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Reactive oxygen species measured in the unprocessed semen samples of 715 infertile patients

Abstract Purpose To determine whether reactive oxygen species (ROS) in semen samples could be measured with the Monolight™ 3010 Luminometer. Methods Using the Monolight™ 3010 Luminometer, the ROS was measured in the unprocessed semen samples of infertile male patients, as well as the luminescence of 190 semen samples. The samples were classified as “luminescence‐detectable” (n = 89) and “luminescence‐undetectable” (n = 101). Thereafter, the luminescence of the semen samples that had been obtained from the 715 infertile patients was measured and compared by using Sperm Motility Analyzing System measurements. Moreover, in order to investigate the ROS measurement consistency, the chemiluminescence values of 84 samples were measured concurrently by using the Monolight™ 3010 Luminometer and the 1251 Luminometer™. Results The semen volume, sperm motility, and progressive motility of the samples were significantly higher in the luminescence‐undetectable samples. The sperm motility, straight‐line velocity, curvilinear velocity, mean amplitude head displacement, beat cross frequency, and progressive motility showed an inverse correlation with the logarithmic‐transformed luminescence level in the luminescence‐detected samples. The integrated chemiluminescence levels in the 84 samples were correlated. Conclusion The substance that was measured in the unprocessed semen with the Monolight™ 3010 Luminometer and stimulated chemiluminescence is ROS.

decline in sperm motility. 5,6 Furthermore, ROS infiltrate into sperm and break down the sperm DNA. 7 These adverse effects of ROS result in a decrease in the natural pregnancy rate 8 and the fertilization rate of assisted reproductive technology. 9 As a result of a negative correlation between the ROS levels with sperm motility and fertilization, the ROS levels in semen could serve as an independent marker of male factor infertility, particularly in cases of idiopathic infertility. 10,11 Numerous studies concerning ROS in semen have been reported. [6][7][8][9][10][11][12] Chemiluminescence assays are widely adopted in ROS measurement. 6,8,[12][13][14][15][16][17] Constant chemiluminescence after the addition of 5-a mino-2,3-dihydro-1,4-phthalazine-dione (luminol) to unprocessed or washed semen is measured with a luminometer. The authors previously had measured ROS in semen samples by using the 1251 Luminometer ™ (LKB Wallac, Turku, Finland); however, cuvettes were unavailable for ROS measurement with the 1251 Luminometer ™ . Nevertheless, it was possible to obtain the Monolight ™ 3010 Luminometer (BD Biosciences Pharmingen, Ltd., San Diego, CA, USA) and the necessary cuvettes.
To the authors' knowledge, no previous study has measured ROS in semen by using the Monolight ™ 3010 Luminometer. Therefore, ROS were measured in the unprocessed semen samples of infertile male patients by using this device. This study aimed to determine whether ROS in whole semen samples could be measured with the Monolight ™ 3010 Luminometer. The semen analyses were performed two or three times before treatment with the Sperm Motility Analyzing System (SMAS ™ ; DITECT, Ltd., Tokyo, Japan). After measurement of the semen volume with a 10 mL serological pipet (FALCON ® ; Corning, Tewksbury, MA, USA), the following parameters were measured by using the SMAS ™ : sperm concentration (×10 6 /mL); sperm motility (%); straight-line velocity (VSL) (μm/s) (measured as the straight-line distance from beginning to end of a sperm track divided by the time taken); curvilinear velocity (VCL) (μm/s) (measured as the total distance traveled by a given sperm divided by the time elapsed); linearity index (LIN), or the ratio of VSL to VCL; mean amplitude of lateral head displacement (ALH) (μm) (measured as the mean width of sperm head oscillation); beat cross frequency (BCF) (Hz), defined as the frequency of the sperm head crossing the average sperm path; and progressive motility (%), or the fraction of spermatozoa that progress at a rate >25 μm/s in liquefied, unprocessed semen.

| Measurement of the chemiluminescence of the semen samples with the Monolight ™ 3010 Luminometer
During each patient's first consultation, immediately after the semen analysis, the results of the chemiluminescence of the sufficiently liquefied semen samples, using the Monolight ™ 3010 Luminometer, a double-tube luminometer, at room temperature (~25°C) in a slightly dark laboratory room, were recorded after the addition of 40 μL of 100 mmol L -1 luminol to 500 μL of whole semen. 16 First, the integrated chemiluminescence between 0 and 200 seconds, without the addition of luminol, was measured. Subsequently, the integrated chemiluminescence over a similar period of time after the addition of luminol to the samples was measured. The integrated chemiluminescence value was calculated as the difference between after and before the addition of luminol to the semen samples. When the integrated luminescence value was negative, the value was defined as zero. When comparing the sperm motile parameter and the lumines-

| Comparison of the data measured with the 1251 Luminometer ™ and the Monolight ™ 3010 Luminometer
The luminometer was changed from the 1251 Luminometer ™ to the Monolight ™ 3010 Luminometer because cuvettes for ROS measurement with the former were unavailable. The chemiluminescence of the semen samples from 84 patients was measured by using both the 1251 Luminometer ™ and the Monolight ™ 3010 Luminometer in order to investigate the compatibility of chemiluminescence between the two devices. Moreover, only 84 cuvettes for the 1251 Luminometer ™ were available; hence, 84 samples were examined.
The 1251 Luminometer ™ was used to measure the whole-semen ROS level according to a previously reported method. 16 When the luminescence was ≥0.1 mv/s at peak value, ROS production in this sample was considered to be detectable ( Figure 1). Additionally, the integrated ROS values were used to clarify the differences between the ROS-detectable and the ROS-undetectable cases. The integrated ROS levels between 0 and 30 min after the addition of luminol were expressed as mV/30 minutes and considered as a new ROS level of the sample.
The integral ROS level, measured with both luminometers, was plotted on a graph and the correlation between the ROS levels, as measured by the two different luminometers, was investigated.

| Statistical analysis
The statistical values are presented as the mean ± standard deviation. A chi-square test was used to confirm bias in disease and the time-course-curve pattern, as well as disease and cases showing disease and emission values above the threshold. A Mann-Whitney U-test was used to compare the luminescence values and semen parameters in the luminescence-detectable and -undetectable groups.
Correlations between the log (luminescence value) and the semen parameter, measured with the SMAS ™ , and those between the luminescence values that had been measured with the two luminometers were investigated by using Spearman's correlation coefficient. Differences were considered to be statistically significant when the P-value was ≤.05. All the calculations were performed with the IBM SPSS statistics for Macintosh software (v. 22.0; IBM Corporation, Armonk, NY, USA).

| Time course of chemiluminescence
When the chemiluminescence was measured with the Monolight ™ 3010 Luminometer, the luminescence level increased for several seconds and then decreased rapidly before the addition of luminol. When multiple measurements were performed on the same specimen, the chemiluminescence values were almost the same (data not shown).
Therefore, the data that were measured by using this study's method were reproducible and hence chemiluminesence measurement was performed once after the semen analysis. Moreover, the relationship between the time-course-curve pattern and the chemiluminescence value was investigated by using 190 sample data and the time course was recorded from the start of the study. The patients were diagnosed as having idiopathic infertility (n = 55), untreated varicocele (n = 80), spermatogenic failure due to cancer chemotherapy (n = 19), treatment of undescended testis (n = 2), or other causes (n = 6) and 29 had an infertile female partner. These 190 samples were divided into three groups, according to the time-course pattern after the addition of luminol. The luminescence level increased rapidly and then decreased slowly for 100-150 s in Group A (n = 62). The integral luminescence value of the samples in this group was 51.62 ± 166.89-fold higher than that observed before the addition of luminol. The luminescence level of the second sample group (Group B, n = 27) increased for several seconds and did not decrease during the measurement. The integral luminescence value of the samples in this group was 24.61 ± 31.84fold higher than that measured before the addition of luminol. In Group C (n = 101), although the peak value increased, the pattern of the time course was similar to that before the addition of luminol ( Figure 2).
The integral luminescence value was 1.68 ± 0.57-fold higher than that measured before the addition of luminol (Table 1). Table 2

| Determinants of the threshold level of chemiluminescence
When the measured luminescence values were arranged in order from the lowest, all the samples exhibiting a luminescence value of F I G U R E 1 Reactive oxygen species (ROS), as measured by the chemiluminescence method with a 1251 Luminometer ™ . 16 When the peak level was ≥.1 mv/s, the ROS formation was considered to be positive. The integral level of ROS production was calculated by subtracting the area under the baseline from the total chemiluminescence values, between 0 and 30 minutes after the addition of 40 μL of 100 mmol of luminol to 500 μL of unprocessed semen, and expressed as mV/30 min 10 6 spermatozoa F I G U R E 2 Time course of luminescence in the unprocessed semen samples. Before the addition of 40 μL of 100 mmol of luminol, the luminescence value increased for several seconds, before decreasing rapidly. After the addition of luminol, a rapid increase and then a slow decline in the luminescence level occurred in Group A, a rapid increase and then the maintenance of luminescence during the measuring time occurred in Group B, and a course similar to that observed before the addition of luminol, but with a slight increase in the integral luminescence level, occurred in Group C    (Figure 7).

| Correlation of the luminescence levels measured by the 1251 Luminometer ™ and the Monolight ™ 3010 Luminometer
The chemiluminescence values of the 84 samples were measured concurrently with the two luminometers and plotted to determine whether a correlation between the devices existed. The integrated luminescence level in the 84 semen samples, as measured by the Monolight ™ 3010 Luminometer, was strongly correlated with that measured by the 1251 Luminometer ™ (conversion formula: y = 31.974X + 1769.6; P < .001, R = 0.824) (Figure 8).

| DISCUSSION
Oxidative stress is one of the major factors that could result in male infertility. 2,3 A study reported that several male patients with infertility of unknown cause had OS. 17 Moreover, testicular damage due to OS is induced by male infertility diseases, such as varicocele and cryptorchism, 18 and by exposure to chemicals, such as anticancer drugs, 18 heavy metals, 19 and phthalates. 18 Numerous studies on the relationship between ROS in semen and sperm motility, as well as fertility, have been published.
The methods of measuring OS in semen include the following: (i) a direct assay that measures the amount of ROS, including chemiluminescence, electron spin resonance, nitroblue tetrazolium test, and thiobarbituric acid assay; and (ii) an indirect assay that measures the effect of OS, including the measurement of antioxidants, lipid peroxidation, and DNA damage. 11 Of these methods, the chemiluminescence assay is used widely to measure ROS in semen. [6][7][8][13][14][15]18,20 To  (Table 2; P = .065). This finding could be because F I G U R E 6 Chemiluminescence (mean ± standard deviation) value above the threshold level of the samples in each group of patients F I G U R E 5 Number of patients with a chemiluminescence value greater or less than the threshold level. The patients are categorized according to the cause of their infertility. P < .001, according to the chi-square test the dose of luminol (100 mmol L -1 , 40 μL) in the current study was higher than that in other reports. 6,8,[13][14][15]21 The authors assumed that increasing the dose of luminol would make ROS detection clearer and the authors adopted the luminol dose that was reported by a specific study. 16 However, in this study's results, the percentage of the semen samples in Groups A and B were 31.9 and 14.1%, respectively. In all the patients, the percentage of samples that had greater than the threshold level was 44.1%. These percentages were almost similar to those in other reports. Moreover, Figure 3 shows that the ranges of logarith-

Conflict of interest:
The authors declare no conflict of interest. Human and Animal Rights: All the procedures that were followed were in accordance with the ethical standards of the responsible committees on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. This article does not contain any study with animal participants that has been performed by any of the authors.