Agarwal and Bui Investig Clin Urol 2017;58:385-399 ...
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Agarwal and Bui Investig Clin Urol 2017;58:385-399. November 2017. https://doi.org/10.4111/icu.2017.58.6.385 Supplementary Table 1. Summary of the commonly used techniques to evaluate seminal oxidative stress Technique Instrument Advantages Disadvantages ORP MiOXSYS Provides a snapshot of the redox balance in real time Levels of all oxidants and reductants are mea- sured Less time consuming Inexpensive materials Simple methodology Both fresh and frozen semen and seminal plasma can be measured Semen age, high viscosity, and repeated centrifugation may alter results of measurements ROS by chemiluminescence Luminometer Robust chemiluminescence High sensitivity and specificity Intracellular and extracellular ROS are detected 15–30 minutes to yield test results Cost and size of equipment Semen age, volume, repeated centrifugation, temperature and background luminescence may alter results of measurements TAC Colorimeter Luminometer Reliable and predictive of antioxidant capacity Total antioxidants in seminal plasma measured Cannot differentiate the amounts of enzymatic and nonenzymatic antioxidants independently Long duration of inhibition time Cost of microplate readers ROS-TAC Statistical analyses Superior to ROS or TAC alone Calculated through statistical analyses Does not directly measure ROS or TAC MDA Colorimeter and fluorometer for MDA-TBA adduct HPLC Assesses lipid peroxidation Rigorous controls required Nonspecific test Only detects post hoc damage ORP, oxidation-reduction potential; ROS, reactive oxygen species; TAC, total antioxidant capacity; MDA, malondialdehyde; TBA, thiobarbituric acid; HPLC, high performance liquid chromatography. Adapted from Agarwal et al. Ther Adv Urol 2016;8:302-318 [22].
Transcript of Agarwal and Bui Investig Clin Urol 2017;58:385-399 ...
Agarwal and BuiInvestig Clin Urol 2017;58:385-399. November 2017. https://doi.org/10.4111/icu.2017.58.6.385 Supplementary Table 1. Summary of the commonly used techniques to evaluate seminal oxidative stress
Technique Instrument Advantages DisadvantagesORP MiOXSYS Provides a snapshot of the redox balance in
real timeLevels of all oxidants and reductants are mea-
suredLess time consumingInexpensive materialsSimple methodologyBoth fresh and frozen semen and seminal
plasma can be measured
Semen age, high viscosity, and repeated centrifugation may alter results of measurements
ROS by chemiluminescence
Luminometer Robust chemiluminescenceHigh sensitivity and specificityIntracellular and extracellular ROS are detected
15–30 minutes to yield test resultsCost and size of equipmentSemen age, volume, repeated
centrifugation, temperature and background luminescence may alter results of measurements
TAC ColorimeterLuminometer
Reliable and predictive of antioxidant capacityTotal antioxidants in seminal plasma measured
Cannot differentiate the amounts of enzymatic and nonenzymatic antioxidants independently
Long duration of inhibition timeCost of microplate readers
ROS-TAC Statistical analyses Superior to ROS or TAC alone Calculated through statistical analysesDoes not directly measure ROS or TAC
MDA Colorimeter and fluorometer for MDA-TBA adduct
HPLC
Assesses lipid peroxidation Rigorous controls requiredNonspecific testOnly detects post hoc damage
ORP, oxidation-reduction potential; ROS, reactive oxygen species; TAC, total antioxidant capacity; MDA, malondialdehyde; TBA, thiobarbituric acid; HPLC, high performance liquid chromatography.Adapted from Agarwal et al. Ther Adv Urol 2016;8:302-318 [22].
Supplementary Table 2. Semen parameters and oxidation-reduction potential (ORP) changes in all patients (n=28) who had repeated tests done over a period of 16.8±7.7 weeks
Variable 1st sample 2nd sample p-valueConcentration (106 sperm /mL) 7.2 (2.93–20.73) 10.55 (5.22–33.3) 0.019Total motility (%) 32 (17.5–47.5) 42.5 (29.25–53.75) 0.008Morphology (normal form %) 2 (1–5.5) 2 (1.25–4.75) 0.57ORP (mV/106 sperm/mL) 6.08 (2.63–15.92) 2.06 (0.7–9.16) 0.007Patients treated with doxycycline (n=9) Endtz (M/mL) 0.8 (0.4–2.3) 0 (0–0.4) 0.024 ORP (mV/106 sperm/mL) 0.77 (0.49–1.05) 0.62 (0.49–0.74) 0.086
Values are presented as median (interquartile range).Adapted from Agarwal et al. Urology 2017;104:84-9 [55], permission of Elsevier.
A B C D
E F G H
sO
RP
(mV
/10
sperm
/mL)
6
20 140
Concentration (10 /mL)6
0
5
4
3
2
1
120100806040
r= 0.643p<0.001
0 200
sO
RP
(mV
/10
sperm
/mL)
6
Concentration (10 /mL)6
0
400
300
200
100
r= 0.883p<0.001
sO
RP
(mV
/10
sperm
/mL)
6
50 350
Total sperm count (10 )6
0
5
4
3
2
1
r= 0.577p=0.002
0
sO
RP
(mV
/10
sperm
/mL)
6
Total sperm count (10 )6
0
400
300
200
100
r= 0.811p<0.001
sO
RP
(mV
/10
sperm
/mL)
6
45
Motility (%)
0
5
4
3
2
1
r= 0.207p=0.31
20sO
RP
(mV
/10
sperm
/mL)
6
Motility (%)
0
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200
100
r= 0.369p=0.035
sO
RP
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/10
sperm
/mL)
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0
Morphology (%)
0
5
4
3
2
1
r=0.273p=0.18
0
sO
RP
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/10
sperm
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Morphology (%)
0
400
300
200
100
r= 0.322p=0.10
100 150 200 250 300 50 55 60 65 70 75 2 4 6 8 10 12
50 100 150 200 400 600 800 40 60 800 5 10 15
CCF 2016C
Supplementary Fig. 1. Correlation of semen static oxidation-reduction potential (sORP) (mV/106 sperm/mL) with semen parameters at 0 min-utes in healthy controls (A, concentration; B, total sperm count; C, motility; D, morphology) and infertile patients (E, concentration; F, total sperm count; G, motility; H, morphology). Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
A B C D
E F G H
0 200
sO
RP
(mV
/10
sperm
/mL)
6
Concentration (10 /mL)6
0
400
300
200
100
r= 0.846p<0.001
50 350
Total sperm count (10 )6
r= 0.506p=0.008
0
sO
RP
(mV
/10
sperm
/mL)
6
Total sperm count (10 )6
0
400
300
200
100
r= 0.772p<0.001
45
Motility (%)
r= 0.234p=0.25
20sO
RP
(mV
/10
sperm
/mL)
6
Motility (%)
0
400
300
200
100
r= 0.293p=0.10
0
Morphology (%)
r=0.342p=0.09
0
sO
RP
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/10
sperm
/mL)
6
Morphology (%)
0
400
300
200
100
r= 0.224p=0.26
100 150 200 250 300 50 55 60 65 70 75 2 4 6 8 10 12
50 100 150 200 400 600 800 40 60 800 5 10 15
20 140
Concentration (10 /mL)6
120100806040
sO
RP
(mV
/10
sperm
/mL)
6
0
20
15
10
5
r= 0.576p=0.002
sO
RP
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/10
sperm
/mL)
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0
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10
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sO
RP
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/10
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CCF 2016C
Supplementary Fig. 2. Correlation of seminal plasma static oxidation-reduction potential (sORP) (mV/106 sperm/mL) with semen parameters at 0 minutes in healthy controls (A, concentration; B, total sperm count; C, motility; D, morphology) and infertile patients (E, concentration; F, total sperm count; G, motility; H, morphology). Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
A B C D
E F G H
20 140
Concentration (10 /mL)6
120100806040
sO
RP
(mV
/10
sperm
/mL)
6
0
6
4
2
r= 0.644p<0.001
0 200
Concentration (10 /mL)6
r= 0.882p<0.001
sO
RP
(mV
/10
sperm
/mL)
6
0
400
300
200
100
50 350
Total sperm count (10 )6
r= 0.574p=0.002
0
Total sperm count (10 )6
r= 0.808p<0.001
45
Motility (%)
r= 0.123p=0.55
20
Motility (%)
r= 0.446p=0.009
0
Morphology (%)
r=0.267p=0.19
0
Morphology (%)
r= 0.315p=0.11
100 150 200 250 300 50 55 60 65 70 75 2 4 6 8 10 12
50 100 150 200 400 600 800 40 60 800 5 10 15
sO
RP
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/10
sperm
/mL)
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0
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sO
RP
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sO
RP
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/10
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/mL)
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sO
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sO
RP
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/10
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sO
RP
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/10
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/mL)
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CCF 2016C
Supplementary Fig. 3. Correlation of semen static oxidation-reduction potential (sORP) (mV/106 sperm/mL) with semen parameters at 120 min-utes in healthy controls (A, concentration; B, total sperm count; C, motility; D, morphology) and infertile patients (E, concentration; F, total sperm count; G, motility; H, morphology). Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
A B C D
E F G H
20 140
Concentration (10 /mL)6
120100806040
r= 0.652p<0.001
0 200
Concentration (10 /mL)6
r= 0.930p<0.001
sO
RP
(mV
/10
sperm
/mL)
6
0
400
300
200
100
50 350
Total sperm count (10 )6
r= 0.585p=0.002
0
Total sperm count (10 )6
r= 0.866p<0.001
45
Motility (%)
r= 0.221p=0.28
20
Motility (%)
0
r= 0.476p=0.005
0
Morphology (%)
r=0.251p=0.22
0
Morphology (%)
r= 0.331p=0.09
100 150 200 250 300 50 55 60 65 70 75 2 4 6 8 10 12
50 100 150 200 400 600 800 40 60 800 5 10 15
sO
RP
(mV
/10
sperm
/mL)
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0
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/10
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/mL)
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sO
RP
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/10
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CCF 2016C
Supplementary Fig. 4. Correlation of seminal plasma static oxidation-reduction potential (sORP) (mV/106 sperm/mL) with semen parameters at 120 minutes in healthy controls (A, concentration; B, total sperm count; C, motility; D, morphology) and infertile patients (E, concentration; F, total sperm count; G, motility; H, morphology). Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
A BS
ensitiv
ity
0.0
1.0
0.8
0.6
0.4
0.2
1.0
1-Specificity
0.0
Cutoff=1.48Sensitivity=60.0% (32.3-83.7)Specificity=75.0% (59.7-86.8)PPV=45.0% (23.1-68.5)NPV=84.6% (69.5-94.1)Accuracy=71.2% (57.9-82.2)
AUC=0.648
0.2 0.4 0.6 0.8
sO
RP
(mV
/10
sperm
/mL)
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Abnormal motility(n=15)
Normal motility(n=44)
500
0.01
1.48
Sensitiv
ity
0.0
1.0
0.8
0.6
0.4
0.2
1.0
1-Specificity
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Cutoff=2.09Sensitivity=46.7% (21.3-73.4)Specificity=81.8% (67.3-91.8)PPV=46.7% (21.3-73.4)NPV=81.8% (67.3-91.8)Accuracy=72.9% (59.7-83.6)
AUC=0.615
0.2 0.4 0.6 0.8
50
10
1
0.1
sO
RP
(mV
/10
sperm
/mL)
6
Abnormal motility(n=15)
Normal(n=44)
motility
500
0.01
2.09
50
10
1
0.1
C D
CCF 2016C
Supplementary Fig. 5. Receiver operating characteristic curve establishing the cutoff in semen (A) and seminal plasma (B). Distribution of static oxidation-reduction potential (sORP) (mV/106 sperm/mL) in subjects with normal and abnormal motility in semen (C) and seminal plasma (D), suggesting a criterion for sORP in distinguishing semen quality based on good (≥40%) and poor (≤40%) motility. AUC, area under the curve; NPV, negative predictive value; PPV, positive predictive value. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
sO
RP
(mV
/10
sperm
/mL)
6
Con
trols
(n=5
1)
200
0.02
1.36
50
10
2
0.1
0.5
Con
trols
with
prov
enfe
rtility(n
=15)
Con
trols
with
unpr
oven
ferti
lity(n
=36)
Pat
ient
s(n
=106
)
Varicoc
ele
(n=3
8)
Idiopa
thic
(n=1
3)
CCF 2016C
Supplementary Fig. 6. Distribution of static oxidation-reduction potential (sORP) (mV/106 sperm/mL) in (1) normal healthy controls; (2) controls with proven fertility; (3) controls with unproven fertility; (4) infertile patients; (5) infertile patients presenting with a clinical varicocele; and (6) those with idiopathic infertility. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
sO
RP
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/10
sperm
/mL)
6
0.5
0.4
0.3
0.2
0.1
0.0
A B
Replicate 1Replicate 2Replicate 3
O1
Sample 1
O2 O3 O1 O2 O3 O1 O2 O3
Sample 2 Sample 3
sO
RP
(mV
/10
sperm
/mL)
6
2.0
1.5
1.0
0.5
0.0Observer 1 Observer 2 Observer 3
CCF 2016C
Supplementary Fig. 7. Static oxidation-reduction potential (sORP) (mV/106 sperm/mL) across samples and observers showing (A) Intraobserver reliability by observing the replicate sORP measures for each of the three samples. Most replicates were similar to each other and across the 3 observers (01–03). (B) Interobserver reliability by comparing sORP-values across observers. The mean sORP for each observer was equivalent with similar standard error of the mean, suggesting that all observers obtained similar sORP-values for each of the 10 samples tested, which were measured in 4 replicates. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
Sensitiv
ity
0.0
1.0
0.8
0.6
0.4
0.2
1.0
1-Specificity
0.0
Cutoff=1.42Sens=60.6% (56.5-64.6)Spec=74.3% (64.6-82.4)PPV=93.3% (90.3-95.6)NPV=24.3% (19.6-29.4)Acc=62.6% (58.9-66.2)
AUC=0.703
0.2 0.4 0.6 0.8
CCF 2017C
Supplementary Fig. 8. Receiver operating characteristic curve of oxidation-reduction potential (ORP) (mv/106 sperm/mL) in distinguishing in-fertile patients from healthy controls in the combined dataset (Cleveland Clinic and Doha, Qatar). Acc, accuracy; AUC, area under the curve; NPV, negative predictive value; PPV, positive predictive value; Sens, sensitivity; Spec, specificity. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2013-2017.
