate OSAS
Park et. al 2013 [66] Enzyme immunoassay Targeted for cortisol salivary morning/night cortisol ratios after
polysomnography significantly decreased with
OSAS severity.
salivary cortisol morning/night ratios:
Control > mild/moderate > severe OSAS
Patacchioli et. al 2014
[69]
Enzyme-linked bioassay Targeted for cortisol and α-amylase Salivary cortisol decreased with the severity of OSA;
Salivary α-amylase activity was not predictive of
OSA or OSA severity.
Raff et. al 2011 [50] Enzyme-linked
immunosorbent assay
Targeted for cortisol CPAP treatment decreases morning cortisol levels
(not evening) in patients with OSAS
Schmoller et. al 2009 [67] Luminescence assay for free
cortisol
Targeted for cortisol CPAP treatment salivary cortisol levels in patients
with OSAS decreases before breakfast and
evening but increases after lunch (afternoon).
Siber-Hoogeboom et. al
2017 [40]
Enzyme-linked
immunosorbent assay
(ELISA)
Targeted for TFF3 and TFF2 Decreased TFF3 is associated with rhochopathy and
OSAS patients compared to control. Level of TFF3
reducation may be associated with severity of
OSAS. No association for TFF2.
(Continued )
6S. BENCHARIT ET AL.
prospectively [35–38,60]. While saliva samples can be collected in
the form of whole saliva, Salivette or cotton roll, or tear sample
strip, the samples should have been immediately centrifuged to
remove tissue of food debris and to be frozen at −80°C for optimal
preservation of the biomarkers. Adding protease or RNAase inhi-
bitors may also be needed for proteomics [60] or transcriptomics
[61]. There is a need for consistent saliva collection, sample treat-
ment, and processing for future meaningful comparison of these
studies [34,35].
Second, there was a strong focus on salivary cortisol and ɑ-
amylase in OSA salivary biomarker studies. This finding is
similar to another systematic review [32,62]. Cortisol and ɑ-
amylase are perhaps the most common salivary biomarkers
examined in OSA patients. There are perhaps a few reasons for
that. First, both biomarkers are relatively easy to examine with
multiple kits commercially available. Second, there is a long
history of examining both biomarkers associated with stress
and other physiological conditions. The majority of included
studies examined the salivary cortisol and ɑ-amylase levels
associated with OSA. It is known that salivary cortisol levels
are greatly varied among individuals and dependent on the
circadian rhythm as well as stress and other psychological
factors [63–65]. In order to reduce these variations, some
studies such as Park et al [54,66] used the morning/night
salivary cortisol ratio. This ratio as well as repeated measuring
of salivary cortisol seems to correlate with the severity of the
symptoms and effects of OSA treatment such as CPAP
[47,50,67]. Other studies used cortisol with other biomarkers
such as ɑ-amylase [45–50,66,68], and testosterone [48,69]. Use
of salivary cortisol seems to be one of the most consistent and
widely used salivary biomarkers in OSA studies.
In terms of responses to OSA treatment, a few studies
demonstrated the effectiveness of salivary biomarkers after
OSA treatments. For example, Celec et. al 2012 [70] examined
patients with OSA before CPAP, and then at 1 month and
6 months after CPAP treatment. They found AGEs and fructo-
samine decreased with the use of CPAP. Similarly, Ghiciuc
et. al 2013 [47] and Ghiciuc et. al 2016 [48] examined obese
subjects with OSA before, and after 3 months and 6 months of
CPAP compared to control. They found that salivary cortisol
and cortisol/testosterone spiked in the morning and level off
in the evening in control subjects. However, the trend is
opposite in obese subjects with OSA (before CPAP treatment).
They also found that the normal cortisol and cortisol/
Table 2. (Continued).
Study
Biomarker detection
technique Target or non-target (NT) Biomarkers
Thimgan et. al 2015 [54] RNA transcriptome analysis Targeted for 96 genes 21 gene upregulated in OSAS compared to control
(and related to sleepyness):
Β-actin,
Arachidonate 5-lipoxygenase, Arachidonate 12-
lipoxygenase, Annexin A1,
Annexin A3,
Annexin A5,
β-2-microglobulin, Caspase 1 (apoptosis-related
cysteine peptidase),
Glyceraldehyde-3-phosphate dehydrogenase,
Intercellular adhesion molecule 1,
Interleukin 1 receptor, type II,
Interleukin 2 receptor-gamma, Integrin alpha M,
Integrin beta 2, Mitogen-activated protein kinase
1,
Mitogen-activated protein kinase 3, Mitogen-
activated protein kinase 14,
Phosphodiesterase 4B cAMP-specific,
Prostaglandin-endoperoxide synthase 2
(prostaglandin
G/H synthase and cyclooxygenase), Tumor
necrosis factor receptor superfamily member 1A,
Tumor necrosis factor receptor superfamily
member
Tóthová et. al 2019 [43] Spectrofluorometry Targeted markers of oxidative stress
thiobarbituric acid reacting
substances (TBARS), advanced oxidation
protein products
(AOPP), advanced glycation end products
(AGEs), and total antioxidant capacity (TAC)
apnea-
hypopnea index
(AHI), TBARS, AOPP, and AGEs decreased with
CPAP
Traxdorf et. al 2016 [53] Enzyme-linked
immunosorbent assay
Targeted for S100B S100B in saliva (and serum)
OSA > Ronchopathy > control
Yan et. al 2019 [52] Immune-enzymatic cortisol
saliva and α-amylase saliva
kits
Targeted for α-amylase and cortisol α-amylase and cortisol increase with severity of
OSAS patients. Hypertension with the OSAS
group has the highest α-amylase level. However,
hypertension increases cortisol level with and
without OSAS.
Zheng et. al 2014 [41] Mass spectrometry (MALDI-
TOF MS) with weak cation-
exchange
(WCX) magnetic beads,
Western blotting
NT Five mass peaks identified as Alpha-2-HS-
glycoprotein precursor, fibrinogens) showed an
upregulation in CVD, whereas six mass peaks
downregulated in CVD (identified as Tubulin
alpha-4A chain).
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 7
testosterone ratio in obese subjects with OSA returns to nor-
mal (like the control subjects) after CPAP treatment. Similar
results for salivary cortisol levels and the responsiveness to
CPAP treatment were also found in studies by Raff et. al 2011
[50] and Schmoller et. al 2009 [67].
ɑ-amylase, one of the most abundant enzymatic proteins in
saliva, is often identified to be related to multiple oral and systemic
diseases [34,37]. However, the relationship between OSA and ɑ-
amylase is not clear. ɑ-amylase was found to be upregulated in the
2D gel-based mass spectrometry in controls compared to OSA
patients [51]. While Park et al [71] found ɑ-amylase level was
elevated in severe OSA compared to mild and moderate OSA,
Patacchi et al [69] found the ɑ-amylase was unrelated to OSA
severity. There may be some underlying mechanism of ɑ-
amylase secretion that is associated with dysfunction with OSA.
However, the mechanism of the pathology is not clear and there-
fore the use ɑ-amylase for OSA is questionable.
The two main flaws of the overall salivary biomarker discovery
for OSA are: 1) many studies fail to follow standardization of the
saliva collection protocol and 2) the majority of the studies
focussed on only amylase and cortisol. Additionally, the research-
ers seemed to neglect the oral/dental condition. There was no
report of oral condition, numbers of teeth, periodontal conditions
as well as other systemic conditions, diabetes, medication etc.
These factors are known to have influence on salivary biomarkers
[37,38]. Salivary biomarker discovery may have some benefits of
oral/dental evaluation at the baseline of the study. Systemic con-
ditions and medications should also be accountable for.
Third, there were some attempts to link OSA with other diseases
and conditions through salivary biomarker. Cytokines and pro-
teases as well as other inflammatory markers may have had
some contributions to OSA and suggested a link between period-
ontal disease and sleep apnea [44–46]. It is important to note that
many cytokines and inflammatory biomarkers especially in saliva
can be transient in nature or secreted in a minute amount. This
physiologic change can be a surrogate for changes in OSA condi-
tion; however, it may be too minute or transient such that cannot
be detected without a highly sensitive tool such as mass spectro-
metry. Otherwise, the signal may have to be amplified for example
by using mRNA analysis. More importantly, the timing of saliva
sample collection and prompt preservation/analysis would be
crucial. A few included studies such as those by Nizam et. al. also
link serum and salivary biomarkers together. For example, there
was a correlation between proMMP-9 concentration in serum and
MMP-9 activity in saliva [45]. OSA research has an advantage of
keeping the subjects overnight and collecting saliva and serum
samples at the same time. Linking salivary biomarkers with serum
biomarkers [72] can be very useful in the understanding of disease
mechanisms as well as to develop new saliva-based diagnostic
tools [33,35,36].
Finally, all included studies targeted known biomarkers, while
only two truly explored novel biomarkers through proteomics
[41,51] and one through transcriptomics [54]. It is interesting to
note that the two mass spectrometry-based proteomics studies
were very different. Liu et al used the 2D gel technology which has
clear limitations in its sensitivity, small number of identified pro-
teins, and potential masking of more abundant proteins [34]. This
technology is not sensitive enough for low concentration proteins;
therefore, it cannot provide a larger number of biomarkers. Low
concentrated proteins may be masked by nonspecific higher con-
centration proteins, such as ɑ-amylase, immunoglobulins, or car-
bonic anhydrase. On the other hand, Zheng et al [41] used weak
cationic exchange beads to identify low abundant proteins.
Fractionation of samples such as Surface-enhanced laser deso-
rption/ionization mass spectrometry (SELDI-TOF/MS) has shown
to be a promising tool for salivary biomarkers [73]. Thimgan et al
[41,54] is the only study using transcriptomics. Transcriptomics can
be a powerful tool for salivary biomarkers especially for diseases
such as OSA that may be related to inflammatory or oxidative
biomarkers/pathways, gene array Multiplex assays, whole tran-
scriptomics, as well as mass spectrometry-based proteomics ana-
lyses would be important to further explore salivary biomarkers
associated with OSA.
Multiple novel and known salivary biomarkers have been
shown to be associated with the presence or severity of OSA as
well as the response to treatment such as CPAP. It is important for
investigators to further develop these biomarkers as a tool possibly
for clinical as well as at home use. OSA affects not only the
compliant adult population but also infants, children, elderly popu-
lation as well as people with disability or anxiety. Saliva sampling
home testing if successful would ease OSA monitoring with
a wider population. Most current research has been done in adults,
mostly Caucasian males. Future research should include female
adults, children, infants, as well as subjects with disability.
It is also important to recognize the limitations of this work.
Studies written in languages other than English were excluded;
therefore, this review may not include every study conducted on
this topic. Further, due to publication bias, research that fails to
find relationships are less likely to be published. Consequently,
evidence that contradicts our findings of relationships between
biomarkers found in saliva and OSA may not have been available,
and therefore would have been missed. We excluded a few articles
such as Jeong et al [74] and Papaioannou et al [75] which were
a technique article and a negative result article. Jeong et al inves-
tigated the effect of adenotonsillectomy on cortisol levels before
and after surgery in OSA diagnosed children [74]. This work
demonstrated changes in salivary cortisol; however, as
a technical article, it was not included in this review.
Papaioannou et al examined the effect of OSA on melatonin and
the circadian rhythm [75]. They compared 22 OSA subjects with 22
healthy controls. Salivary melatonin as a marker for circadian
rhythm was obtained every 30 minutes from 19:30 to 22:30. No
statistical difference was found between the two groups.
Although this was the only paper that discussed melatonin as
a possible biomarker, we did not include it as there were no
significant findings from the paper to suggest melatonin has
a distinctive role in the circadian rhythms of OSA patients. While
these limitations are common in many systematic reviews, most
salivary biomarker systematic reviews often suffer most from the
lack of good clinical studies [34].
5. Conclusion
Salivary biomarkers have been used to evaluate OSA conditions
and treatment responses. Most current studies focus on known
salivary biomarkers including cortisol and ɑ-amylase. There is
a need for standardization of saliva collection protocol and
8S. BENCHARIT ET AL.
sample processing. Future research should explore novel sali-
vary biomarkers through proteomics and transcriptomics.
Linking salivary biomarkers to severity and symptoms as well
as known serum biomarkers associated with OSA has the poten-
tial for the development of novel noninvasive saliva-based
diagnostic tools of OSA in an out-patient setting or for patients
with difficulty to obtain serum samples.
6. Expert Opinion
Patients with OSA often require a life-long monitoring of the
severity of the condition as well as the effectiveness of parti-
cular OSA treatment. While PSG, the gold standard for OSA
diagnosis, can be done, the cost and labor for PSG as well as
the compliance of the application prevent the use of PSG on
the day-to-day basis. At home devices like HSAT can be used
in certain groups of patients. However, this still leaves a large
population of patients with OSA such as elderly, dementia,
disable patients or children that may use HSAT. The non-
invasive nature of saliva has therefore become a prime target
that can be developed as a non-invasive and potentially more
compliance real time at home monitoring of OSA conditions.
While saliva can be obtained non-invasively and readily
available in all patients, the current technology and knowl-
edge of salivary biomarkers associated with OSA are still in an
infantile stage. As we can see from this systematic review that
there are several biomarkers that may have potential to trans-
late into a real time OSA monitoring device. Unfortunately,
there was a clear lack of consistency in saliva collection and
processing as well as biomarker detection. There is a real need
to standardize the collection of saliva, the immediate sample
processing, as well as the methods of storage as discussed
early. This lack of sample collection standardization posts of
a question of study’s validity and applicability of the results.
The results of this systematic review point out at least three
future research opportunities. First, we can utilize the period
of PSG that is often done for most patients with OSA to collect
saliva samples to create baseline for individual patients as well
as a sample depository that can be used for future research.
This presents a tremendous opportunity for long-term studies
in a larger sample size with hopefully better technology in the
future. Institutions should consider saliva sample depository
alongside blood and urine samples. Second, there is currently
a lack of interest in searching for novel salivary biomarkers
associated with OSA. Multi-OMICS as well as gene array or
muliplex array technology should be considered and
employed to better understand OSA and changes in saliva
composition. There is a real need for salivary biomarker dis-
covery in the OSA research field. Finally, saliva unlike blood is
affected by oral and systemic conditions as well as are not
tightly regulated. Saliva composition can vary greatly among
different individuals and even within the same individual
throughout the day. However, salivary OMICS profiling is the-
orecticlly consistent within an individual during certain time of
the day. This should be understood and studied.
Transcriptomic, proteomic, and metabolomic profiling
changes associated with OSA can lead to not only novel
biomarkers, but may also allow the utilization of the profiling
itself as a monitoring tool.
Funding
This paper was not funded.
Author Contributions
S BENCHARIT ROLES Conceptualization, Methodology, Project administra-
tion, Supervision, Writing – original draft, Writing – review & editing
RG REDENZ ROLES Screening the articles, Extracting Data, Data cura-
tion, Formal analysis, Investigation, Methodology, Software, Writing – ori-
ginal draft, Writing – review & editing
ER BRODY ROLES Performing article searches, Formal analysis,
Investigation, Writing – original draft
H CHIANG ROLES Conceptualization, Methodology, Screening the arti-
cles, Writing – original draft
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Reviewer Disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
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Appendix I
Medline Ovid: (Saliva/or saliva.mp. or salivary.mp.) AND (sleep Apnea
Syndromes/or (sleep apnea or sleep apnea).mp.), restricted to English
language articles.
Embase: (Saliva/or saliva.mp. or salivary.mp.) AND (sleep Apnea
Syndromes/or (sleep apnea or sleep apnea).mp.), restricted to English
language articles.
Dental and Oral Sciences Source (DOSS): DE ‘SLEEP apnea syndromes’ OR
((sleep apnea or apnea or obstructive sleep apnea)) AND (DE ‘SALIVA’) or saliva
OR salivary
Web of Science: (saliva OR salivary) AND sleep apnea, restricted to
English language articles.
CINAHL Complete: ((MH ‘Sleep Apnea Syndromes’) OR ‘sleep
apnea’) AND (MH ‘Saliva’) OR ‘saliva’, restricted to English language
articles.
ProQuest Dissertations and Theses: Sleep apnea AND saliva, restricted
to English language articles.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 11
