MiOXSYS® and OxiSperm® II assays appear to provide no clinical utility for determining oxidative stress in human sperm—results from repeated semen collections

Oxidative stress in semen contributes up to 80% of all infertility diagnosis. Diagnostics to measure oxidative stress in semen was recently added to the 6th edition WHO methods manual, although diagnostic predictive values need to be interpreted with caution as there are still several research questions yet to be answered.


INTRODUCTION
Sperm counts are declining globally, with infertility affecting one in 20 men, and a male factor contributing to 30%-50% of all couples seeking assisted reproductive technology (ART) treatment. 1Currently, the most widely used tool for the diagnosis of male infertility is the semen analysis. 39However, while sperm count, motility, and morphology reveal useful information for providing a starting point for the evaluation of male infertility, none of these parameters is themselves a direct measure of fertility potential. 25creased semen and sperm reactive oxygen species (ROS) and associated oxidative stress are shown to contribute to up to 80% of all male infertility diagnoses. 37More recently, diagnostics to measure semen oxidative stress have been added to the recently released 6th edition of the WHO laboratory manual for the examination and processing of human semen, under advanced examinations. 39Importantly, the 6th edition highlights that their diagnostic predicative values need to be interpreted with caution, 39 as there is still a number of research questions left to be answered before their potential clinical utility may be realised.These include: the fundamental knowledge of their natural fluctuations in men and their ability to correctly determine sperm oxidative stress. 16e MiOXSYS ® system is a point-of-care device requiring only 30 μl of neat semen in order to assess oxidative stress based on galvanostatic measure of electron availability in semen. 6It is relatively cheap and takes between 2 and 5 min to run. 16To date, MiOXSYS ® remains the most extensively studied system, reported as being highly sensitive and predictive of male infertility when assessing abnormal semen parameters 2 and sperm DNA fragmentation. 19However, most of the published literature supporting its clinical effectiveness has been generated by a single research group. 16To date, only one published study has evaluated whether semen redox-potential as measured by MiOXSYS ® fluctuates within individuals over time, reporting in two repeat semen samples that levels varied based on baseline semen analysis results; concentration and motility. 7Additionally, no studies have assessed the system against measurements of sperm oxidative stress such as sperm lipid peroxidation and/or oxidised DNA damage.
OxiSperm ® II (manufactured by Halotech ® ) measures redox activity through the nitro blue tetrazolium (NBT) assay via a membrane-based gel.In this assay, the yellow NBT molecules are reduced to the insoluble blue crystal (formazan), in the presence of oxidoreductases [i.e., superoxide anion, P450-and cytochrome b5-reductases, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate]. 9Briefly, at the end of the reaction, the membrane displays a varying intensity of blue colour depending on the level of NBTreactivity which can then be used to qualitatively categorise a sample as having low, medium, or high redox activity based on the colour scale provided.A further benefit of this assay is the ability to measure redox activity in neat semen, seminal plasma and in sperm cells independently.As such, this second generation assay helps to remove some of the intrinsic biases associated with the original OxiSperm ® assay, where high levels of reductase present in seminal plasma could lead to false positive results. 9While the original OxiSperm ® test has previously been shown to be an indicator of male infertility (medium to high reactivity), 17,22,23,33 the clinical utility of the newer OxiSperm ® II assay is yet to be confirmed by validation studies.
The aim of this current study was first to evaluate whether oxidative stress, when measured using commercially available methods purported for measuring levels in human semen, fluctuates within and between individual men over time.Understanding the extent of this variation will inform the establishment of decision limits/reference ranges/thresholds to be employed clinically when evaluating redox activity.Second, we aimed to determine whether a relationship exists between measures of the sperm function (hyperactivation and binding to hyaluronan) and oxidative stress (sperm DNA fragmentation and lipid peroxidation) when redox levels are measured using the MiOXSYS ® and OxiSperm ® II assays.

Participant characteristics
All men completed a general health questionnaire, providing information such as age, chronic health conditions (i.e., diabetes), current medication use, smoking status, alcohol consumption (frequency/per/week and units consumed) and previously fathered pregnancies at the first semen collection.The questionnaire also included the SF-36 short-form health status survey which provides scores from 0 to 100 about general physical and mental health with a high score indicating better overall health. 15For subsequent collections men filled in a shortened questionnaire, which included the SF-36 and indications for any changes in chronic conditions, medication use, smoking status, alcohol consumption or fathering of a pregnancy since the previous semen collection.Participant height was measured with a stadiometer (cm) and weight (kg) was measured with electronic scales.Body mass index (BMI) was calculated using the formula weight/height 2 and categorised based as being underweight (< 18.5 kg/m 2 ), normal weight (18.5-24.9kg/m 2 ), overweight (25.0-29.9kg/m 2 ) and obesity (> 30.0 kg/m 2 ).Waist circumference was measured using a standard measuring tape set around the waist at a level midway between the lower rib and the iliac crest.Participants were instructed to fully exhale, with the abdomen relaxed.

Semen anaysis
Participants were asked to abstain from ejaculation for 2-7 days prior to semen collection.Semen samples were produced in one of the private, clinical rooms in the University of Adelaide's Clinical Research Facility and collected in a sterile container or produced at home and brought in within 45 min of ejaculation.Only approved lubricant (Ovoil, Vitrolife, Goteborg, Sweden) was used in the study at participant request.After liquefaction at room temperature, a standard semen analysis was performed within 1 h of ejaculation as per WHO V guidelines for the assessment of human semen. 38men volume was measured using a 10 ml serological pipette and semen pH measured using pH strip indicators ranging from pH 4.5 to 10 (Merck, Darmstadt, Germany).Sperm concentration and motility were measured on the CASA ® semi-automatic semen analyser (Microptic, Spain, Barcelona according to the Kruger strict criteria. 38The proportion (expressed as %) of sperm with normal morphology was then calculated.

Redox-potential: MiOXSYS ® system
The MiOXSYS ® system (Aytu Bioscience, CO, USA) was used to detect total static oxidative-reductive potential (sORP), or redox-potential, within a semen sample as per manufacture's instructions. 5The sORP (mV) value for samples was automatically generated on the MiOXSYS ® reader and divided by the samples sperm concentration to calculate normalised sORP/10 6 sperm/ml.High and low controls supplied separately by the manufacturer were run monthly, ensuring consistency in machine detection, with the lowest detectable limit being 0.001 sORP (mV).Samples having values greater than or equal to 1.47 sORP/10 6 sperm/ml were classified as having high redox potential. 29

NBT-reactivity: OXISPERM II ® test
OxiSperm ® II (Halotech DNA, Madrid, Spain) was used as per manufacturer's instructions.The protocol allows for the detection of the redox activity in neat semen, seminal plasma and sperm separately.Seminal plasma was prepared, by spinning the neat semen at 6,000×g for 10 min and transferring the seminal plasma fraction to a clean tube.The remaining sperm pellet was resuspended in 50 μl of PBS and re-spun at 6,000×g for 10 min a further two times.Following the last spin, the sperm pellet was re-suspended in 7.5 μl of the provided sperm reactivity induction solution and left at room temperature for 5 min.Five microlitres of each sample (neat, seminal plasma and spermatazoa) was added to membrane base and allowed to sit at room temperature for 15 min.Colour reactivity of the membrane gel for each semen component was compared with the colour of the scheme and classified as NBT-LOW (1+), NBT-MEDIUM (2+) or NBT-HIGH (3+).Negative controls were run at each analysis by placing the same volume of PBS onto the control membrane.

Sperm hyperactivation
Sperm hyperactivation was assessed on the CASA ® semi-automatic system (Microptic) based on the vigorous motility and flagella movements of spermatazoa.Washed motile sperm was allowed to capacitate in G-IVF PLUS (Vitrolife) at 37

Sperm binding: HBA ® hyaluronan binding assay
Sperm binding was assessed using the HBA ® hyaluronan binding assay (CooperSurgical Fertility Solutions, Knardrupvej, Denmark) according to Jakab et al. 24 Briefly, 10 μl of semen was loaded into the assay chamber and a Cell-Vu ® gridded cover-slip was installed.The sample was incubated for 15 min at 20-30 • C. A total of 200 motile sperm was then counted and classified as either bound (head attached and tails moving) or unbound (freely moving).Scores of 80% or higher were classified as normal binding and those below this threshold were classified as displaying reduced binding. 24
Sperm DNA fragmentation index (DFI) percentage was calculated with the following equation: (fragmented + degraded /200) × 100; in which fragmented DNA included small and absent halos.A pooled control comprised of 16 randomly selected participant samples was stained and analysed at the time of each test to ensure consistency within the assay and reagents; a maximum standard error of 3% was considered an acceptable range.
Lipid peroxidation was assessed on a BD FACSCanto™ II Flow Cytometer (BD Bioscience, NSW, Australia), which had CS&T research beads run daily to ensure fluorescence was kept consistent on measurement days.Then, 10,000 cells per sample were examined and non-specific events gated out.Positive controls of 3,000 μM of hydrogen peroxide spiked preparation were run monthly.Negative controls consisted of spermatazoa incubated in G-IVF medium alone.Lipid peroxidation was expressed as both the ratio shift in red to green fluorescence (a lower red to green ratio indicative of higher lipid peroxidation 30 ) and the proportion of sperm that had high lipid peroxidation. 14Average shift in sperm red to green ratio due to hydrogen peroxide exposure was −1.72 from untreated or a 4.67-fold increase in lipid peroxidation compared with unexposed sperm, indicating that the dye was able to differentiate sperm lipid peroxidation induced from oxidative stress.Details of our gating strategy are presented in Figure S1.Except for total sperm motility, NBT-reactivity (OxiSperm ® II) and lipid peroxidation ratio, all GLMs and correlation analysis were run on data transformed by natural logarithm which normalised the data.In all cases, statistical significance was inferred when P < 0.05.

Participant characteristics and baseline semen results
Men recruited into the study were of prime reproductive age, averaging 27 years (ranging 19-37 years), non-smokers (only 2/31 participants) who consumed approximately 1-3 standard alcoholic drinks per week (Table 1).Men in the study were also relatively healthy, with a median general health score of 75/100, median emotional wellbeing score of 72/100, an average BMI of 24.6 kg/m 2 (range 19.0-33.5 kg/m 2 ) and 26% (8/31) taking prescription medication (predominantly antidepressants) (Table 1).It was also noted Of the samples collected, 56.7% (67/118) reported at least one abnormal semen characteristic (Figure S2A), with decreased progressive motility (< 32%) being most prevalent (31%) (Figure S2B).When assessing samples produced from the same man, 58% of men were found to have at least one abnormal semen parameter over their repeat collections (Figure S2B).samples tested) and this was contributed by high levels of reactivity in their seminal plasma (96%) and not their sperm (4%) (Figure S2A,B).

Relationship between high MiOXSYS and Oxisperm II with sperm function and oxidative stress
Using the clinical cut-offs of ≥1.47 sORP/10 6 sperm/ml for MiOXSYS ® and 2+ and 3+ (medium-high NBT-reactivity) for OxiSperm ® II, we The relationship between high semen redox potential (MiOXSYS ® ) with measures of sperm function and oxidative stress when assessing sORP/10 6 sperm/ml.Normalised high redox potential (> 1.47 sORP/10 6 sperm/ml) or low redox potential (< 1.47 sORP/10 6 sperm/ml) on measures of sperm function (A) concentration 10 6 /ml, (B) progressive motility, (C) total motility, (D) normal morphology, (E) hyperactive motility, (F) sperm binding and sperm oxidative stress, (G) DNA fragmentation, (H) proportion of lipid peroxidation and (J) lipid peroxidation ratio of red:green fluorescence.Data are representative of N = 118 samples from 31 men and analysed by a general linear model with participant added as a random factor.
next assessed their potential relationships with sperm function and oxidative stress.Samples with normalised sORP/10 6 sperm/ml readings above 1.47 were found to have lower sperm concentration (P < 0.001), progressive motility (P = 0.008), total sperm motility (P = 0.001) and normal sperm morphology (P = 0.012) and higher sperm DNA fragmentation (P < 0.001) (Figure 2).No differences were seen for sperm hyperactive motility, sperm binding or sperm lipid peroxidation (P > 0.05) (Figure 2).However, as normalised sORP is adjusted by sperm concentration we also sought to determine whether any relationship existed between high semen redox potential as measured by baseline sORP mV reading.Using the median sORP mV value from our population (38.2), we reassessed outcomes following classification as either high > 38.2 or low < 38.2 sORP mV.There was no effect of high semen redox potential, expressed as sORP mV, with any measure of sperm function or oxidative stress (P > 0.05) (Figure 3).There were no differences between low (1), medium (2) or high (3) levels of NBT-reactivity in neat semen (OxiSperm ® II) on sperm function (concentration, progressive motility, total motility, normal morphology, hyperactive motility, sperm binding) or oxidative stress (DNA fragmentation and lipid peroxidation) (P > 0.05) (Figure 4).No differences were also found for most measures of sperm function and oxidative stress using NBT-reactivity in seminal plasma (P > 0.05) (Figure S3) and sperm (P > 0.05) (Figure S4).However, sperm DNA fragmentation was found to be reduced in those samples with seminal plasma NBT-reactivity of medium (2+) or high (3+) compared with those that scored low (1+, P < 0.05) (Figure S3) and normal sperm morphology was reduced in those samples with medium/high (2+) NBT-reactivity in spermatazoa compared with those that scored low (+1, P < 0.05) (Figure S4).Further, lipid peroxidation ratios were increased in those samples with medium/high (2+) NBT-reactivity in sperm compared with those that scored low (+1, P < 0.05, Figure S4).

Correlations between clinical measures of ROS (MiOXSYS and OxiSperm II) to sperm function and oxidative stress
Next, we assessed whether MiOXSYS ® and OxiSperm ® II values correlated with measures of sperm function and oxidative stress, irrespective of their suggested clinical cut-offs.The only relationship observed between semen absolute sORP (mV), as measured by MiOXSYS ® , and any sperm function or oxidative stress indicator, was a weak positive association with the proportion of sperm positive for lipid peroxidation (0.167, P < 0.05) (Table 2).Normalised sORP/10 6 sperm/ml was strongly negatively associated with sperm concentration (−0.749,P < 0.001) and moderately negatively associated with total sperm count (−0.584,P < 0.001, Table 2).This is not surprising given sperm concentration is the denominator used to determine normalised sORP.Further, normalised sORP/10 6 sperm/ml was moderately negatively associated with total sperm motility (−0.385,P < 0.001) and weakly negatively associated with progressive sperm motility (−0.224,P < 0.01), sperm normal morphology (−0.282,P < 0.01), sperm binding (−0.176,P < 0.05) and sperm DNA fragmentation (−0.277,P < 0.01) (Table 2).
Next, since normalised sORP/10 6 sperm/ml is calculated by dividing sORP (mV) by sperm concentration, we wanted to examine whether these associations persisted after adjusting for sperm concentration.
Following adjustment for sperm concentration, the negative associations observed previously between normalised sORP/10 6 sperm/ml and sperm function and oxidative stress were no longer present (P > 0.05) (Table 3).However, new correlations were revealed, with normalised sORP/10 6 sperm/ml weakly positively associated with abstinence (0.203, respectively, P < 0.05) (Table 3) and proportion of sperm positive for lipid peroxidation (0.224, P < 0.05) (Table 3).To further investigate whether the adjustment for sperm concentration in the calculation of normalised sORP/10 6 sperm/ml was the primary driver of the association of MiOXSYS ® with measures of the sperm function and oxidative stress, we again performed partial correlations of the sperm concentration adjusting for normalised sORP/10 6 sperm/ml.In these analyses, the sperm concentration was strongly positively associated with total sperm count (0.643, P < 0.001),  S2).

DISCUSSION
High concentrations of ROS in sperm are considered one of, if not, the main contributor to male idiopathic infertility. 3,37Despite this, there remains no widespread, point-of-care assay that can be employed in clinics to give a rapid assessment of sperm oxidative stress.A potential explanation for this is because fundamental research questions remain, with their ability to accurately measure sperm oxidative stress and whether natural fluctuations in oxidative stress occur within and between men over time are not fully understood. 16Utilising two commercially available tests marketed as clinical diagnostics for the redox activity in semen (MiOXSYS ® and OxiSperm ® II), we aimed to examine whether natural fluctuations in semen redox levels occur over time as well as understand their associations with measures of sperm function and oxidative stress over a 6-month period.We found that while normalised redox potential (≥1.47 sORP/10 6 sperm/ml), as measured by MiOXSYS ® , was associated with lower sperm concentration, motility, morphology and higher DNA fragmentation, these associations were solely driven by sperm concentration (10 6 /ml).Further supporting sperm concentration as independently driving these  This study initially set out to use two commercial assays to measure oxidative stress in human semen in order to understand the normal variation that occurs within and between individuals from the general population to help determine their clinical decision limits.Unfortunately, in this study we found MiOXSYS ® and OxiSperm ® II to have significant limitations.First, it appears that the clinical utility of the MiOXSYS ® system may be exaggerated by adjusting to the sperm concentration.Using the sperm concentration as a denominator when expressing the sORP results was originally introduced as it was believed that sperm would be the major source of electrons in the ejaculate. 4However, a recent publication by Joao et al. 26 instead shows that the presence of sperm in seminal plasma is unrelated to the baseline sORP (mV) readings, and as a result, using the ratio correction method to normalise data overestimates the sORP/10 6 sperm/ml value in patients with low sperm concentration.This is because normalisation to sperm concentration does not take into account the observation that sperm concentration itself has independent associations with measures of sperm function and oxidative stress. 11In this study, we found that all the associations of normalised redox potential (sORP/10 6 sperm/ml) with sperm function and oxidative stress were being driven by its adjustment with sperm concentration, and the independent negative associations low sperm concentration had with count, motility, morphology and hyperactive motility and positive associations with DNA fragmentation.As such, for MiOXSYS ® to be clinically meaningful, baseline sORP (mV) readings need to be the measure used for determining its ability to predict both male infertility and sperm oxidative stress.In our study, sORP (mV) was found to be largely unrelated to measures of sperm function and oxidative stress, although we do acknowledge a weak positive association with sperm lipid peroxidation.
A key reason why MiOXSYS ® may not be able to accurately predict sperm function and oxidative stress may come down to its underlying biochemistry and biological application.Oxidation-reduction measures the potential for electrons to move from one chemical species to another thereby being reduced or oxidised, respectively, with oxidisers having a positive ORP value and reducers having a negative ORP value. 36Therefore, ORP is dependent on the total concentrations of reductants and oxidants in a particular system with positive ORP suggestive of a larger oxidant concentration. 36ROS molecules including superoxide anion, hydroxyl radicals, hydrogen peroxide, nitric oxide, peroxynitrites all contribute to the oxidant level, while molecules including: thiols, vitamin C, tocopherol, β-carotene, lycopene, uric acid, bilirubin and flavonoids contribute as reductants. 27Importantly, ROS are not the only molecules that contribute to the oxidant levels of biological fluids. 31It has since been established that semen sORP (mV) readings are not influenced by sperm, 26 which leads to the question of 'how is the redox status of a fluid that is created instantaneously at ejaculation by sex accessory glands (seminal plasma) going to be predictive of the oxidative stress status of sperm?' .This is because the majority of oxidative damage to sperm occurs between spermiogenesis and epididymal maturation prior to mixing with seminal plasma at ejaculation. 8,10,12Additionally, seminal plasma is a potent source of some of the most highly specialised antioxidants and scavenging enzymes of any biological fluid [i.e., glutathione peroxidase (GPx5), extracellular superoxide dismutase (SOD), uric acid, vitamin C, tyrosine and polyphenols] which act to protect against oxidative damage to sperm. 12As such, we should consider whether there is any benefit at all to focus on developing point-of-care devices that measure redox status of seminal plasma if what we really need to understand is the status of sperm?
Another important consideration when developing new methods for measuring ROS/oxidative stress in semen for evaluating male infertility is which component of the ejaculate is most informative?One might argue that the oxidative stress status of the motile sperm fraction would be the most important, given these sperm are those most likely to go on to fertilise the egg and contribute to fetal development.
However, one may also contend that given recent advances in better understanding the complex role seminal plasma plays in promoting fertility and fecundity through its beneficial effects on the female immune system, 35 that a measure of oxidative stress of the entire ejaculate might be more informative.
There are also many factors that limit oxidation reduction potential interpretation, including; temperature, pH, irreversible reactions, slow electrode kinetics, non-equilibrium, small exchange currents and presence of multiple redox couples. 28Recently, a study by Garcia-Segura et al. 21reported that despite correcting for semen pH, semen redox-potential remained unrelated to measures of sperm DNA fragmentation as assessed by the single-cell gel electrophoresis assay.
Therefore, in this study it appears that MiOXSYS ® provides no additional diagnostic information for the assessment of male infertility, or sperm oxidative stress, beyond that of a standard semen analysis.
Similar to the MiOXSYS ® system, the inability of the OxiSperm ® II assay to determine sperm oxidative stress is also likely related to the biochemistry of the assay.While it is correct that the NBT will undergo reduction in the presence of superoxide, any molecule capable of giving up an electron is equally able to reduce NBT, including NADH and phenazine methosulphate. 32Further, in vitro studies using human sperm have shown that exposure to the sperm mitochondrial ROS generators arachidonic acid or 4-hydroxynonenal were unable to modify NBT reactivity. 13However, only the potent redox cycler menadione was able to modify NBT reactivity in human sperm, although high background readings were also present. 13As such, it cannot be stated with certainty that the high levels (3+) of NBT reactivity are the result of high superoxide concentrations in the ejaculate.The lack of association between OxiSperm ® II and sperm function and oxidative stress observed in this study is consistent with previous studies using the original OxiSperm ® assay.In these, it was reported that NBT-reactivity did not correlate with male infertility, sperm parameters, fertility rates, sperm DNA damage nor pregnancy and live birth rates, with only 31%-76% of infertile men displayed medium to high redox activity (2+ and 3+). 17,22,23,33Further, superoxide production in sperm, as measured using the MitoSOX red probe, has been previously reported as being predictive of human sperm DNA damage as measured by Halosperm ® and the Comet assay, 34 suggesting the ability of OxiSperm ® II to correctly identify superoxide concentrations is limited.
Although, the newer OxiSperm ® II assay offers the ability to assess the redox activity in individual components of the ejaculate (neat semen, seminal plasma, and spermatazoa), the qualitative nature of the assay creates considerable risk of bias, thereby limiting the utility of the assay.This bias is likely to take the form of subjective differences in colour perception between operators, with the potential to result in different interpretations as to what is classified as low (1+), medium (2+) or high (3+). 16Even though ∼58% of participants in our study had at least one abnormal semen analysis over the collection period, the majority of the high (3+) OxiSperm ® II observations came from the seminal plasma fraction.This is consistent with evidence showing that high levels of reductase present in seminal fluid reduce the NBT to formazan, creating false positive results. 9As a result, it appears that the newer OxiSperm ® II assay may also be unable to correctly determine sperm oxidative stress which acts to limit its clinical application.
Studies examining between-and within-individual variation in semen redox status are also limited.Using chemiluminescence, Zorn et al. 40 reported ROS levels as being relatively consistent in 25 infertile men who had repeat measures taken over a 6-month period.In contrast, a longitudinal study (21-month period) conducted in healthy, fertile men found ROS concentrations to be quite variable, reporting these fluctuations as appearing independent to sperm count, motility and morphology. 18The investigators concluded that the variation was likely attributable to changes in ejaculation frequency and seasonal/lifestyle variation.Interestingly, in our study, frequency of ejaculation and BMI were stable within individuals over time, as was good general health, which likely contributed to the low level of variation we observed in semen redox-potential and activity over time.
An important limitation of our study was only accessing relatively healthy men from the general population, thereby limiting the number of infertile men in our cohort.However, we did not want to bias our findings by only evaluating men seeking infertility treatment.Despite this limitation, approximately 30% of all samples analysed in our study were found to have an abnormal characteristic in their basic sperm parameters.Further, it must be noted that majority of high (3+) redox activity readings in neat semen in our study were due to increased seminal plasma redox activity and not sperm redox activity, raising the possibility that our samples were biased towards those with low-sperm redox activity.Finally, further studies are required to determine whether similar observations to those described in our study are also observed in sub fertile populations.

CONCLUSION
Appropriately designed and suitably powered studies are required to determine the clinical utility of both MiOXSYS ® and OxiSperm ® II assays, as the balance of evidence suggests that these tests may have been prematurely introduced clinically, before a rigorous understanding of their underlying biochemistry was known.As such, it appears from this study that these assays provide no additional clinical utility beyond that of a standard semen analysis.The development of new robust point-of-care devices for accurately determining oxidative stress in spermatazoa is therefore warranted.
This project was approved by Human Research Ethics Committee at the University of Adelaide (H-2020-163), and all protocols followed The National Statement on Ethical Conduct in Human Research (2007)-Updated 2018.All participants provided informed consent and were reimbursed for their time.In total, 31 English speaking men between the age of 18 and 45 years were recruited from the general population (2020-2021), providing four semen samples over a 6-month period, at least 4 weeks apart (The ROSS cohort).Twenty-nine participants provided all four semen samples, while two participants provided only a single semen sample, equating to a total of 118 samples.All samples were analysed at the Adelaide Health and Medical Sciences Building at the University of Adelaide.Exclusion criteria were men with a history of vasectomy or vasectomy reversal, men with undescended testicles or genetic conditions affecting their fertility (i.e., Prader-Willi, Klinefelter's Syndrome etc.), and men with known infectious status, such as HIV/AIDS.
Prism v9.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com)and IBM SPSS Statistics for Windows, version 26 (IBM Corp., New York, USA) were used for all statistical analyses.In determining whether parameters varied within and between participants, and which contributed greater to the overall variation observed, a repeated measure one-way ANOVA test was performed.Corresponding F-values were used, with values closer to 1 indicating less variation.To determine the relationship between clinical cut-offs for MiOXSYS ® (1.47 sORP/10 6 sperm/ml and 38.2 sORP mV) and OxiSperm ® II NBT-HIGH (3+) with measure of the sperm function (count, motility, morphology, hyperactivation, binding) and oxidative stress (DNA fragmentation and lipid peroxidation) a general linear model (GLM) was performed.High/low concentrations of ROS for each assay were added as a fixed factor, participant ID as a random factor and the measures of sperm function/oxidative stress as the dependent variable.To further understand relationships between MiOXSYS ® and OxiSperm ® II and sperm function and oxidative stress, partial correlations controlling for participant ID initially, and then sperm concentration and normalised sORP/10 6 sperm/ml were used to analyse quantitative data relationships between MiOXSYS ® , sperm concentration, sperm function and oxidative stress, while point biserial correlations were performed on associations of OxiSperm ® II (count data) with sperm function and oxidative stress.
When assessing clinical measures of oxidative stress, 67%(21/31)   of participants recorded at least one semen sample with high redox potential (≥1.47 sORP/10 6 sperm/ml), while 33% (39/118) of all collected samples were above the suggested cut-off (FigureS2A,B), as measured by MiOXSYS ® .The range in baseline sORP mV readings was high across participants (0.1-200) with a median value 38.2 (Table1).For OxiSperm ® II, 74% of men recorded at least one sample with high levels of NBT reactivity (3+) in their neat semen (31% of all semen F I G U R E 1 Variations in semen redox potential between and within individual men as measured using the MiOXSYS ® system.Semen oxidative redox potential sORP (mV) as (A) box and whisker plots for each individual (the box depicts the 25th, 50th and 75th percentiles and the whiskers minimum and maximum values) or (B) line plots for each participant over the collection period.Normalised sORP/10 6 sperm/ml [sORP(mV) divided by sperm concentration 10 6 /ml] as (C) box and whisker plots for each individual (the box depicts the 25th, 50th and 75th percentiles and the whiskers minimum and maximum values) or (D) line plots for each participant over the four collections.Data are representative of N = 116 samples from 29 men and analysed by a repeated measures one-way ANOVA with corresponding F-values closer to one indicating less variation.

F I G U R E 3
The relationship between high semen redox potential (MiOXSYS ® ) with measures of sperm function and oxidative stress when assessing sORP (mV).High redox potential (> 38.2 sORP mV) or low redox potential (< 38.2 sORP mV) on measures of sperm function (A) concentration 10 6 /ml, (B) progressive motility, (C) total motility, (D) normal morphology, (E) hyperactive motility, (F) sperm binding and sperm oxidative stress, (G) DNA fragmentation, (H) proportion of lipid peroxidation and (J) lipid peroxidation ratio of red:green fluorescence.Data are representative of N = 118 samples from 31 men and analysed by a general linear model with participant added as a random factor.

F I G U R E 4
The relationship between high neat semen redox activity (OxiSperm ® II) with measures of sperm function and oxidative stress.Effect of differing levels of NBT-reactivity (1-low, 2-medium and 3-high) on measures of sperm function (A) concentration 10 6 /ml, (B) progressive motility, (C) total motility, (D) normal morphology, (E) hyperactive motility, (F) sperm binding and sperm oxidative stress, (G) DNA fragmentation, (H) proportion of lipid peroxidation and (J) lipid peroxidation ratio of red:green fluorescence.Data are representative of N = 118 samples from 31 men and analysed by a general linear model with participant added as a random factor.moderately positively associated with total motility (0.356, P < 0.001) and normal morphology (0.400, P < 0.01), moderately negatively associated with DNA fragmentation (−0.310,P = 0.001) and weakly positively associated with progressive motility (0.230, P = 0.008) (Table correlations and not redox-potential, was the observation that baseline sORP mV values had little to no relationship with sperm function or oxidative stress.Further, low (1+), medium (2+) or high (3+) NBTreactivity (OxiSperm ® II) and measures of sperm function or oxidative stress (DNA fragmentation and lipid peroxidation) were only minimally associated, suggesting these assays may have little to no diagnostic potential for accurately measuring sperm oxidative stress in humans.

Table 1
also shows the means, medians and ranges of all semen parameters, sperm function tests and oxidative stress indicators measured.

MiOXSYS (sORP mV) MiOXSYS (sORP/10 6 sperm/ml)
Correlations between MiOXSYS ® and OxiSperm ® II to sperm function and oxidative stress Data are representative of N = 118 samples from 31 men and analysed by partial correlations adjusting for participant ID for MiOXSYS ® and point biserial for NBT-reactivity (OxiSperm ® II).Bold values indicate the statistical significance.
TA B L E 2

10 6 sperm/ml)
Correlation between MiOXSYS ® , sperm function and oxidative stress after adjusting for sperm concentration Data are representative of N = 118 samples from 31 men and analysed by partial correlations adjusting for participant ID and sperm concentration (10 6 /ml).Bold values indicate the statistical significance.
TA B L E 3