scientific conference posters - durviz sl

MiOXSYS®

Scientific Conference Posters2015-2016

MiOXSYS® Scientific Conference Posters 2015-2016

ASRM 2015

Effect of time on ORP in semen and seminal plasma 1

ASA 2016

ORP is related to abnormal semen parameters in male 2 factor infertility

Measurement of ORP as a newer tool indicative of OS in 3 infertile men with leukocytospermia

AUA 2016

Validation of ORP in fresh/frozen samples with MiOXSYS® 4

ESHRE 2016

ORP predictor for sperm morphology in infertile men 5

Seminal fluid static ORP is lower in seminal fluid that meets 5 all who criteria for fertility

ORP of spermatozoa selected for intracytoplasmic 6 sperm injection

ASRM 2016

ORP a novel test for evaluating male infertility 7

ORP a novel marker of varicocele pathophysiology 8

Infertile men have a redox imbalance that distinguishes 9 them from fertile men

ORP can differentiate fertile from infertile men 10

Evaluation of intra and inter observer reliability 11

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1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 2Medical Physiology, Stellenbosch University, Tygerberg, South Africa,3Redox Biology Laboratory, School of Life Science, Ravenshaw University, Orissa, India, 5Soroka Medical Center, Ben-Gurion University, Beer Sheva, Israel,

6Department of Urology, Cleveland Clinic, Cleveland, OH

Ashok Agarwal, PhD1, Stefan S. Du Plessis, PhD1,2, Rakesh Sharma, PhD1, Luna Samanta,PhD1,3, Avraham Harlev, MD1,4

Gulfam Ahmad, PhD1,5, Sajal Gupta, MD1 and Edmund S. Sabanegh, MD6

ESTABLISHING THE OXIDATION-REDUCTION POTENTIAL IN SEMEN AND SEMINAL PLASMA

OBJECTIVE: Oxidation-reduction potential (ORP) is a novel measure of oxidative stress or redox imbalance in biological samples. Static ORP (sORP) provides an integrated measure of the balance between total oxidants and reductants in a biological system, whereas capacity ORP (cORP) equates to the amount of antioxidant reserves. sORP has been shown to correlate well with illness and injury severity that accompanies the state of oxidative stress; cORP correlates with the ability to respond to illness or injury. Our objectives were to evaluate whether 1) ORP could be measured in semen and seminal plasma samples and 2) ORP levels correlate with sperm motility.

DESIGN: Prospective study measuring ORP in both semen and seminal plasma.

MATERIALS AND METHODS: Semen samples (n=18) from normal control subjects were divided into two fractions and the seminal plasma was isolated from one fraction (300g, 7min). Sperm count and motility were assessed manually. sORP (mV/106 sperm) and cORP (µC/106 sperm) were measured in both fractions (MiOXSYS, Aytu BioScience). Values are reported as Mean ± SEM. Spearman correlation and Receiver Operating Characteristic curves (ROC) were used for statistical analysis.

RESULTS: sORP and cORP levels in semen correlated significantly with the levels in seminal plasma. A significant negative correlation existed between sperm motility and sORP in both semen (r=-0.609; P=0.004) and seminal plasma (r=-0.690; P=0.002). Furthermore, a sORP cutoff of 4.73mV/106 sperm in semen (sensitivity = 100%, specificity = 89.5%, AUC=0.947) and 4.65mV/106 sperm in seminal plasma (sensitivity = 100%, specificity = 93.8%, AUC = 0.969) was highly predictive of abnormal sperm motility.

CONCLUSIONS: RedoxSYS® accurately measured sORP and cORP in both semen and seminal plasma samples. Based on high sensitivity as assessed by ROC analysis, the sORP levels can be used to screen infertile men with oxidative stress. These results are being validated in a larger cohort of infertile men.

ABSTRACT

Male factor infertility is a significant medical condition accounting for up to half of all cases of couple infertility. It affects approximately one in 20 men in the general population and no identifiable cause can be found in over 25% of infertile males. Oxidative stress is well established as a leading contributing factor to male infertility and evidence now suggests that reactive oxygen species (ROS)-mediated damage to sperm is a significant contributing pathology in 30-80% of cases. The quality of semen is considered a key indicator in male fertility, yet a significant proportion of male infertility remains unexplained in part because of the lack of standardized tests available to clinicians and researchers to assess oxidative stress.Currently, the most common method for measuring oxidative stress is by measurement of the levels of ROS by chemiluminescence assay in semen and total antioxidant capacity (TAC) by colorimetric assay in the seminal plasma and subsequently calculating a composite ROS-TAC score. However, the current methods used to measure oxidative stress involve sophisticated instruments, are cumbersome, require larger sample volume, and increase the turn-around-time for these tests. Redox imbalance is caused by a higher production of ROS and reactive nitrogen species or a decrease in endogenous protective antioxidants. Surrogate markers have historically been used to measure redox imbalance in patients (e.g., antioxidants like glutathione, lipid peroxidation, free radical production, protein oxidation, and/or enzyme activity). Relying upon measurements of individual markers often results in an unreliable, variable, and conflicting measurement of a patient’s redox balance. Oxidation-reduction potential (ORP) is a measure of oxidative stress or redox imbalance in biological samples. Static ORP (sORP) provides an integrated measure of the balance between total oxidants and reductants in a biological system, whereas capacity ORP (cORP) equates to the amount of antioxidant reserves. sORP has been shown to correlate well with illness and injury severity that accompanies the state of oxidative stress; cORP on the other hand correlates with the ability to respond to illness or injury. The objectives of this study were to evaluate whether 1) ORP could be measured in semen and seminal plasma samples and 2) ORP levels correlate with sperm motility.

INTRODUCTION

After obtaining, Institutional Review Board approval, a prospective study was carried out on 18 normal healthy men. All subjects provided written consent to be enrolled in this study. After liquefaction a standard semen analysis was performed. Semen samples were subsequently divided into two fractions and the seminal plasma was isolated from one fraction (300g, 7min) before measuring the sORP and cORP in both fractions with theMiOXSYS system (Aytu BioScience).

Semen AnalysisFollowing liquefaction, manual semen analysis was performed according to WHO 2010 guidelines to determine sperm concentration and motility. Smears were stained with a Diff-Quik kit for assessment of sperm morphology. Sample was tested for Leukocytospermia, i.e. >1 X 106 WBC/mL when the round cell concentration was >1 X 106 and. confirmed by the peroxidase or the Endtz test.

Oxidation-Reduction (ORP) MeasurementORP was measured using the RedoxSYS test comprised of a RedoxSYS analyzer (Figure 1) MiOXSYS sensor (Figure 2). The sensor was inserted face-up and with the sensor electrodes facing the MiOXSYS Analyzer. Using a micropipette, 30µL of sample (semen or seminal plasma) was released onto the sensor’s application port. Once the sample reached the reference cell of the sensor, the testing automatically began and audible beeps indicated completion of the test. The sORP (in millivolts or mV) and cORP (in microcoulombs or µC) were recorded. Analysis was performed in triplicate and the average values were corrected for sperm concentrations andexpressed as sORP (mV/106 sperm) and cORP (µC/106 sperm).

Statistical AnalysisSpearman correlation test was used for statistical analysis to compare qualitative variables. A P value of < 0.05 was considered statistically significant. A Receiver Operating Characteristic curve (ROC) was used to determine the preliminary predictive power of sORP values to identify semen samples with poor motility.

MATERIALS and METHODS

1. Semen sORP levels (3.30±3.835 mV/106 sperm) correlated significantly with the sORP levels in seminal plasma (3.21±3.690 mV/106 sperm) (Figure 2a).

2. Semen cORP levels (0.92±2.955 µC/106 sperm) correlated significantly with the cORP levels in seminal plasma (1.09±3.192 µC/106 sperm) (Figure 2b).

3. A significant negative correlation existed between sperm motility and sORP in semen (r=-0.609; P=0.004) (Figure 2c).

4. A significant negative correlation existed between sperm motility and sORP in seminal plasma (r=-0.690; P=0.002) (Figure 2d).

5. Furthermore, a sORP cut-off of 4.73mV/106 sperm in semen (sensitivity = 100%, specificity = 89.5%, AUC=0.947) and 4.65mV/106 sperm in seminal plasma (sensitivity = 100%, specificity = 93.8%, AUC = 0.969) was highly predictive of abnormal sperm motility, i.e. < 40% total motility (Figures 3 and 4).

RESULTS

1. The MiOXSYS test can accurately measure ORP in semen and seminal plasma.2. Both semen and seminal plasma can be used to measure sORP and cORP levels without differing significantly. Individual values were significantly correlated. 3. Higher sORP measures were associated with lower levels of sperm motility4. Based on high sensitivity, as assessed by ROC analysis, the sORP levels might be used to screen infertile men with motility abnormalities.

CONCLUSIONS

ORP Levels in Semen and Seminal Plasma

sORP(mV/106 sperm)

cORP(µC/106 sperm)

2.79±0.662.67±0.71

0.994<0.001

SemenSeminal PlasmaCorrelation (r)P value

0.20±0.080.31±0.16

0.959<0.001

Amer

ican

Center for Reproductive Medicine

Centrum Excellentiae Est.

1993

The MiOXSYS (Aytu BioScience) test used for measuring oxidation-reduction potential (ORP). The test consists of the analyzer (A) and disposable single use sensor strips (B).

Figure 1A B

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ORP values between Semen and Seminal Plasma were highly correlated suggesting that either semen or seminal plasma could be used to assess oxidative stress (sORP, A) or antioxidant capacity (cORP, B).

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Higher sORP values measured in semen (A) and seminal plasma (B) were correlated to lower levels of motility.

Semen samples with a measured sORP value higher than 4.73 sORP/106 cells may have poorer motility rates. 100% of all samples with poor motility were identified as having poor motility by their sORP values.

Seminal plasma with a measured sORP value higher than 4.65 sORP/106 cells may have poorer motility rates. As with semen measures, 100% of all samples with poor motility were identified as having poor motility by their sORP values measures in seminal plasma.

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Cutoff = 4.73Sensitivity = 100.0% (15.8−100.0)Specificity = 89.5% (66.9−98.7)PPV = 50.0% (6.8−93.2)NPV = 100.0% (80.5−100.0)Accuracy = 90.5% (69.6−98.8)

AUC = 0.947

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Cutoff = 4.65Sensitivity = 100.0% (15.8−100.0)Specificity = 93.8% (69.8−99.8)PPV = 66.7% (9.4−99.2)NPV = 100.0% (78.2−100.0)Accuracy = 94.4% (72.7−99.9)

AUC = 0.969

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ASRM 2015

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Introduction

Semen samples from twenty four infertile men attending our andrology clinic were collected after informed consent. Semen analyses were first performed according to the WHO guidelines. A well-mixed aliquot of 30µl was then used to measure sORP and cORP using the MiOXSYS analyzer after calibration. All values reported as mean ± SEM and the Spearman correlation coefficient (r) carried out using GraphPad Prism software.

Results Results (Continued) Oxidative stress (OS) is known to impair the physiological function of spermatozoa leading to male infertility. OS occurs due to imbalance resulting from higher production of reactive oxygen species (ROS) and lower activity of the antioxidant enzymes in semen. The assessment of such imbalance is complicated and often difficult to investigate in the clinical setting. Oxidation-reduction potential (ORP) is a newer and simpler tool to assess the status of oxidative stress. The present study measures both static-ORP (sORP as mVolt) and capacity-ORP (cORP current as µC) in fresh semen samples using a new electrochemical system, MiOXSYS™ to correlate ORP (as a measure of oxidative stress) with semen parameters of infertile men.

Semen Oxidative Reduction Potential (ORP) is related to Abnormal Semen Parameters in Male Factor Infertility

Jaideep S. Toor, Lucelia Daniels, Faysal A. Yafi, Wayne J.G. Hellstrom and Suresh C. Sikka. Department of Urology, School of Medicine, Tulane University, 1430 Tulane Avenue, New Orleans, LA-70112.

Fig. 1. The MiOXSYS system measures oxidative-reduction potential based on capacity to either release or accept electrons from chemical reaction. This system measures (a) sORP that reflects the existing balance between total oxidants and reductants in the sample and is expressed in millivolts, and (b) cORP that measures antioxidant capacity reserves in the semen samples by applying resistance to a small current sweep through the sample and is expressed in micro-coulombs

Objectives

Materials and Methods

To understand the potential impact of sORP and cORP in semen of infertile men, (a) who have shown improvement in their semen parameters over time, and (b) who have consistently low semen parameters after follow-ups.

Conclusions

MiOXSYS sensors

Internal low and high positive controls

Insert sensor

correlation with sperm concentration, total sperm count, sperm motility and morphology. 2a: sORP levels were measured in fresh whole semen from infertile men with improved/normal sperm concentration (NC), low sperm concentration (LC), improved/normal total sperm count (NTC), low total sperm count (LTC), improved/normal motility (NM), and low motility (LM) using the new electrochemical system, MiOXSYSTM. “Infertile” bar shows composite data of all groups taken together. All data are expressed as mean±SEM values. Significant differences show p-value<0.05 (*). Figure 2b to 2e represent scatter plots depicting significant negative correlation between sORP and respective semen parameters. The solid line represents the linear regression of points and dotted line represents 95% confidence interval for the mean values. Rho- and p- values are for the Spearman’s rank correlation test.

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Figure 2: Shows sORP/million sperm/ml levels in whole semen of infertile men and its

Sperm Concentration Total Sperm Count Sperm Motility

Normal sperm concentration

Low sperm concentration

Normal total sperm count

Low total sperm count

Normal motility

Low motility

Sperm concentration (million/ml) 62.5±10.0 5.66±1.4 56.49±9.7 4.35±1.1 31.49±5.8 32.88±9.7 Total sperm count (million) 161.1±27.7 15.46±3.5 149.2±25.7 12.07±2.1 97.51±23.9 86.79±25.3

% Total motility 45.6±3.2 44.26±4.3 44.65±3.3 41.71±4.3 60.29±1.9 33.68±2.1 Morphology (% Normal sperm) 23±2 19±1.3 23±1.8 17±1.6 22±1.6 20±1.7

Abstinence (days) 4±0.6 4±0.7 4±0.7 4±0.7 4±0.5 4±0.7

Age (years) 32±1.39 37±1.84 33±1.31 37±2.06 31±1.30 36±1.9

Table 1: Baseline parameters (Mean±SEM ) in different groups of infertile men (no significant differences obs. in the WBC count among all groups)

3a 3b

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3a:cORP levels were measured in fresh whole semen from infertile men with improved/normal sperm concentration (NC), low sperm concentration (LC), improved/normal total sperm count (NTC), low total sperm count (LTC), improved/normal sperm motility (NM), and low motility (LM) using the new electrochemical system, MiOXSYSTM. Infertile bar shows composite data of all groups taken together. 3b: sORP/cORP ratio in semen of infertile males in the above mention groups. All data are expressed as mean±SEM values. Significant differences are represented as the p-value <0.05 (*). 3c: Represents scatter plot depicting significant negative correlation between cORP in fresh whole semen from infertile males and sORP/million sperm/ml levels (one data point is outside the axis limits). The solid line represents the linear regression of points and dotted line represents 95% confidence interval for the mean values. Rho- and p- values are for the Spearman’s rank correlation test.

Figure 3: Shows cORP levels (µC) and sORP/cORP ratio in whole semen of infertile men with normal (improved) and low semen parameters over time, as well as its negative correlation with sORP.

1. sORP in the semen samples of infertile men correlated well with their sperm concentration, motility, and morphology.

2. A significantly high sORP/cORP ratio as assessed by MiOXSYS in infertile patients with low sperm concentration and motility suggests the role of increased oxidative stress.

3. This newer system (MiOXSYS) measuring ORP could potentially serve as an important additional indicator in the workup of male factor infertility.

4. These results provide evidence that sORP and cORP levels in semen of infertile men can differentiate and predict responders vs non-responders to infertility therapy.

ASA 2016

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Measurement of Oxidation-Reduction Potential (ORP) as a newer tool indicative of Oxidative Stress in Infertile Men with Leukocytospermia

Suresh C. Sikka, Jaideep S. Toor, Lucelia Daniels, Faysal A. Yafi, and Wayne J.G.Hellstrom. Department of Urology, Tulane University, New Orleans, Louisiana.

Conclusions • Our previous and current studies showed TLR-4 translocation from cytoplasm to nucleus and overexpression of COX-2, indicative of

active inflammation in parallel with high WBC content during leukocytospermia. • Both TLR-4 and COX-2 are potential biomarkers for GUI and male factor infertility. • MiOXSYS System sORP data suggest higher oxidation-reduction potential and one-step indicator of increased oxidative stress in semen

samples of infertile men with leukocytospermia . • This newer ORP parameter as measured by MiOXSYS could potentially serve as an important additional indicator in the workup of male

factor infertility. Much larger standardized studies in a multicenter format are needed to establish such diagnostic role of MiOXSYS in evaluation of OSS in routine clinical practice.

Results

Fig.3: Quantitative measurement of fluorescence intensity

Fig.4: sORP and cORP levels (expressed as /million sperm/ml) were measured in fresh whole semen from infertile men with and without leukocytospermia using the new electrochemical system, MiOXSYSTM. sORP showed positive correlation

with high WBC in leukocytospermia compared to non-leukocytospermia

Our Publication: Differential expression of novel biomarkers (TLR-2, TLR-4, COX-2, and Nrf-2) of inflammation and oxidative stress in semen of leukocytospermia patients. Andrology (2015) 3; 848–855

Introduction

• Leukocytospermia (LCS, a common cause of male infertility) is characterized by the presence of >1X10^6/mL white blood cells (WBC) in the semen sample.

• LCS caused by prostatitis and genitourinary inflammation (GUI), manifested by inflammatory chemokines and increased oxidative stress (OS) in the semen results in decreased sperm motility, high sperm DNA damage, decreased sperm function, i.e., fertility.

• Oxidation-reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (ROS) and reductants (antioxidants) that reflects oxidative stress status (OSS) in a biological system.

• However, the current methods to measure OSS are time consuming, expensive, need fresh sample, have many limitations and thus are not routinely performed.

• This preliminary study measures both static-ORP (sORP as mVolt) and capacity-ORP (cORP as µC) in fresh semen samples using the new electrochemical system, MiOXSYS™.

Materials and Methods • Semen samples from LCS and non-LCS infertile patients in our clinic

were collected, evaluated for semen parameters, and processed for:

(a) Expression/localization of TLR-4 and COX-2 in washed sperm by immunofluorescence microscopy. “Image-J” software (NIH) was used to quantify fluorescence intensity. (b) 30µl aliquot was placed on the special sensor in the calibrated MiOXSYS analyzer to measure sORP and cORP. All values were normalized to sperm concentration and expressed as Mean + SEM units/million sperm/mL).

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Fig.3: TLR-4 trans-location from the plasma membrane to the nucleus of LCS samples (lower panel) compared to non-LCS (upper panel)

Fig.4: COX-2 expression is up-regulated in LCS samples (lower panel) compared to non-LCS (upper panel)

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Validation of oxidation-reduction potential in freshand frozen semen samples with MiOXSYSTM System

Ashok Agarwal*, Rakesh Sharma, Stefan Du Plessis and Edmund SabaneghAmerican Center for Reproductive Medicine and Department of Urology, Cleveland Clinic, Cleveland, OH

Amer

ican



1993

MATERIAL AND METHODSStudy Samples and Semen AnalysisThis study was reviewed and approved by the Institutional Review Board; semen samples were collected from healthy normal men (n = 20) after 2-3 days of sexual abstinence. Semen samples were analyzed according to the 2010 World Health organization (WHO) guidelines. After complete liquefaction, a 5µL aliquot of a well-mixed sample was loaded on a MicroCell counting chamber (Vitrolife, San Diego, CA) and manually evaluated for sperm concentration (x 106/mL), percentage motility, number of round cells per high power field by using 20x phase contrast optics. Samples that were highly viscous or had >1x106/mL round cells were excluded from the study.

Sample processing and induction of oxidative stress by cumene hydroperoxide Each sample was divided into three aliquots: aliquot 1 - incubated with sperm and medium (control); aliquots 2 and 3 were exposed to oxidative stress induced by freshly prepared cumene hydroperoxide (CH: 5 and 50µmoles) in PBS and incubated for 20 minutes at 37°C (1:9 CH : semen vol./vol.). All fractions were snap frozen (-80°C) without any cryoprotectant for 24 hours for measurement of ORP in frozen samples.

Measurement of oxidation-reduction potential Oxidation reduction potential is a measure of the transfer of electrons from a reductant (or antioxidant) to an oxidant and was measured using a novel galvanostat-based technology - the MiOXSYS System (Figure 1 A-C); Aytu Bioscience, Englewood, CO). The MiOXSYS System was calibrated according to the manufacturer’s instructions. Briefly, the specific sensor was pre-inserted into the Analyzer and a 30µL of well-mixed liquefied semen at room temperature was applied at the exposed end. The measurement begins automatically and takes 3 to 4 minutes. After thawing the aliquots, ORP was measured again in all three fractions in triplicate using the method as described above. The average values for sORP and cORP were recorded. Data were normalized to sperm concentration in order to account for differences in sperm concentration. Finally, these data were presented as mV/106 sperm/mL for sORP and µC/106 sperm/mL for cORP.

Statistical Analysis Summaries of the categorical variables are reported as percent frequency; quantitative variables as mean ± SEM along with the 95% confidence intervals. Paired t test was used for measuring percent change in sORP and cORP in pre-freeze and post-thaw samples. A P-value of < 0.05 was considered statistically significant.

CONCLUSIONS1. MiOXSYS System can measure the ORP in both fresh and frozen semen samples in real time.

2. ORP technique is sensitive to changes in ORP following induction of oxidative stress by cumene hydroperoxide.

3. Induction of oxidative stress by CH, and measurement of ORP in real time supports the dynamics of the MiOXSYS System in evaluating sORP and cORP and validates the measurements in human semen.

4. Further studies utilizing larger sample size and in multicenter format using semen samples from infertile men are important to verify its clinical utility as an alternative to current oxidative stress markers. This will introduce an easy, simple, and real-time measure of oxidative stress and ORP in Andrology and IVF laboratories.

RESULTSDescriptive information is provided in Tables 1 and 2.

Effect of cumene hydroperoxide on sperm motility and seminal ORP1. Exposure to 5 and 50µmoles of CH resulted in a dose-dependent decline in pre-freeze sperm motility compared to controls (41± 2 and 34 ± 2 in groups receiving 5 and 50µmoles of CH vs. 52 ± 2 in control). 2. CH increased the available oxidants resulting in an increase in sORP/106/mL sperm. Conversely, the cORP/106/mL levels showed a significant decline in the available antioxidant reserves following exposure to both 5 and 50µmoles of CH (Table 1).

Change in pre-freeze sperm motility after exposure to cumene hydroperoxide1. Compared to the controls, exposure to 5 and 50µmoles of CH resulted in a decline in motility (95% CI: -13.32, -8.15; P<0.001 and -21.52, -14.86; P<0.001, respectively) in pre-freeze samples.

Change in pre-freeze and post-thaw ORP after exposure to cumene hydroperoxide1. Compared to control, exposure to 50µmoles of CH significantly (P = 0.04) increased the sORP/106 sperm/mL (Table 2; Figure 2A). 2. Following thawing, a significant increase (P = 0.025) was seen in sORP/106

sperm/mL between control and 50µmoles of CH (Table 2; Figure 2C). The change in cORP/106 sperm/mL from pre-freeze to post-thaw in control as well as in samples exposed to both 5 and 50µmoles of CH was significant (P < 0.05) (Table 2, Figure 2F).

ABSTRACTINTRODUCTION AND OBJECTIVES: Oxidative stress in seminal ejaculates is quantified by measuring individual markers such as reactive oxygen species, lipid peroxidation and total antioxidants. However, they have limited diagnostic capabilities. Oxidation-reduction potential (ORP) is a measure of oxidative stress or redox imbalance in a biological sample. Static ORP (sORP) is a balance between total oxidants and reductants in a biological system and correlates well with illness and severity of illness that accompanies oxidative stress. cORP correlates with the ability to respond to illness or injury. The goal of this study was to determine whether ORP can be measured in fresh and frozen semen following exogenous induction of oxidative stress using the Analyzer. METHODS: Semen samples were collected from 18 healthy normospermic subjects after 2-3 days of abstinence. Oxidative stress was induced with cumene hydroperoxide (5 and 50 µm). Sperm motility and viability were assessed before freezing. ORP was measured using novel galvanostat-based technology the MiOXSYS System. A 30 µL aliquot of the test sample was applied to the sensor. The Sensor is inserted into the Analyzer; measurement begins automatically. sORP (mV/106 sperm) and cORP (µC/106 sperm) were quantified in both fresh and snap-frozen fractions. Values are reported as Mean ± SEM.RESULTS: Both 5 and 50 µM cumene hydroperoxide significantly decreased motility and viability in the prefreeze semen samples (P<0.001). sORP was 2.21 ± 0.40 (95% CI: 1.38, 3.04) in the prefreeze controls and 2.73 ± 0.54 (95% CI: 1.62, 3.84) in the frozen samples. sORP significantly increased after freezing from 2.29 ± 0.41 (95% CI: 1.43, 3.14) in the controls to 3.03 ± 0.60 (95% CI: 1.78, 4.28) with 50 µm cumene hydroperoxide. The change in sORP was higher in the controls vs. the sperm treated with 50 µm (P<0.001) in both pre- and post-freeze semen samples. The change from pre- to post freeze sORP was comparable.CONCLUSIONS: The MiOXSYS System can measure redox potential in both fresh and frozen semen specimens. It is a simple and real-time measure of redox potential that can be used by fertility laboratories.

INTRODUCTIONThe imbalance between ROS production and scavenging by antioxidants causes oxidative stress, which deteriorates sperm quality, fertilizing ability, chances of natural pregnancy as well as outcomes of assisted reproductive techniques. Oxidative stress can be quantified by measuring the levels of ROS in the seminal ejaculate and total antioxidant capacity (TAC) in the seminal plasma. Earlier, we demonstrated that a combined ROS-TAC score provides a better measure of oxidative stress compared to evaluating individual ROS and TAC parameters at any given time. However, this often results in an unreliable, highly variable and conflicting evaluation of a patient’s redox balance and also involves the use of sophisticated instruments, for example, luminometer, flow cytometer, plate reader, etc.). Such tools are cumbersome, require large volume of fresh seminal fluid, and have high turn-around-time for test results, too. In addition, they (1) are unable to provide sufficient diagnostic value on their own; (2) do not provide a complete picture of redox imbalance as miss other important factors that contribute to a semen sample’s overall redox balance; (3) require multiple manual inputs, which can lead to complex errors; and (4) must be performed in multiple, time-consuming steps. Another major limitation is that accurate ROS levels cannot be measured in frozen semen samples by the chemiluminescence method.

We have recently evaluated another measure of oxidative stress based upon oxidation-reduction potential (ORP), which is an integrated quantitative measure of the balance between total oxidants (ROS) and total reductants (antioxidants) in a biological system. It provides an overall picture of all known and unknown elements that generate redox imbalance and has been shown to correlate with the ability to respond to related illness or injury. Static ORP is a “snapshot” of the current redox balance; a higher sORP reading is indicative of oxidative stress. Capacity ORP or cORP reflects the amount of antioxidant reserves available in a given system.

The goals of this study were to examine the feasibility of using the MiOXSYS System to: (a) measure ORP in semen before and after freezing, and (b) examine the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP.

Figure 1. Measurement of oxidation-reduction potential (ORP) by A: Analyzer by placing the sensor; B: gently inserting the sensor; C: view when the sensor is in place.

Figure 2. Comparison of oxidation-reduction potential (ORP) following exposure to 5 and 50µmoles of cumene hydroperoxide (CH). A: pre-freeze sORP/106 sperm/mL; B: pre-freeze cORP/106 sperm/mL; C: post-thaw sORP/106 sperm/mL; D: post-thaw cORP/106 sperm/mL; E: change in pre-freeze and post-thaw sORP/106 sperm/mL; and F: change in pre-freeze and post-thaw cORP/106 sperm/mL.

The gray boxplots demonstrate the percentiles (5th, 25th, median, 75th, and 95th) of the data distributions at each concentration of CH, while the red dots and lines show the mean and its 95% confidence interval. The blue lines show the changes for individual subjects between CH concentrations.

Table 1.

Pre- freeze and post-thaw seminal sORP and cORP after exposure to cumene hydroperoxide

Table 2.

Effect of freezing on seminal sORP and cORP after exposure to cumene hydroperoxide

CH = cumene hydroperoxide

Parameter Treatment Pre-freeze(mean ± SEM)

95% CI Post-thaw

(mean ± SEM) 95% CI P value

sORP/106 sperm/mLControl 2.21 ± 0.40 1.38, 3.04 2.29 ± 0.41 1.43, 3.14 0.76

5µmoles of CH 2.49 ± 0.48 1.49, 3.49 2.68 ± 0.50 1.66, 3.14 0.3450µ moles of CH 2.73 ± 0.54 1.62, 3.84 3.03 ± 0.60 1.78, 4.28 0.22

cORP/106 sperm/mLControl 1.27 ± 0.32 0.62, 1.93 0.96 ± 0.31 0.33, 1.59 0.009

5µ moles of CH 1.10 ± 0.29 0.49, 1.70 0.82 ± 0.27 0.26, 1.37 <0.00150µ moles of CH 1.10 ± 0.36 0.34, 1.85 0.83 ± 0.31 0.19, 1.48 0.020

Parameter Treatment Mean ± SE 95% CI P value

sORP/106 sperm/mL

Control vs. 5moles of CH 0.28 ± 0.15 (-0.03, 0.58) 0.075 vs. 50µmoles of CH 0.24 ± 0.17 (-0.10, 0.59) 0.16

Control vs. 50µmoles of CH 0.52 ± 0.24 (0.03, 1.01) 0.04

cORP/106 sperm/mL

Control vs. 5µmoles of CH 0.18 ± 0.11 (-0.40, 0.05) 0.125 vs. 50µmoles of CH 0.00 ± 0.15 (-0.30, 0.30) >0.99

Control vs. 50µmoles of CH 0.18 ± 0.15 (-0.48, 0.13) 0.24

sORP/106 sperm/mLControl vs. 5µmoles of CH 0.40 ± 0.26 (-0.15, 0.94) 0.15

5 vs. 50µmoles of CH 0.35 ± 0.25 (-0.17, 0.87) 0.17

Control vs. 50µmoles of CH 0.75 ± 0.31 (0.10, 1.39) 0.025

cORP/106 sperm/mLControl vs. 5µmoles of CH 0.14 ± 0.08 (-0.31, -0.02) 0.09

5 vs. 50µmoles of CH 0.02± 0.10 (-0.20, 0.24) 0.86

Control vs. 50µmoles of CH 0.12 ± 0.09 (-0.31, -0.06) 0.18

sORP/106 sperm/mLControl 0.08 ± 0.24 (-0.43, 0.58) 0.76

5µmoles of CH 0.19 ± 0.20 (-0.22, 0.61) 0.34

50µmoles of CH 0.31 ± 0.24 (-0.20, 0.81) 0.22

cORP/106 sperm/mLControl 0.32 ± 0.11 (-0.55, -0.09) 0.009

5µmoles of CH 0.28 ± 0.06 (-0.40, -0.16) <0.001

50µmoles of CH 0.26 ± 0.10 (-0.48, -0.05) <0.02

Pre-freeze changes

Post-thaw changes

Changes in pre-freeze to post-thaw

AUA 2016

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ElBardisi, H1,2, Arafa, M1,2, Agarwal, A3, AlSaid, S1, Majzoub, A1,3,4, Keyvanifard, F1, Abohalawa, M1, AlRumaihi, K1, Bar-Or, D5, Bjugstad, KB5 1. Urology Department - HMC, Doha, Qatar, 2. Andrology Department, Cairo University, Egypt, 3. Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA, 4. Department of Urology, Glickman Urological and Kidney Institute, 5. Swedish Medical Center, Englewood, CO USA.

Seminal Fluid Static Oxidation-Reduction Potential is Lower in Seminal Fluid that Meets All WHO Criteria for Fertile Men

Background and Study Question Discrete measures of free radicals, antioxidant activity, and oxidative damage suggest an ambiguous relationship between the redox system and male fertility. sORP measures the balance between all oxidants and antioxidants providing a comprehensive status of the redox system. In previous studies, sORP was elevated in brain trauma, stroke, and multi-trauma patients, indicating a state of oxidative stress; thus sORP levels may be used to clarify the relationship between the redox system and semen parameters associated with male infertility. Further, a sORP cut-off value may identify semen samples that meet the WHO normal reference values. Identify a cut-off value for sORP that can be used to identify semen that meet the normal reference range of WHO parameters from ones that fail one or more. Study Design and Methods Table 1 shows the patient demographics. The study subjects were grouped into those that had all normal semen parameters by WHO 2010 guidelines (Meets) and those who failed to meet one or more criteria (Fails). Exclusion criteria included azoospermia, presence of STD or chronic disease, use of prescription or OTC medications or antioxidants. Samples were collected and semen parameters assessed using the WHO guidelines (5th edition, 2010) (Table 2). sORP was measured (mV) using the MiOXSYS system and normalized to concentration (mV/106 sperm/mL).

Table 1. Patient Demographics N Mean StDev

Age 400 35.76 7.88 BMI 352 31.62 26.99 Abstinence (days) 400 4.35 2.21 Hormone Tests

Estradiol (E2) 154 182.58 76.09 Luteinizing hormone

(LH) 243 4.41 2.71

Follicle stimulating horm (FSH) 241 6.15 17.83

Protactin (PRL) 242 235.15 175.67 Testosterone 248 31.62 219.16

Table 2. WHO reference limits and N’s for those that met the reference (Normal) and those that failed (Abnormal)

Characteristic Lowest normal limit Normal (N) Abnormal (N)

Volume (mL) > 1.5 mL 93 14 Total sperm # >39x106 74 33 Concentration (X 106/ mL) >15X106/mL 74 33

Total Motility >40% 60 47 Progressive Motility >32% 6 101

Morphology >4% 53 54

Table 3. ROC results based on a cut-off of >1.635 sORP to predict semen samples that fail WHO parameters.

AUC (95% CI) 0.779 * (0.74-0.82)

Sensitivity (95% CI) 59.45% (54.2-64.5)

Specificity (95% CI) 91.43% (76.9-98.2)

Positive Predictive Value (95% CI) 98.6% (96.1-99.7)

Negative Predictive Value (95% CI) 17.8% (12.5-24.2)

Accuracy 62%

Figure 1. Those that Meet all WHO criteria for normal sperm have significantly lower sORP values than those that fail one or more parameters (* p< 0.05)

Figure 2. Semen with more abnormal parameters have higher levels of oxidative stress. Semen that fail 4 or more semen parameters have significantly higher sORP values. (* p< 0.05)

Figure 3. Abnormal progressive motility alone does not elevate sORP values, but the addition of abnormal morphology induces a significant elevation. Progressive motility (97.3% of samples) was the most common abnormal parameter followed by morphology (59% of samples). (* p< 0.05)

Semen that fail one or more parameters for normal semen have higher levels of oxidative stress. Abnormal morphology contributes to increasing sORP values. Semen with an sORP value that was greater than 1.635

had a 98.6% chance of being abnormal on one or more of the semen parameters. This represents an initial clinical cut-off value for interpreting the results of the MiOXSYS System, an adjunct measure

to routine semen analyses.

Arafa, M1,2, ElBardisi, H1,2, Agarwal, A3, AlSaid, S1, Majzoub, A1,3,4, AlRumaihi, K1, Bar-Or, D5, Bjugstad, K.B5. 1. Urology Department - HMC, Doha, Qatar, 2. Andrology Department, Cairo University, Egypt, 3. Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA, 4. Department of Urology, Glickman Urological and Kidney Institute, 5. Swedish Medical Center, Englewood, CO USA.

Oxidation-Reduction Potential: A New Predictor for Sperm Morphology in Infertile Men

Background and Study Question Oxidative stress has been associated with infertility in men, and specifically with morphology. sORP measures the balance between all oxidants and antioxidants providing a comprehensive status of the redox system. In previous studies, sORP was elevated in brain trauma, stroke, and multi-trauma patients, indicating a state of oxidative stress; thus sORP levels may be used to clarify the relationship between the redox system and semen parameters associated with male infertility. To measure the ORP in semen samples of infertile men and assess its predictive value for abnormal sperm morphology. Study Design and Methods Table 1 shows patient demographics. Data was collected in 3 sets (Group 1; n = 107; Group 2; n = 197; Group 3; n = 96). Exclusion criteria included azoospermia, presence of STD or chronic disease, use of prescription or OTC medications or antioxidants. Samples were collected and semen parameters assessed using the WHO guidelines (5th edition, 2010) (Table 2). sORP was measured (mV) using the MiOXSYS system and normalized to concentration (mV/106 sperm/mL).

Table 1. Patient Demographics N Mean StDev

Age 400 35.76 7.88 BMI 352 31.62 26.99 Abstinence (days) 400 4.35 2.21 Hormone Tests

Estradiol (E2) 154 182.58 76.09 Luteinizing hormone

(LH) 243 4.41 2.71

Follicle stimulating horm (FSH) 241 6.15 17.83

Protactin (PRL) 242 235.15 175.67 Testosterone 248 31.62 219.16

Table 2. WHO reference limits and N’s for those that met the reference (Normal) and those that failed (Abnormal)

Characteristic Lowest normal limit Normal (N) Abnormal

(N)

Volume (mL) > 1.5 mL 93 14 Total sperm # >39x106 74 33 Concentration (X 106/ mL) >15X106/mL 74 33

Total Motility >40% 60 47 Progressive Motility >32% 6 101

Morphology >4% 53 54

Table 3. sORP cut-off value to identify abnormal morphology (* p< 0.05) ORP Cut-Off = 3.29 mV

Data Set 1

Data Set 2

Data Set 3

Combined Data Sets

AUC 0.90 * 0.77 * 0.83 * 0.82 * Sensitivity 58.2 51.0 62.5 55.6 Specificity 96.2 87.0 85.0 89.1 PPV 94.1 82.1 83.0 85.7 NPV 68.5 60.2 65.9 63.1 Accuracy 77 % 68 % 72 % 71 %

Figure 1. Oxidative stress was reduced in those sample that were within the normal range for the WHO parameters. Smaller graphs show the results for each data set (1- blue; 2- red, 3- green) for morphology, motility, concentration, total sperm counts and volume. Combined data shown in the larger graph. In all cases, those that fell within the normal range had significantly lower sORP values. Volume was the only parameter that had no significant differences. (* p< 0.05).

Semen with abnormal parameters have higher levels of oxidative stress. By measuring sORP, a cut-off value of 3.29mV/sperm concentration was able to reliably predict abnormal morphology.

This study suggests that measuring sORP might provide users with an independent measure for confirming sperm morphology.

ESHRE 2016

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Oxidation-reduction Potential (ORP) of Spermatozoa Selectedfor Intracytoplasmic Sperm Injection (ICSI) After Exposure to

Polyvinylpyrrolidone (PVP) and Hyaluronic Acid (HA)Ashok Agarwala, Shubhadeep Roychoudhurya,b Sandro Estevesc,

Israel Maldonado Rosasd, Rakesh Sharmaa and Sajal Guptaa

aAmerican Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH 44195, USA, bDepartment of Life Science and Bioinformatics,

Assam University, Silchar 788011,India, cIngenes Institute of Fertility and Genetics, Carretera Mexico – Toluca 5420, 05340 Mexico City, Mexico,dANDROFERT, Center for Male Reproduction, Av. Dr. Heitor Penteado 1464, Campinas, SP 13075-460, Brazil

AbstractStudy question: Are there differences in the ORP of human spermatozoa exposed to PVP and HA prior to ICSI? Summary answer: After exposure for 20 minutes and 1 hour, the lowest levels of ORP were noted in PVP-treated spermatozoa compared with the controls and HA-treated group.What is known already: PVP is used in ICSI to facilitate sperm immobilization. It may, however, adversely impact the sperm function including DNA fragmentation, delays in calcium oscillations and loss of membrane integrity. Use of HA, a physiologic alternative to PVP, has been shown to help deselect sperm with DNA fragmentation, which may result in better embryo development after ICSI. ORP is a comprehensive measure of oxidative stress as it provides the overall balance between all available oxidants and antioxidants in a given sample. Static ORP (sORP) measures the current state of activity between oxidants and antioxidants.Study design, size, duration: Human semen samples from healthy consented donors (n = 11) with normal semen parameters according to the 2010 WHO guidelines were used for sperm preparation by density gradient centrifugation. Washed specimens were incubated with either PVP (Origio, Denmark) or HA (SpermCatch, Nidacon, Sweden). Controls were treated with sperm wash medium only (Quinn’s Advantage medium with HEPES; Origio, Denmark). sORP was measured after 20 minutes and 1 hour of exposure.Participants/materials, setting, methods: Sperm preparation adjusted to 5x106/mL was added to PVP, HA or control group in a 3:10 ratio (vol./vol.). ORP was measured using a novel galvanostat-based technology with a MiOXSYS Analyzer (Aytu Bioscience, Englewood, CO) in triplicate at room temperature. Data were normalized and represented as mV/106 sperm for sORP. Paired-t test was used to compare the data and differences were considered statistically significant at the level of P<0.05. Main results and the role of chance: Sperm sORP levels differed significantly (p<0.05) among the three groups after both 20 minutes and 1 hour of exposure. The lowest sORP levels were noted in PVP-treated group followed by the HA-treated group and control group after 20 minutes and 1 hour of exposure. Prior to any treatment with PVP or HA (Time 0), the sORP level in the control group (mean ± standard deviation) was 57.60 ± 0.63 mV/106 sperm. After 20 minutes, sORP levels were as follows: PVP-treated = 46.27 ± 0.37; HA-treated = 50.88 ± 0.15 and control = 57.49 ± 0.79, respectively. These values were not significantly different after 1 hour: PVP-treated = 47.04 ± 1.01; HA-treated = 50.80 ± 1.23 and control = 57.30 ± 2.66, respectively.Limitations, reasons for caution: Samples were obtained from normozoospermic men. Further experiments involving sperm from infertile men may be helpful in confirming our findings. Embryo development and implantation potential of resulting embryos from sperm populations with different ORP were not assessed.Wider implications of the findings: Levels of oxidative stress as represented by sORP levels were lowest in PVP-treated group, possibly because PVP contains a synthetic serum replacement--a chelating agent not present in HA. The findings indicate that PVP may have antioxidant properties and support continued use of PVP for sperm manipulation in ICSI processes.

ResultsThe ORP of human sperm selected for ICSI after exposure to PVP, HA or culture medium (control) are presented in Figure 2. The sORP levels differed significantly among the three groups both after 20 minutes and 1 hour of exposure. The lowest sORP levels were noted in the PVP-treated group followed by the HA-treated group and control group at both time points. Prior to any treatment with PVP or HA (Time 0), sORP level in the control group wase 57.60 ± 0.63 mV/106 sperm/mL. After 20 minutes incubation in PVP, HA and culture media (control), sORP levels were 46.27 ± 1.96 mV/106 sperm/mL, 50.88 ± 1.38 mV/106 sperm/mL and 57.49 ± 2.55 mV/106 sperm/mL, respectively. These values did not change significantly after 1 hour of exposure, and were 47.04 ± 1.01 mV/106 sperm/mL, 50.80 ± 1.23 mV/106 sperm/mL and 57.30 ± 2.66 mV/106 sperm/mL in PVP, HA and control groups, respectively. Statistical significance was set at a p value <0.05.

IntroductionDuring ICSI, a small amount of the medium containing PVP is usually unavoidably injected into the oocyte cytoplasm. Concerns about the safety of PVP and its possible adverse impact on embryo development have been raised. A few reports indicate that HA might represent a good physiologic alternative to PVP. In vitro, sperm bound to HA are believed to be those that have completed plasma membrane remodeling, cytoplasmic extrusion, and nuclear maturation. Oxidative stress has been recognized as an important contributory factor to male infertility and can lead to sperm dysfunction and has been associated with impaired IVF outcomes and other co-morbidities such as miscarriage and increased cancer risk. In ICSI, seminal plasma is removed during semen processing but toxic oxygen metabolites (generated by immature spermatozoa and leukocytes) might still attack the sperm devoid of protective seminal plasma antioxidants. Additionally, the detrimental effect of reactive oxygen species (ROS) on sperm functional competence can be exaggerated by the in vitro sperm processing techniques such as centrifugation and prolonged incubation that usually precede ICSI. Assessment of oxidation-reduction potential (ORP) presents a novel and comprehensive measure of seminal oxidative stress as it provides the overall balance between all available oxidants and antioxidants in a given sample. Static ORP (sORP) measures the current state of activity between oxidants and antioxidants; higher values indicate an imbalance in the activity of oxidants relative to antioxidants. To our knowledge, the ORP of sperm selected for ICSI after exposure to PVP and HA has not been measured yet.

Our objectives were therefore to assess the ORP of human sperm selected for ICSI after exposure to PVP and HA. The measurement of ORP in prepared sperm selected for ICSI following exposure to PVP or HA will further help identify the media with low oxidative stress.

Materials and MethodsFresh human semen samples (n=11) from normozoospermic men were used. Sperm were prepared by density gradient centrifugation and the washed sperm preparations were adjusted to 5x106 sperm/mL. A sperm aliquot was then loaded into a microdroplet of either PVP or HA or sperm wash medium in a 3:10 ratio (vol./vol.). The sORP was measured after 20 minutes and 1 hour of exposure using a galvanostat-based technology--the MiOXSYS System (Aytu Bioscience, Englewood, CO) in triplicate at room temperature (Figure 1). A (30µL) aliquot of the sperm sample in PVP or HA or control was applied to the sample application port on the inserted sensor. The sORP value was divided by sperm concentration to obtain normalized sORP, which was expressed as mV/106 sperm/mL and expressed as mean ± standard deviation (SD). Paired t-test was used to compare the groups and differences were considered statistically significant at a level

ConclusionsThe ORP is a comprehensive measure of oxidative stress as it provides the overall balance between all available oxidants and antioxidants in a given sample. Lower levels of sORP in PVP PVP-treated group than in the HA-treated group and the control group are novel and suggest that PVP might alleviate sperm oxidative stress as it contains a synthetic serum replacement that acts as a chelating agent. Our results support the continued clinical use of PVP for selection and manipulation in ICSI processes.

.

Figure 1(A-C): Sperm Count and ORP Measurement

Figure 2. Sperm motility

Oxidation reduction potential of human sperm selected for ICSI after exposure to of PVP, HA or culture medium (control) for 20 minutes and 1 hour. Differences in sORP were significantly different among the groups both at 20 minutes and 1 hour (p < 0.05).

Measurement of ORP. A: MiOXSYS Analyzer (placement of the sensor strip); B: Insertion of the sensor strip; C: Sensor in place.

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1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH2Department of Life Science and Bioinformatics, Assam University, Silchar 788011, India

Ashok Agarwal, PhD1, Shubhadeep Roychoudhury, PhD1,2, Rakesh Sharma, PhD1

OXIDATION REDUCTION POTENTIAL – A NOVEL TEST FOR EVALUATING MALE INFERTILITY

ABSTRACTObjective: Oxidative stress (OS) is associated with male infertility. The measurement of oxidation-reduction potential (ORP) represents the balance of all known and unknown contributors of OS. It is not limited to a specific constituent as in individual markers such as reactive oxygen species, total antioxidants, or lipid peroxidation. The MiOXSYS System is a novel tool for measuring ORP in the area of infertility. It provides an alternative for measuring seminal oxidative stress in male infertility patients by facilitating rapid measurement in small sample volumes in real time. The objective was to establish a reference value for ORP in semen to distinguish between normal semen and abnormal semen (at least one of the sperm parameters abnormal: volume, concentration, total count, motility, morphology) quality that may be used in the qualitative diagnostic testing of male infertility.

Design: Measurement of ORP in fertile and infertile men.

Materials and Methods: Semen samples were obtained from 51 normal healthy controls and 106 infertile men. Controls were recruited based on normal semen analysis according to 2010 WHO guidelines. ORP was measured using the MiOXSYS System. Receiver operating characteristic curve was plotted to predict a preferred cutoff value of ORP in semen in order to distinguish controls from infertile males.

Results: ORP was elevated in the semen of infertile patients (6.22±1.10 mV/106 sperm/mL) compared to controls (1.59±0.29 mV/106 sperm/mL; p<0.05). A ORP cutoff value of 1.36 mV/106 sperm/mL identified normal and abnormal semen quality with a sensitivity 69.6%, specificity 83.1%, positive predictive value 85.3% and negative predictive value 65.9%.

The accuracy of the test was 75.2% (AUC =0.770). In the control group the median sORP level was below the ORP cutoff of 1.36, whereas it was above this cutoff in the group of infertile patients.

Conclusions: We have standardized the ORP test in semen using the new MiOXSYS System as a novel method for oxidative stress testing. Results distinguish between healthy normal men and male factor infertility patients. These findings suggest that semen ORP level of 1.36 mV/106/mL can serve as a preferred cutoff value for distinguishing the prevalence of OS in male factor infertility.

Male infertility is a relatively common medical condition affecting up to 12% of men globally. Semen parameters are poor surrogate measure of a man’s ability to fertilize. Advanced tests of sperm function have been proposed as alternative methods that can enhance the diagnostic accuracy of male infertility particularly in cases of unexplained infertility, one or more abnormal semen parameters, recurrent pregnancy loss, or failure of intrauterine insemination. Oxidative stress (OS) resulting from a number of endogenous and exogenous stressors is believed to play a central role in the pathogenesis of male infertility. A delicate balance in the redox system is required for essential physiologic functions of the spermatozoa such as chromatin compaction in maturing spermatozoa, capacitation, hyperactivation, acrosome reaction and sperm-oocyte fusion. The World Health Organization (WHO) has also acknowledged OS as an important parameter that plays a significant role in male infertility, and thus its assessment and management are critical for patient care.

Currently available assays for OS measure only known or a discrete quantity of oxidants (ROS by chemiluminescence assay), antioxidants (TAC assay) or post-hoc damage (MDA assay). Such tests are also tedious, time consuming, and require special technical skills and large sample volumes. Since OS describes a state of the redox system in which the activity of the oxidants exceeds the capabilities of the antioxidants to quench them, a measure that comprises all known and unknown oxidants and all antioxidant activity in a semen sample would be the best indicator to describeits role in male infertility.

Oxidation-reduction potential (ORP) is a measure of the relationship between oxidants and antioxidants that provides a comprehensive measure of the redox system and thus of OS. A novel technology based on a galvanostatic measure of electrons—the MiOXSYS System has been developed that easilyand readily measures ORP in semen.

The objective of our study was to establish a reference value for ORP to distinguish between normal semen and abnormal semen (at least one of the sperm parameters abnormal: volume, concentration, total count, motility, morphology) quality that may be used in the qualitative diagnostic testingof male infertility.

INTRODUCTION

Following institution approval, semen samples were obtained from 51 healthy controls with proven and unproven fertility and 106 infertile patients between August 2015 and March 2016. The control group was composed of 51 healthy men based only on the normal semen parameters according to the WHO 5th edition guidelines (World Health Organization, 2010). Participants with proven (n=15) and unproven fertility (n = 36) were included in this group. The infertile group was composed of patients presenting to the male infertility unit complaining of primary or secondary infertility. The exclusion criteria for both patients and controls were: presence of azoospermia on semen analysis, evidence of obstructive pathology or ejaculatory dysfunction suggested by a low semen volume with/without an acidic pHand incomplete semen collection.

The study population was divided according to the results of the semen analysis into an abnormal semen group and a normal semen group. The abnormal semen group had a semen volume <1.5mL, and/or sperm concentration <15 x106 sperm/mL, and/or total sperm count <39 x106 sperm, and/or total motility <40%, and/or normal morphology <4%. In the normal semen group,these parameters fell within the 2010 WHO normal reference ranges.

Semen AnalysisSemen specimens were collected by masturbation after 48 to 72h of sexual abstinence, and the sperm parameters were analyzed after complete liquefaction at 37°C for 20 minutes. Each sample was evaluated for both macroscopic parameters such as color, pH, ejaculate volume, age of the sample and viscosity. An aliquot of the sample (5µL) was examined for sperm concentration, total sperm count, sperm motility and round cell concentration. If the round cell concentration was >1X106 /mL, the aliquot was examined for the presence of white blood cells using the Endtz or the peroxidase test (World Health Organization, 2010). Air dried smears were preparedfor morphological evaluation; 4% normal morphology was used as a cut-off.

Measurement of Oxidation-reduction Potential Oxidation reduction was measured using a novel galvanostat-based technology (MiOXSYS System;Aytu Bioscience, Englewood, CO, USA). Briefly, a 30µL sample suspension was loaded on the sample port of the disposable MiOXSYS sensor (Figure 1). The sensor was inserted in the MiOXSYS analyzer. The test started when the sample filled the reference electrode, thereby completing the electrochemical circuit. After a short period, the ORP values, in millivolts (mV), are displayed on the screen. MiOXSYS System measures ORP, which is a “snapshot” of the current balance of the redox system. A higher ORP level indicates an imbalance in the activity of all available oxidants relative to all available antioxidants in the seminal ejaculate - a state of OS. Both absolute (millivolts) and normalized ORP values (millivolts /106 sperm/mL) were calculated.


Background information on the study population is presented in Table 1 along with a comparisonof semen parameters between the control and infertile groups.

1. The mean ORP (mV/106 sperm/mL) in the semen of the infertile patients was 6.22±1.10 mV/106 sperm/mL whereas that of the control group was 1.59±0.29 mV/106 sperm/mL (p = 0.004). Out of the 157 samples, 65 were found to have normal semen parameters and 92 were found to have abnormal semen parameters. ROC curve analysis of the ORP (mV/106 sperm/mL) test results predicting normal versus abnormal semen quality values was performed to calculate test sensitivity, specificity, and positive and negative predictive values (Figure 2).

2. The distribution of subjects in the control and infertile groups above or below the established cutoff value of 1.36 mV/106 sperm/mL is depicted in Figure 3.

3. The median ORP (mV/106 sperm/mL) level was below the established cutoff value of 1.36 mV/106 sperm/mL in the control group, whereas it was above this cutoff in the group of infertile patients (p = 0.004). Only 1 of the 51 (less than 2%) participants in the control group fell below this cutoff. In men undergoing evaluation for male-factor infertility (n = 106), 44.3% (47/106) presented with sperm concentration <15 x 106/ mL in the seminal ejaculates. The distribution of ORP (mV/106 sperm/mL) within different sub-groups of controls and infertile patients was also assessed and is presented in Figure 4.

4. Compared to the control group, the ORP (mV/106 sperm/mL) levels were elevated in the infertile patients presenting with a clinical varicocele (p = 0.0003) and those with idiopathic infertility (p = 0.007).

5. The association of ORP (mV/106 sperm/mL) with semen variables was examined in order to establish the ability of the ORP test to predict the diagnosis of OS (Figures 5-7).

RESULTS

Amer

ican



1993

Figure 1

Measurement of oxidation reduction potential by the MiOXSYS System.

A: MiOXSYS Analyzer showing the sensor socket and the sensor module. B: Sensor showing the reference cell and the sample port where the sample is loaded.

Figure 2A receiver operating characteristic (ROC) curve was used to identify the ORP (mV/106 sperm/mL) criterion i.e. cutoff, sensitivity, and specificity, positive predictive value, negative predictive value, accuracy and area under curve (AUC) that best predicted the normal and abnormal semen parameters as well as differentiated normal healthy controls from male factor infertility patients.

Figure 3

Distribution of ORP in controls and patients, showing the established cutoff value.

Figure 4

Box Plot distribution of ORP in:

A. Normal healthy controlsB. Controls with proven fertility C. Controls with unproven fertilityD. Infertile patientsE. Infertile patients presenting with a clinical varicoceleF. Infertile patients with idiopathic infertility.

Figure 5 Figure 6

Figure 7

Correlation of ORP with sperm parameters in all subjects.

A. Concentration B. Total sperm count C. Motility D. Morphology

Correlation of ORP with sperm parameters in normal healthy controls.


Correlation of ORP with sperm parameters in infertile patients


1. ORP test in semen using the MiOXSYS System is an alternative method for measuring OS and distinguishing normal men from male factor infertility patients.

2. Sperm parameters related to male infertility are under a state of oxidative stress.

3. An ORP cutoff value of 1.36 mV/106 sperm/mL in semen can distinguish between normal men and patients with male factor infertility.

CONCLUSIONS

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ASRM 2016

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American Center for Reproductive Medicine, Department of Urology, Cleveland Clinic, Cleveland, OH

Ashok Agarwal, PhD., Ahmad Mazjoub, MD., Shubhadeep Roychoudhury, PhD., and Rakesh Sharma, PhD

OXIDATION REDUCTION POTENTIAL – A NOVEL MARKER OF VARICOCELE PATHOPHYSIOLOGY

ABSTRACTObjectives: Oxidative stress (OS) provides a comprehensive, measure of OS. Oxidation reduction potential (ORP) has recently been proposed as a rapid and accurate alternative to current laboratory assays used to measure OS in the evaluation of infertile men. We investigated the application of this system in a group of infertile men with clinical varicocele.

Design: Prospective case-control pilot study.

Materials and Methods: Thirty-eight patients with clinical varicocele presenting with primary infertility were subdivided into 3 groups according to clinical grade. Semen analysis was based on current WHO 5th edition (2010) guidelines. ORP measurements were performed using the MiOXSYS System. Fifteen patients with idiopathic infertility and 13 fertile controls were included. Wilxcon’s rank sum test was used to compare the groups with respect to quantitative variables. A value p<0.056 was considered significant.

Results: The patients mean age ± SEM was 33 ±1.2 years. Compared with controls, grade 3 varicocele patients and men with idiopathic infertility had a lower sperm concentration, % total motility and % normal morphology (Table 1). Grade 2 varicocele patients had a significantly lower sperm concentration and % normal morphology while grade 1 varicocele patients had a significantly lower % normal morphology than the control group. ORP levels were higher in all the infertile groups than in the controls. However, these results were statistically significant only among the patients with grade 3 varicocele. A direct relationship was seen between the ORP level and varicocele grade (p<0.05).

Conclusions: Patients with clinical varicocele have elevated ORP values that are highest in men with Grade 3 varicocele. The ORP levels could serve as an indicator of sperm dysfunction in men with varicocele. We speculate that ORP measurement may be utilized before and after varicocele surgery to assess surgical success.

Varicocele (n = 38) Idiopathic Control Grade 1 Grade 2 Grade 3 Infertility (n=15) (n =13) (n =10) (n =15) (n=13)

Volume (mL) 2.82 ± 0.4 3.25 ± 0.41 3.15 ± 0.44 3.22 ± 0.33 4.01 ± 0.88

Concentration (106/mL) 55 ± 9.9 32.06 ± 8.1 14.37 ± 5.12* 19.76 ± 8.45* 29.58 ± 11.22*

Total motility (%) 55.8 ± 2.02 49.53 ± 6.19 45.54 ± 5.22 40.93 ± 3.02* 32 ± 4.079*

Morphology (%) 7.8 ± 0.71 3.15 ± 0.64* 2.5 ± 0.56* 3.73 ± 0.77* 3.69 ± 0.84*

ORP (mV/106sperm) 2.52 ± 0.47 3.16 ± 1.3† 5.59 ± 2.3† 11.25 ± 3.9† 7.53 ± 2.7

Values are mean ± SD; *Significantly different compared to control group; †Significantly different compared to grade 1 varicocele.

Table 1: Comparison of semen analysis and sORP levels between study groups.

Male infertility is a relatively common medical condition affecting up to 12% of men globally. Varicocele is the most common correctable cause of male infertility. Oxidative stress is a contributor of varicocele. The World Health Organization (WHO) has acknowledged OS as an important parameter that plays a significant role in male infertility.

Individual markers of oxidative stress such as ROS measured by chemiluminescence assay, antioxidants (TAC assay) or post-hoc damage (MDA assay) are common. However such tests are also tedious, time consuming, and require special technical skills and large sample volumes. A measure that comprises all known and unknown oxidants and all antioxidant activity in a semen sample would be an ideal marker of male infertility.

Oxidation-reduction potential (ORP) provides a comprehensive measure of the redox system and of OS. We have established the ORP levels of 1.36 mVolts/106 sperm/mL that differentiates infertile men from healthy men. The objective of our study was to establish the ORP levels in infertile men with clinical varicocele and compare then with infertile men with idiopathic infertility.

INTRODUCTION

Following institution approval, semen samples were obtained from 15 healthy men according to the WHO 5th edition guidelines (World Health Organization, 2010). The infertile group was composed of 51 patients presenting to the male infertility with varicocele (n=38) and 13 subjects with idiopathic infertility. The exclusion criteria for both patients and controls were: presence of azoospermia on semen analysis, evidence of obstructive pathology or ejaculatory dysfunction suggested by a low semen volume with/without an acidic pH and incomplete semen collection.

Semen analysis Semen specimens were collected by masturbation after 48 to 72h of sexual abstinence, and the sperm parameters were analyzed after complete liquefaction at 37°C for 20 minutes. Each sample was evaluated for both macroscopic parameters such as color, pH, ejaculate volume, age of the sample and viscosity. An aliquot of the sample (5µL) was examined for sperm concentration, total sperm count, sperm motility and round cell concentration. If the round cell concentration was >1X106 /mL, the aliquot was examined for the presence of white blood cells using the Endtz or the peroxidase test (World Health Organization, 2010). Air dried smears were prepared for morphological evaluation; 4% normal morphology was used as a cut-off.

Measurement of oxidation-reduction potential Oxidation reduction was measured using a novel galvanostat-based technology (MiOXSYS System; Aytu Bioscience, Englewood, CO, USA). Briefly, a 30µL sample suspension was loaded on the sample port of the disposable MiOXSYS sensor (Figure 1). The sensor was inserted in the MiOXSYS analyzer. ORP values, in millivolts (mV), are displayed on the screen. MiOXSYS System measures ORP, which is a “snapshot” of the current balance of the redox system. A higher ORP level indicates an imbalance in the activity of all available oxidants relative to all available antioxidants in the seminal ejaculate - a state of OS. Absolute (millivolts) ORP was normalized for sperm concentration and ORP values (millivolts /106 sperm/mL) were calculated.


A comparative analysis of semen parameters and ORP is shown in Table 1. 1. Men with varicocele have significantly poor semen parameters. 2. Infertile men with grade 3 varicocele had the highest levels of ORP compared to those with grade 1 and 2 as well as with idiopathic infertility. 3. Men with idiopathic infertility have elevated ORP levels compared to the control group, 4. The distribution of subjects in the control and infertile groups above or below the established cutoff value of 1.36 mV/106 sperm/mL is depicted in Figure 2.

Figure 1. Measurement of oxidation reduction potential by the MiOXSYS System. A: MiOXSYS Analyzer showing the sensor socket and the sensor module and B: Sensor showing the reference cell and the sample port where the sample is loaded.

Figure 2. Distribution of ORP in: A: controls with proven fertility, B: Infertile patients presenting with a clinical varicocele, and C: Idiopathic infertility.

ORP values are presented as median and 25th, 75th percentile.

RESULTS

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1. Men with clinical varicocele have semen parameters that are under a state of oxidative stress and have elevated levels of ORP.

2. ORP is a single measure of oxidative stress in semen specimens that can be easily measured in both men with clinical varicocele and idiopathic infertility.

CONCLUSIONS

Table 1: Comparison of semen analysis and ORP levels between study groups. Varicocele (n = 38) Idiopathic Control Grade 1 Grade 2 Grade 3 Infertility (n=15) (n =13) (n =10) (n =13) (n=13)

Volume (mL) 2.82 ± 0.4 3.25 ± 0.41 3.15 ± 0.44 3.22 ± 0.33 4.01 ± 0.88

Concentration (106/mL) 55 ± 9.9 32.06 ± 8.1 14.37 ± 5.12* 19.76 ± 8.45* 29.58 ± 11.22*

Total motility (%) 55.8 ± 2.02 49.53 ± 6.19 45.54 ± 5.22 40.93 ± 3.02* 32 ± 4.079*

Morphology (%) 7.8 ± 0.71 3.15 ± 0.64* 2.5 ± 0.56* 3.73 ± 0.77* 3.69 ± 0.84*

ORP (mV/106 sperm/mL) 2.52 ± 0.47 3.16 ± 1.3† 5.59 ± 2.3† 11.25 ± 3.9† 7.53 ± 2.7Values are mean ± SD; *Significantly different compared to control group; †Significantly different compared to grade 1 varicocele.

Controls withproven fertility

(n=15)

0.02

0.1

0.5

2

10

50

200

1.36

OR

P (

mV/

10

6 s

perm

/mL)

Varicocele(n=38)

Idiopathic(n=13)

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MiOXSYS SystemThe MiOXSYS System (Aytu Bioscience, Inc.) measures theredox balance in semen or seminal plasma samples bymeasuring ORP. The MiOXSYS provides a snapshot of currentredox balance, referred to as the static ORP (sORP). A highersORP reading is indicative of an imbalance between oxidantand reductant activity which refers oxidative stress.

Figure 1: (a) Setup of the ORP instrument. (b) View of thesensor showing the application port for loading of the sample.(c) View of the sensor.

RESULTSInfertile donors had higher sORP values then fertile. The oddsof being infertile with a sORP value greater than 1.38mV/sperm concentration was 9.76 times higher than if thesORP was lower. 63% of donors had abnormal morphology,including 38% of fertile and 97% of infertile.

(a) (b)

Figure2: (a) Fertile donors have significantly lower sORP valuesthan infertile (t(79)=4.26, p<0.001). (b) Comparing the sORPvalues based on the number of abnormal parameters, it wasfound that when there are 2 abnormal parameters there is asignificant jump in sORP values (F(2, 77)= 6.76, p<0.05). Only 1person was found to have 3 abnormal parameters and no onewas abnormal in all 4 parameters.

RESULTS

Figure 4: The fertile donors have significantly fewer head(t(80)= 11.27, p< 0.001) (A), neck (t(80)= 8.17, p< 0.001) (B),and tail (t(80)= 7.50, p< 0.001)(C) abnormalities than infertiledonors. sORP increased 0.12 mV/106/mL for each 1% increasein abnormal neck/midpiece and decreased by 0.008mV/106/mL per million sperm in the semen sample.

RESULTS

Table 2: Abnormal morphology was found in nearly all infertilesamples. As a single abnormal parameter, it was found in nearlyas many fertile and infertile samples (94.4% vs 100%). When itwas paired with any other abnormal parameters, it was alwaysfound in infertile samples. Fertile samples were found either thisabnormal morphology alone OR with abnormal motilitymeasures.MR indicated that sperm neck abnormalities specificallycontributed to sORP, when head and tail morphology, andmotility were held constant.

Figure 3: when the total sperm # increases the sORP valuesdecrease as indicated by the transition to green; as % of neckabnormalities increase the sORP also increases as indicated bythe transition to red. If you have higher neck abnormalities andlow sperm number you have the highest sORP values indicatedby the deep red corner, if you have low neck and high numbers,you have the lowest sORP indicated by the deep green corner.

MATERIAL AND METHODSParticipants were recruited from an international fertilityclinic; 34 men with diagnosed primary infertility and 47 whohad fathered a child in the last 24 months. Semen sampleswere analyzed respecting WHO Manual 5th Edition criteria.

Table 1: WHO semen parameters cut-off values

Data were analyzed using t-tests, receiever operatingcharacteristics (ROC), ANOVAs, and Multiple Regression (MR)analysis. Power analysis, set at α=0.05, 1-β=0.90, revealedsatisfactory samples sizes for the study. Significance wasaccepted at p<0.05. Redox balance were determined bymeasuring static oxidation reduction potential (sORP, MiOXSYSSystem).

Infertile Men Have A Redox Imbalance That Distinguishes Them From Fertile MenA. Ayaz1, K.B. Bjugstad2, D. Bar-Or3, A.Armagan4

1 Yildiz Technical University, Istanbul, Turkey 2 Aytu Bioscience, Englewood, CO, 3Trauma Research, Swedish Medical Center, Englewood, CO, 4 Urology, Bezmi Alem Vakif University, Istanbul, Turkey

OBJECTIVEThe Redox system is carefully balanced between the activitiesoxidant to reductants based on biological need. In fertility, alimited amount of oxidant activity is required to support spermcapacitation and embryonic stem cell differentiation. However,excessive amount of this activity causes oxidative stress whichdamages lipids, proteins and result in DNA fragmentation. Thegoal of the study was to determine if the imbalance in theredox system in infertile men was statistically significant todistinguish them from fertile men, and to identify anycorresponding semen parameters that may account for thisdifference.

# of Failed Parameters

Fertile (n=47) Infertile (n=34)

N (%)

Most common

failed parameter

N (%) Most common failed parameter

0 19 (40.4%) 1 (2.9%)

1 18 (38.3%)

94.4% morphology

23 (67.6%) 100% morphology

2 10 (21.3%)

90.0% total and

progressive motility

9 (26.5%)

88.9% morphology and total numbers

3 0 1 (2.9%)morphology, total and progressive

motility4 0 0

CONCLUSION

• Infertile patients have higher sORP value when compared tofertile donors.

• Abnormal sperm parameters, especially two parameters,were found to be correlated with increased sORP values.One patient has 3 abnormalities and no patient had fourabnormalities.

• Morphological abnormality was found almost all infertilepatients. When combined with other abnormal parameters,only infertile group has both abnormalities. Fertile grouphas either morphology or other abnormality.

• Independent of other variables, as the % of neckabnormalities increased, sORP/Concentration was alsoincreased. As the total number of sperm in a semen sampleincreased, sORP /Concentration tended to decrease but thisdecrease was very small.

Infertile men have a significant imbalance in the semen redoxsystem , such that by measuring sORP, infertile patients andfertile donors can be distinguished by their sORP values.Measurement of sORP can be used to diagnose male infertilitypatients with abnormal semen parameters and it may help tooptimize antioxidant treatment as well as evaluation ofassisted reproduction.

Semen parameters 1980 1987 1992 1999 2010

Volume (mL) Not defined ≥ 2 ≥ 2 ≥ 2 ≥ 1.5

Concentration (106 sperm) 20-200 ≥ 20 ≥ 20 ≥ 20 ≥ 15

Total sperm count (106

sperm)Not

defined ≥ 40 ≥ 40 ≥ 40 ≥ 39

Motility (%) ≥ 60 ≥ 50 ≥ 50 ≥ 50 ≥ 40Progressive motility (%) ≥ 2 ≥ 25 ≥ 25 ≥ 25 ≥ 32

Morphology (%) 80.5 ≥ 50 ≥ 30 ≥ 14 ≥ 4

Leukocytes (106/mL) < 4.7 < 1.0 < 1.0 < 1.0 < 1.0

Vitality (%) Not defined ≥ 50 ≥ 75 ≥ 75 ≥ 58

(a)

(b)

(c)(a)

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Introduction

The study included 365 males from infertile couples and 50 fertile controls which had achieved pregnancy in the last 24 months. Exclusion Criteria for All Participants- included azoospermia, the use of antioxidant therapy, current or past pyospermia, clinically palpable varicocele, history of occupational chemical or radiation exposure, the presence of female factor infertility, abstinence less than 2 days, and/or a sperm concentration less than 1 million sperm per milliliter (<106/mL) Semen analysis was done according to the 5th Edition WHO manual (2010) after at least 2 days of abstinence. Static ORP (sORP) was measured using the MIOXSYS System (Figure 1) Wilcoxon’s rank sum test was used for group comparisons with respect to quantitative variables. A receiver operating characteristic (ROC) analysis was used to identify the sORP criterion that best differentiated fertile from infertile men.

Results Results Male factor can be the sole or contributing factor in roughly half of infertility cases. No identifiable cause can be found in over 25% of infertile males. Semen analysis is routinely used to evaluate the male partner in infertile couples and to assess the reproductive toxicity of environmental or therapeutic agents. Despite it being a routine analysis, it is a subjective interpretation of semen quality and is a poor predictor of fertility evidenced by the presence of unexplained infertility in men with normal semen analysis. These limitations call for an unbiased, quantitative measure to accurately differentiate fertile from infertile men and validate the subjective values of semen quality. Oxidation-reduction potential (ORP) is a simple objective measure, newly applied to assess the status of oxidative stress in semen and has been evaluated in comparison with the WHO parameters (5th Edition, 2010). ORP may hold potential value in fertility assessment, as oxidative stress has been linked with infertility. The goals of this study were to determine if ORP relates to measures of semen quality identified by the WHO manual (5th Edition, 2010) and to determine if ORP can distinguish and predict semen form infertile men.

ARAFA, M1,2, ELBARDISI, H1,, AGARWAL, A3, ALSAID, S1, MAJZOUB, A1,3,4, , BJUGSTAD, K.B5, ALRUMAIHI, K1 1. UROLOGY DEPARTMENT - HMC, DOHA, QATAR, 2. ANDROLOGY DEPARTMENT, CAIRO UNIVERSITY, EGYPT, 3. CENTER FOR REPRODUCTIVE MEDICINE, GLICKMAN UROLOGICAL AND KIDNEY INSTITUTE, CLEVELAND CLINIC, CLEVELAND, OH, USA, 4. DEPARTMENT OF UROLOGY, GLICKMAN UROLOGICAL AND KIDNEY INSTITUTE, 5. AYTU BIOSCIENCE, ENGLEWOOD CO USA

Materials and Methods

Conclusions

Measuring sORP provides an objective measure that relates to semen quality.

sORP, at a cutoff value of 1.42 mv/106 sperm/mL, provides an objective and repeatable measure for identification of patients more likely to be infertile.

This new test can rapidly help clinicians identify infertile patients with confidence, so that treatment strategies can be initiated immediately. This in turn helps the clinic improve the chances of pregnancy.

Infertile Patients Fertile Controls p values

N 365 50

Age 35.8 +/- 0.3 32.3 +/- 1.0 p< 0.002*

Abstinence 4.4 +/- 0.1 3.7 +/- 0.2 p< 0.02*

BMI 31.5 +/- 1.6 25.5 +/- 0.5 p= 0.13ns

Table 2. Basic demographic data for infertile patients and fertile controls. Data are presented as the mean +/- SEM. Wilcoxon rank sum test was used to compare groups; p values are presented. *significant difference, ns- no significant difference; Fertile controls were significantly younger and had a shorter period of abstinence, but overall health (body-mass index; BMI) was similar between the two groups.

WHO Cut-off For Good Quality

Infertile (failed N)

Fertile (failed N)

Total Sperm Number > 39x106 103 3

Total Motility > 40% 149 3

Progressive Motility > 32% 321 36 Normal Sperm Morphology > 4% 184 8

Overall Quality 70 0 Table 1. Cutoff values for each parameter defined by the WHO manual for semen quality and respective N’s for each group that failed to meet a criteria. N’s represent the number of samples that failed criteria for that individual parameter Overall quality represents the number of semen samples that failed all four parameters, suggesting a poor quality of sperm.

Figure 2. sORP can reliable identify semen form fertile controls and predict which semen were from infertile patients. A cutoff value of >1.42 mv/106 sperm/mL has a specificity of 78% (identifying the fertile controls) and Positive predictive value of 95.7%, predicting which semen were from infertile patients. The accuracy was accuracy of 62.7% with an an area under curve (AUC) of 0.71 (p< 0.05).

Infertile Patients Fertile Controls p values

sORP (mV/106 sperm/mL) 24.1 +/- 4.1 1.3+/- 0.1 p< 0.001

Volume 3.1 +/- 0.0 2.9 +/- 0.2 p= 0.44

Total Sperm Number 101.8 +/- 5.2 172.1 +/- 17.9 p< 0.001

Total Motility 42.9 +/- 1.2 59.9 +/- 1.8 p< 0.001

Progressive Motility 14.1 +/- .7 27.0 +/- 1.4 p< 0.001

Morphology 7.0 +/- 0.4 10.0 +/- 0.7 p< 0.001

Table 3. Infertile Patients have higher sORP measures than Fertile Controls. Infertile patients were also significantly different in the total number of sperm, the percent of motile sperm, and in sperm morphology. These measures of semen quality combined with higher sORP values would suggest that oxidative stress may play a role in semen quality.

Figure 1. The MiOXSYS System. This system measures ORP in semen using disposal electrode sensors and an analyzer. The system uses only thirty (30) microliters of semen and provides an ORP value which is then normed to semen concentration. ORP values are presented as mv/106 sperm/mL

Infertile patients had:

-Higher sORP values -Fewer total sperm -Lower motility -Lower normal sperm morphology

sORP can distinguish between infertile patients and fertile controls sORP cutoff of greater than 1.42mv/106/mL can predict if a semen sample came from infertile patients with a predictive value of 95.7%

Discussion

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EVALUATION OF INTRA- AND INTER-OBSERVER RELIABILITY OF THE OXIDATION-REDUCTIONPOTENTIAL TEST FOR OXIDATIVE STRESS IN MALE FACTOR INFERTILITY

Ashok Agarwal, PhD.,1 Shubhadeep Roychoudhury, PhD.,1 Rakesh Sharma, PhD.,1 Sajal Gupta, MD.,1

Ahmad Majzoub, MD.,2 Kim Bjugstad, PhD.,3 and Edmund S Sabanegh., MD.2 1American Center for Reproductive Medicine, Department of Urology, Cleveland Clinic, Cleveland, OH, 2Cleveland Clinic, Cleveland, OH, 3Aytu Bioscience, Englewood, CO

Table 1: The intra- and inter-observer reliability of the ORP test

Observer 1(O1)

Observer 2 (O2)

Observer 3(O3)

Average

7.98% 5.72% 11.96% 8.39%

0.018(0.013)

0.014(0.012)

0.034(0.023)

0.022(0.018)

1.23 (1.49) 1.22 (1.57) 1.17 (1.48) 1.21 (1.51)

O1 vs O2 O1 vs O3 O2 vs O3 Average

0.986 0.993 0.993 0.991

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ABSTRACTObjective: Oxidative stress (OS) plays a role in male infertility. Because OS is a state in which oxidant activity exceeds antioxidants protection, a measure of both would be the best indicator of OS in male infertility. ORP is a measure of the relationship between oxidants and antioxidants. Measuring ORP is a novel approach to testing infertility as such the repeatability of an ORP test is crucial for its clinical application. The objective of this study was to evaluate the intra-observer and inter-observer reliability of the ORP test.Design: Test-retested design measuring ORP in normozoospermic men. Materials and Methods: Semen ORP was measured using the MiOXSYS System. For intra-observer reliability, 3 samples were measured 3 times by 3 observers using the same analyzer. The reliability was determined using the average coefficient of variation (%CV) across samples within an individual observer. For inter-observer reliability, 10 samples were measured 4 times by 3 observers. The extent to which there was agreement between observers was determined by the difference between observer means, correlation, and %CV. Results: Results of the intra- and inter-observer reliability experiments are summarizedin Table 1. The overall %CV was 8.39% suggesting a strong level of intra-observer reliability. The %CV across observers was 3.61%. Similar ORP values across observers, high correlations between them, and a low %CV supports a strong level of inter-observer reliability.Conclusions: Our results of the intra- and inter-observer reliability experiments confirm the reproducibility of the ORP test which is important for its application in clinical setting.

INTRODUCTIONMale infertility is a relatively common medical condition affecting up to 12% of men globally. Oxidative stress (OS) resulting from a number of endogenous and exogenous stressors is believed to play a central role in the pathogenesis of male infertility. A delicate balance in the redox system is required for essential physiologic functions of the spermatozoa such as chromatin compaction in maturing spermatozoa, capacitation, hyperactivation, acrosome reaction and sperm-oocyte fusion. Currently available assays for OS measure only known or a discrete quantity of oxidants (ROS by chemiluminescence assay), antioxidants (TAC assay) or post-hoc damage (MDA assay). OS describes a state of the redox system in which the activity of the oxidants exceeds the capabilities of the antioxidants to quench them.

We have recently introduced the oxidation-reduction potential (ORP), a measure of the relationship between oxidants and antioxidants that provides a comprehensive measure of the redox system and thus of OS. The ORP test is novel in the area of infertility. It is a novel technology based on a galvanostatic measure of electrons — the MiOXSYS System – that easily and readily measures ORP in semen.

The goal of this study was to evaluate the intra-observer and inter-observer reliability of the test in an effort to standardize this this test.

RESULTSThe reliability of the ORP test was determined by assessing the differences between ORP (mV/106sperm/mL) measurements using intra- and inter-observer settings (Table 1).

Intra-observer reliability1. The intra-observer reliability was based on 27 ORP measurements made by three experienced observers. The %CV for the three observers tested were: Observer 1 = 7.98%; Observer 2 = 5.72%, and Observer 3 = 11.96%

2. The average %CV was 8.39%, suggesting a strong level of intra-observer reliability. On average, the difference between any two replicates for any one observer was less than 0.1 mV/106 sperm/mL (Table 1, Figure 4A).

Inter-observer reliability1. A total of 120 measurements of ORP (mV/106sperm/mL) were taken by three experienced observers from ten semen samples measured in four replicates.

2. Overall, there was no significant difference between the ORP (mV/106sperm/mL) values obtained across observers [F(2.62)= 1.22, p = 0.30; (Table 1, Figure 4B).

3. Correlations between observers exceeded 0.97 in all cases.

4. The %CV across observers was 3.61%.

5. There was high agreement in ORP (mV/106sperm/mL) values obtained between observers for the same samples, indicating a high inter-observer reliability.

Figure 3. Schematics for assessing A: intra-observer and B: inter-observer reliability the reliability of the MiOXSYS System.

Figure 4. Differences in ORP between A: Replicates and B: observers.

Figure 4. Di�erences in ORP between A: Replicates and B: observers.

Figure 2. Placing the sensor A: on the MiOXSYS System B: gently inserting the sensor and C: view when the sensor is in place.

Figure 1. View of the sensor showing the sample port for loading the sample.

CONCLUSIONS1. The results of the intra- and inter-observer reliability experiments confirm the reproducibility of the MiOXSYS System for use in a clinical setting.

2. It is important to validate the new test/ instrument irrespective of its ease of use and simplicity. This is especially important when a system is used in a clinical setting to report patient results.

Table 1: The intra- and inter-observer reliability of the ORP test

MATERIALS AND METHODSAfter complete liquefaction, initial semen analysis was done to measure sperm concentration which is necessary in the calculation of oxidation reduction potential.

Measurement of oxidation-reduction potential The MiOXSYS System, is composed of a galvanostat-based analyzer and disposable sensors, measures the amount of oxidative or reductive stress (redox balance) in semen samples by measuring the ORP.

After semen analysis and within one hour of collection, a 30-µL aliquot of liquefied semen was subjected to the ORP test using a novel galvanostat-based technology--the MiOXSYS System (Aytu Bioscience, Englewood, CO) (Figure 1 and 2A-C). Testing began automatically when the sample was detected by the sensor’s reference electrodes. The sample analysis was completed in less than three minutes. ORP is a “snapshot” of the current balance of the redox system. ORP data is normalized to sperm concentration to control for the sperm number in a semen sample and represented as mV/106 sperm/mL. Each sample was run in duplicate.

Intra-observer reliabilityThree semen samples were measured three times by three experienced observers using the same analyzer for all measures (Figure 3A). The reliability of an individual observer was assessed by calculating the coefficient of variation (%CV) for each sample, where the mean ORP and standard deviation (SD) for each of the three samples were calculated from the three replicates. The %CV for each sample was then generated. The average %CV across all three samples within an individual observer was calculated as an indicator of individual intra-observer reliability. Overall intra-observer reliability was determined by the average %CV of all three observers.

Inter-observer reliabilityTen semen samples were measured four times (replicates A – D) by three experienced observers (Observers 1 – 3) for each sample in order to calculate the extent to which there was agreement between observers (Figure 3B). This was determined by the difference between observer means, and correlations among observers. ANOVA was used to verify whether the average ORP (mV/106sperm/mL) for each observer was significantly different from any other observer (p < 0.05). Pearson’s r correlations between observers were used to verify whether the ORP (mV/106sperm/mL) values for each sample were consistent across observers. The %CV was calculated from the mean ORP (mV/106sperm/mL) and SD for each observer.

%CV

Average (SD) differencesbetween replications

(mV/106/mL)

Average (SD) (mV/106/mL)

Correlations between observers

Inter-Observer Reliability

Intra-Observer Reliability

Observer 1(O1)

Observer 2 (O2)

Observer 3(O3)

Average

7.98% 5.72% 11.96% 8.39%

0.018(0.013)

0.014(0.012)

0.034(0.023)

0.022(0.018)

1.23 (1.49) 1.22 (1.57) 1.17 (1.48) 1.21 (1.51)

O1 vs O2 O1 vs O3 O2 vs O3 Average

0.986 0.993 0.993 0.991

%CV

Average (SD) differencesbetween replications

(mV/106/mL)

Average (SD) (mV/106/mL)

Correlations between observers

Inter-Observer Reliability

Intra-Observer Reliability

A A

B

B

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