Semen Quality and Infertility Status Can Be Identified Through Measures of Oxidation–Reduction Potential

Accepted: 24 June 2017

DOI: 10.1111/and.12881

ORIGINAL ARTICLE

Semen quality and infertility status can be identified through measures of oxidation–reduction potential

1Urology Department, HMC, Doha, Qatar Summary 2Andrology Department, Cairo University, Cairo, Egypt Standard analyses for evaluating semen quality require technical expertise and are in- 3American Center for Reproductive terpretive in nature. Oxidative stress (OS) alters many of the semen parameters; thus, Medicine, Cleveland Clinic, Cleveland, OH, a measure of OS could be an indicator of semen quality. Static oxidation-reduction USA 4Aytu BioScience, Englewood, CO, USA potential (sORP) is a universal measure of OS traditionally used in environmental ap- plications but is increasingly used in biomedical studies. sORP was measured to deter- Correspondence Mohamed Arafa, Male Infertility Unit, Urology mine how well it associates with semen quality and if it differentiates semen from Department, Hamad General Hospital, Doha, infertile patients and fertile donors. All study participants (Infertile, n = 365 and Fertile, Qatar. Email: [email protected] n = 50) underwent standard semen analyses, and sORP was measured in unprocessed semen. In infertile patients, sORP increased with decreased total sperm number, motility and morphology. sORP values were higher in samples with abnormal quality (low number, motility and/or normal morphology) compared with those of normal quality. Infertile patients had higher sOPR values compared to fertile donors. A sORP cut-off value of 1.38 mV/106 sperm/ml can differentiate normal from abnormal semen samples, while a cut-off value of 1.41 mV/106 sperm/ml, can differentiate between infertile and fertile semen samples. In conclusion, sORP provides a quick and unbiased indicator of semen quality that can be a beneficial addition to semen analysis to determine semen quality and fertility status.

KEYWORDS infertility, oxidation-reduction potential, semen analysis, semen quality, spermatozoa

1 | INTRODUCTION infertility (Wang & Swerdloff, 2014). A study of 378 couples trying to conceive naturally confirmed that no single semen parameter inde- Semen analysis is the corner stone for assessment of male fertility. pendently predicted fertility. However, data derived from the combi- According to the World Health Organization’s (WHO) criteria for nation of parameters was able to give an insight into the likelihood of semen quality (WHO, 2010), there are several parameters of semen success (Buck Louis et al., 2014). It would be beneficial, then, to have a quality; however, sperm number and motility (total and progressive) single measure of quality that reflects the combined influence of mul- carry the most weight as the status of these parameters. tiple parameters. Measuring imbalances in the reduction–oxidation It is generally accepted that the time and expertise required to do (redox) system might qualify as this holistic quality measure. a full semen analysis, especially in a smaller laboratory, is often not The redox system balances the activity of oxidants and antioxi- cost effective (Wyrobek et al., 1997). It is tempting then, to rely on dants (reductants) based on physiological need (Costantini & Verhulst, the results of only one or two semen parameters; however, Wang and 2009; McCord, 2000). In males, oxidants like reactive oxygen species Swerdloff stated that a single parameter cannot be used as a valid bio- (ROS) and reactive nitrogen species have physiological roles in tes- marker of fertility due to presence of multiple factors contributing to ticular immunity, spermatogenesis, motility, capacitation and other

Andrologia. 2017;e12881. wileyonlinelibrary.com/journal/and © 2017 Blackwell Verlag GmbH | 1 of 6 https://doi.org/10.1111/and.12881 2 of 6 | ARAFA et al. functions (Doshi, Khullar, Sharma, & Agarwal, 2012; Ko, Sabanegh, azoospermia, antioxidant therapy, pyospermia, palpable varicocele, & Agarwal, 2014). When the production of oxidants exceeds what is occupational chemical/radiation exposure, sperm concentration physiologically needed and the available antioxidants cannot mitigate <1 million sperm/ml or presence of female factor infertility. All female their effects, a state of oxidative stress occurs. Oxidative stress has partners had proven normal fertility status through a detailed medical been associated with poor morphology, low motility and decreased report from their gynaecologists after performing full clinical examina- number of spermatozoa (Benedetti et al., 2012; Hosseinzadeh Colagar, tion and investigations for fertility. Karimi, & Jorsaraei, 2013; Khosrowbeygi & Zarghami, 2007; Walczak- Jedrzejowska, Wolski, & Slowikowska-Hilczer, 2013). Thus, it could be 2.2 | Fertile donor population used as a biomarker for overall semen quality. Previous attempts to use oxidative stress as an indicator of Men with proven fertility, that is having established a pregnancy in the semen quality measured single features of the redox system mea- last 24 months, were recruited through an advertisement placed by suring only ROS, specific antioxidants or post hoc oxidative damage the same tertiary medical centre. Controls provided a proof of preg- (Agarwal, Mulgund, Sharma, & Sabanegh, 2014; Benedetti et al., 2012; nancy in the past 24 months including child’s birth certificate and a Haghighian, Haidari, Mohammadi-Asl, & Dadfar, 2015). This has led to medical report from the spouse’s gynaecologist stating that the preg- ambiguous overall conclusions. A better measure of the redox system nancy was spontaneous. The same exclusion criteria were applied. is the oxidation–reduction potential (ORP), which provides a measure of the activity of both the oxidants and antioxidants (Costantini & 2.3 | Data collection Verhulst, 2009; Shapiro, 1972). Measuring ORP as an indicator of the redox balance has been available since the 1930’s when it was first General demographics were collected on all participants including age used as an indicator of water quality and later accepted by the WHO and body mass index (BMI). for drinking water standards (Schmelke, 1933; WHO, 2006, 2011). Each participant provided a semen sample after ≥2 days of sex- When applied to biomedical studies, changes in ORP values reflect ual abstinence. Standard semen analysis was performed using the 5th the function of the redox system (Shapiro, 1972). Edition WHO manual (WHO, 2010). Sperm concentration and motility Traditional methods for measuring ORP use re-usable electrodes were performed manually using a hemocytometer. Morphology was contained within glass probes submerged in large sample volumes. assessed by a single experienced technician based on spermatozoa The major limitations of this technology have been electrode contam- stained using the Diff-Quik protocol. Kruger’s strict criteria were used ination and sample volume (Copeland & Lytle, 2014). Because of this, for morphology assessment. the application of ORP to biomedical assessment has been slow, with ORP was measured in duplicate in unprocessed post-liquefied few studies (Rael et al., 2007, 2009; Zhi et al., 2013). Recently, the semen to assess test reproducibility using the MiOXSYS System development of a novel technology has removed the limitations by (Agarwal et al., 2016). The MiOXSYS System is comprised of an elec- creating a single-use electrode sensor that requires only 30 μl of sam- trochemical analyser (Aytu BioScience, cat. # 100229) and single-use ple. This has allowed expansion into biomedical fields in which oxida- disposable semen sensors (cat. # 100283). Because the analyser ap- tive stress is suspected (Agarwal, Sharma, Roychoudhury, Du Plessis, plies a low voltage steady current measured in millivolts (mV), ORP is & Sabanegh, 2016; Bjugstad, Rael, et al., 2016; Bjugstad, Fanale, 2016; represented as the static ORP (sORP, mV). Oxidative stress reflects Rael, Bar-Or, Kelly, Carrick, & Bar-Or, 2015). the relationship between spermatozoa (producers of free radicals) and In this study, we have applied this technology to better explore seminal plasma (an antioxidant reservoir); thus, raw sORP values (mV) the relationship of ORP with semen quality. To that end, we aimed were normalised to sperm concentration—a value that reflects both to determine if ORP measures are related to individual parameters of semen volume and sperm number. Data for sORP are presented as semen quality in infertile men, specifically total sperm number, motil- mV/106 sperm/ml throughout. ity and morphology and if ORP can distinguish between semen from infertile men and men of proven fertility. 2.4 | Statistics

Comparisons of groups were performed using Fisher’s exact test or chi- 2 | MATERIALS AND METHODS square test for categorical variables such as frequency distribution of abnormal sperm count, abnormal motility and abnormal morphology. The prospective study was approved by the Institutional Review Wilcoxon’s rank sum test was used for group comparisons with respect Board, and appropriate consents were signed by the patients before to quantitative variables such as age, abstinence, volume, sperm con- enrolment in the study. centration, total sperm count, per cent motility, sperm morphology and sORP between fertile and infertile group. Relationships between sORP and semen parameters or sORP and fertility were determined using 2.1 | Infertile patient population Spearman’s correlation. For group comparisons, samples were grouped Men attending a specialised male fertility unit in a tertiary medi- into “normal” or “abnormal” semen parameters and fertile and infertile cal centre were recruited. The exclusion criteria were as follows: men. Receiver operator characteristic (ROC) analyses with Youden’s ARAFA et al. | 3 of 6

TABLE 1 Basic demographic data and sORP differences between infertile patients and fertile donors

Infertile patients Fertile donors p values

N 365 50 Age (years) 35.5 ± 0.4 32.3 ± 1.0 <.002* Median = 34 (IQR 31–40) Median = 32 (IQR 27–35) BMI 31.5 ± 1.6 25.5 ± 0.5 <.001* Median = 27.7 (IQR 25.2–31.2) Median = 24.8 (IQR 23.4–27.6) Abstinence (days) 4.4 ± 0.1 3.7 ± 0.2 <.01* Median = 4 (IQR 3–5) Median = 3 (IQR 3–4) Volume (ml) 3.1 ± 0.08 2.9 ± 0.2 .44 Concentration (×106/ml) 36.2 ± 1.6 58.7 ± 3.8 <.001* Total sperm number (×106) 110.8 ± 5.5 172.1 ± 17.9 <.001* Total motility (%) 45.5 ± 1.2 59.9 ± 1.8 <.001* Progressive motility (%) 27.0 ± 1.4 15.2 ± 0.7 <.001* Morphology (%) 7.5 ± 0.5 10.0 ± 0.8 <.001* sORP/concentration (mV/106/ml) 5.00 ± 0.56 1.26 ± 0.15 <.001* (min–max = 0.06–131.67) (min–max = 0.6–5.75)

Values are mean ± SE. *p value <.05 was considered significant by Wilcoxon rank sum test for pairwise comparison between fertile donors and infertile men.

Indices were used to determine cut-off values for sORP. The area under Using the collective status of sperm number, motility and mor- the curve (AUC), sensitivity, specificity, positive predictive value (PPV) phology, data from infertile patients were grouped into normal and the negative predictive value (NPV) with their respective 95% con- semen quality (n = 41; met WHO parameters) and abnormal semen fidence intervals (95% CI) were reported. Summaries of quantitative quality (n = 324; failed one or more). Those with abnormal quality variables are in the form mean ± SE. Tests were performed at a signifi- semen had significantly higher sORP values than those with nor- cance level p = .05. Analyses were performed using R version 2.15.1. mal quality (5.49 mV/106/ml versus 1.03 mV/106/ml, respectively, p value <.02). ROC analysis indicated that sORP can accurately identify abnor- 3 | RESULTS mal semen quality, as indicated by a significant coverage of area under the curve (AUC = 0.78, p < .0001). The associated sORP criteria for Final data set used for statistical analyses was 365 infertile patients identifying and predicting abnormal quality were >1.38 mV/106/ml and 50 fertile controls. Infertility was primary in 254 (69.6%) patients (Figure 2) with sensitivity of 63.3%, specificity 87.8%, PPV 97.6% and and secondary in 111 (30.4%) with a mean duration 3.2 ± 1.2 years. NPV 23.2% and accuracy of 66%. The odds ratio that a semen sample Table 1 presents the demographic data and overall semen analysis re- with a sORP value greater than 1.38 mV/106/ml was abnormal in qual- sults. sORP did not significantly correlate with age (r = −0.03), BMI ity was 10.05 (p < .001). (r = −0.04) or abstinence (r = −0.0008). When comparing fertile donors to infertile patients’ group, we Duplicate sORP measures were highly correlated with each other found that on average the fertile donors were significantly younger, (r = +0.99, R2 = 0.99, p < .00001). The average difference between du- had a shorter abstinence, but had similar BMI values (Table 2). As plicates was 0.49 mV/106/ml. As a result of the high reliability, repli- correlations between sORP and these variables, age and abstinence, cate data were averaged, and the mean sORP values were used in all were not significant (age: r = −0.03), abstinence: r = −0.0008, p > .05 subsequent analyses. both), no further analyses were carried out. As would be expected, sORP was negatively correlated with sperm concentration fertile controls also had significantly higher concentration, total (r = −0.909; p < .001); total sperm count (r = −799; p < .001) and per sperm numbers, higher sperm motility, and better sperm morphology cent motility (r = −0.554; p < .001). It was directly correlated with than the infertile patients (Table 2). Only volume was similar between sperm abnormalities (r = 0.645; p < .001) and spermatozoa with head the groups. defects (r = 0.500; p < .001; Fig. S1). sORP values measured in infertile patients (n = 365), regardless of In infertile patients, 28% had abnormal sperm number, 41% failed semen quality, were significantly higher compared to those measured total motility, 88% failed progressive motility, and 50% had poor mor- in donors of proven fertility (n = 50; ORP 5 versus 1.26 mV/106/ml, p phology. Semen sORP values differentiated between individual pa- value <.02). In a ROC analysis, sORP was able to significantly discrimi- rameters of quality. Significantly higher sORP values were found in nate between semen samples (AUC = 0.68, p < .001). The optimal cut- semen with low total sperm numbers, with low motility levels and low off value was >1.41 mV/106/ml. Those samples from fertile donors normal semen morphology (Figure 1). reliably fell below this sORP cut-off (specificity 78.0). The odds ratios 4 of 6 | ARAFA et al.

FIGURE 1 Higher sORP values were measured in abnormal parameters. (a) Semen with abnormally low numbers of spermatozoa had higher sORP values. (b, c) Semen with abnormally few motile spermatozoa was also related to higher sORP values. (d) Semen with fewer morphologically normal spermatozoa had higher sORP values. *, significant at p < .05

4 | DISCUSSION

Semen quality is a surrogate indicator of fertility; however, quality is usually based on the interpretation of an assembly of measured parameters. The current study is the largest one discussing sORP and semen analysis up till now with 365 infertile patients and 50 fertile donors. The data presented suggest that sORP could be a reliable measure of semen quality. sORP increased as individual semen parameters decreased. When the parameters were assessed collectively for semen quality, sORP could distinguish between those of abnormal and normal semen quality at the cut-off value of 1.38 mV/106/ml. Further, sORP could discriminate between semen from infertile patients and fertile donors quality at the cut-off value of 1.41 mV/106/ml. Our results also confirm that, in general, individual semen parameters are poor predictors of infertility (Buck Louis et al., 2014; Wang & Swerdloff, 2014). sORP was better than total sperm number, total motility and morphology in identifying semen from infertile patients. Progressive motility was the only individual parameter that, in the current data set, could also identify infertile patients; however, FIGURE 2 sORP values can distinguish and identify those semen progressive motility may not always be so reliable in other data sets. samples that have abnormal quality. The ROC curve represents Furthermore, assessment of progressive motility is subjective, and op- the area under the curve (AUC) that was occupied by different erator dependent, therefore, its reliability as fertility marker is ques- sORP cut-off values (AUC = 0.78*). The optimal sORP cut-off was 1.38 mV/106/ml (circle marker). Black dashed line in A is at the 1.38 tionable. Moreover, in establishing the WHO criteria, Cooper et al. cut-off level. *, significant at p < .05 found that both total and progressive motility were lower in proven fertile donors (Cooper et al., 2010), suggesting a more complex rela- that semen sORP values greater than the cut-off of 1.41 mV/106/ml tionship between motility and infertility. were from infertile patients was 4.75 (p < .001). Because sORP is a measure of oxidative stress and oxidative Comparing sORP to individual WHO parameters for predicting stress affects sperm numbers, motility and morphology (Agarwal semen from infertile patients indicated that progressive motility had et al., 2014; Benedetti et al., 2012; Haghighian et al., 2015), it would a slight advantage over sORP with a higher accuracy but a lower PPV be reasonable to use sORP as a marker to support the interpretation (Table 2). All other individual parameters were significant but, at the of standard semen analyses. Previous studies attempted to relate WHO cut-off values, were poor predictors (Table 2). oxidative stress and fertility using measures from only one side of ARAFA et al. | 5 of 6

TABLE 2 Predicting semen samples from infertile patients and from fertile donors

Total number Total motility Progressive motility Normal morphology sORP (Youden’s Index) (WHO Cut-off) (WHO Cut-off) (WHO Cut-off) (WHO Cut-off)

Cut-off for >1.41 mV/106/ml 39 × 106 40% 32% <4% infertility Sensitivity 57.3% (52.0–62.4) 28.2% (23.7–33.1) 40.8% (35.7–46.1) 88.0% (84.2–91.1) 50.4% (45.2–55.7) Specificity 78.0% (64.0–88.5) 92.0% (80.8–97.8) 94.0% (83.5–98.7) 28.0% (16.2–42.5) 84.0% (70.9–92.8) PPV 95.0% (91.2–97.5) 96.3% (90.7–99.0) 98.0% (94.3–99.6) 89.9% (86.3–92.8) 95.8% (92.0–98.2) NPV 20.0% (14.6–26.3) 14.9% (11.1–19.4) 17.9% (13.4–23.0) 24.1% (13.9–37.2) 18.8% (13.9–24.6) Accuracy 60% 35.9% 47.2% 80.7% 54.5%

Data are presented with 95% confidence intervals (95% CI). the redox balance, only ROS or only antioxidants, resulting in con- ability to measure those small site-specific elevations through semen fusion (Agarwal et al., 2014; Benedetti et al., 2012; Hosseinzadeh sORP helps to focus the relationship between oxidative stress, semen Colagar et al., 2013; Khosrowbeygi & Zarghami, 2007; Walczak- quality, and by extension-infertility. Jedrzejowska et al., 2013). sORP reflects the balance between both The major limitation of the study is that the data comes from a oxidants and antioxidants. In addition to having good discriminative single fertility centre. As the test is newly introduced for the assess- and predictive power, measuring sORP occurs in a fraction of the ment of semen, its reproducibility could be confirmed by comparing time needed for a full semen analysis (<5 min) and does not require results from other centres. Another limitation is the lack of comparison technical expertise. of ORP to other methods of assessment of oxidative stress; however, Using sORP as a measure of oxidative stress in semen was re- as other methods are not validated and only measures one part of the cently investigated by Agarwal and coworkers in two studies and also oxidative equation, no sound comparison is expected. Although the reported strong correlations of sORP to different semen parameters. study results revealed that sORP was slightly inferior to progressive The first study was performed on 33 infertile patients and 26 con- motility in differentiating fertile from infertile semen samples, the trols (Agarwal et al., 2016). They used a cut-off value of 1.48 mV/106/ objectivity and reproducibility of sORP favours its use in conjunction ml to differentiate normal and abnormal semen parameters which are with conventional semen parameters for fertility prediction. slightly higher than the cut-off value in the present study. However, these authors used motility only as an indicator of semen quality while in our study abnormal semen was defined as failure to meet WHO 5 | CONCLUSIONS 2010 criteria in any of the count, motility, progressive motility or normal morphology. The second study included 106 patients and 51 sORP increased when individual parameters of semen quality were healthy controls (Agarwal et al., 2017). In this larger study, the cut-off abnormal. sORP was also able to distinguish between semen with value differentiating infertile from control group was 1.36 mV/106/ml overall abnormal quality from those with normal quality, indicated by which is again slightly different from the cut-off value presented in decreased sperm number, normal morphology and/or sperm motility. our study. However, Agarwal et al. included healthy controls with no The difference between normal quality and abnormal quality was large proven fertility, while in the present study the controls have proven enough to identify a reliable sORP cut-off value where semen with a fertility within the past 2 years. Another major difference between the sORP value greater than 1.38 mV/106/ml were 10 times more likely present study and the previous report by Agarwal and coworkers is to have abnormal semen quality in infertile men. Similarly, the differ- the number of patients (365 versus 106 and 33 patients). However, all ence in sORP values between infertile patients and fertile donors was the studies agreed on the correlation of sORP with semen parameters also large, such that a semen sample with a sORP value greater than with nearly the same cut-off values. 1.41 mV/106 sperm/ml was four times more likely to have come from The sORP values measured in semen did not change because of an infertile patient than a fertile donor. Using sORP measures in con- age or BMI; thus, the sORP values reflect the state of oxidative stress junction with standard semen analysis could substantiate the result of in the male reproductive system and not an indirect systemic measure the analysis and augment any conclusions drawn on a man’s fertility of the body, which would distort the interpretation regarding oxidative status. stress in infertility. With our study, we could confirm the recent study by Agarwal et al. (2016) that sORP is not affected by age. The immuno- ACKNOWLEDGEMENTS privilege status of the testes supports the notion that changes in semen sORP reflect site-specific changes in oxidative stress (Benedetti et al., The authors acknowledge the efforts of Mr. Farid Keyvanifard and Mr. 2012; Chen, Deng, & Han, 2016). Elevations in oxidative stress at the Mohamed AboHalawa for their role in the andrological assessment of site of spermatogenesis and maturation are more likely to translate semen samples. The authors thank Aytu Bioscience, Englewood, CO, into changes in semen and sperm quality than systemic elevations. The for the supply of MiOXSYS Analyzers for this clinical study. 6 of 6 | ARAFA et al.

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