Table 2.
Relevant cohort studies assessing/validating Luminol, MiOXSYS and OxiSperm (NBT).
| Reference | Population | Assay | Primary Finding |
|---|---|---|---|
| [31] Homa, S.T.; Vessey, W.; Perez-Miranda, A.; Riyait, T.; Agarwal, A. Reactive oxygen species (ros) in human semen: Determination of a reference range. Journal of assisted reproduction and genetics 2015, 32, 757–764. | Men attending routine semen analysis; n = 94 normal semen parameters, n = 100 abnormal semen parameters but low leucocytes and n = 41 any semen parameters with leucocytospermia |
Luminol | Significantly different between Groups 1, 2 and 3 19.75 ± 8.12, 95.03 ± 33.63 and 890.17 ± 310.23 RLU/sec/106 sperm, respectively Cut off < 24.1 RLU/sec/106 (specificity 87.2%, sensitivity 80.5%) |
| [32] Ochsendorf, F.R.; Thiele, J.; Fuchs, J.; Schüttau, H.; Freisleben, H.J.; Buslau, M.; Milbradt, R. Chemiluminescence in semen of infertile men. Andrologia 1994, 26, 289–293. | n = 49 consecutive infertile men, and n = 20 controls | Luminol | ROS cut off 1.5 × 105 counted photons per min−1/2 × 106 sperm |
| [33] Athayde, K.S.; Cocuzza, M.; Agarwal, A.; Krajcir, N.; Lucon, A.M.; Srougi, M.; Hallak, J. Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J. Androl. 2007, 28, 613–620. | n = 114 fertile men seeking vasectomy and n = 47 subfertile men | Luminol | Without leucocytes ROS cut off of 5 × 103 counted photons per min/2 × 105 sperm (Specificity 76.0%, sensitivity 73.3%) With leucocytes ROS cut off of 1.25 × 104 counted photons per min/2 × 105 sperm (Specificity 66.7%, sensitivity 71.9%) |
| [34] Fingerova, H.; Oborna, I.; Novotny, J.; Svobodova, M.; Brezinova, J.; Radova, L. The measurement of reactive oxygen species in human neat semen and in suspended spermatozoa: A comparison. Reproductive Biology and Endocrinology 2009, 7, 118. |
n = 91 infertile men and n = 34 men with proven fertility |
Luminol | ROS = 0.26 RLU/103 proven fertile vs. 1.1 RLU/103 for semen abnormalities |
| [35] Desai, N.; Sharma, R.; Makker, K.; Sabanegh, E.; Agarwal, A. Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil. Steril. 2009, 92, 1626–1631. |
n = 54 infertile men and n = 51 fertile donors |
Luminol | ROS ≥ 1.85 × 10 counted photons per min/2 × 105 sperm highly predictive of infertility (77.8% sensitivity and 82.4% specificity) |
| [36] Venkatesh, S.; Shamsi, M.B.; Dudeja, S.; Kumar, R.; Dada, R. Reactive oxygen species measurement in neat and washed semen: Comparative analysis and its significance in male infertility assessment. Archives of gynecology and obstetrics 2011, 283, 121–126. | n = 65 infertile men with abnormal semen parameters, n = 17 infertile with normal semen parameters and n = 43 fertile controls | Luminol | ROS = 3.44 × 104 RLU/min/20 million sperms for men with abnormal semen parameters vs. 7.9 × 103 RLU/min/20 million for infertile normal semen and 3 × 102 RLU/min/20 million for fertile controls |
| [37] Agarwal, A.; Tvrda, E.; Sharma, R. Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reprod. Biol. Endocrinol. 2014, 12, 45. | n = 79 tetratozoospermic men and n = 56 healthy donors | Luminol | ROS cut off of 93 RLU/sec/106 sperm (specificity of 70.4% and sensitivity of 61.4%) |
| [38] Novotny, J.; Aziz, N.; Rybar, R.; Brezinova, J.; Kopecka, V.; Filipcikova, R.; Reruchova, M.; Oborna, I. Relationship between reactive oxygen species production in human semen and sperm DNA damage assessed by sperm chromatin structure assay. Biomedical papers 2013, 157, 383–386. | n = 39 men from infertile couples and n = 23 fertile men | Luminol | Control group = 2.92 (2.32, 3.60), normospermia = 3.78 (3.09, 4.40) and semen abnormality = 4.02 (3.79, 4.29) log RLU/min/2 × 105 sperm |
| [39] Agarwal, A.; Ahmad, G.; Sharma, R. Reference values of reactive oxygen species in seminal ejaculates using chemiluminescence assay. J. Assist. Reprod. Genet. 2015, 32, 1721–1729. |
n = 92 controls and n = 258 infertile men |
Luminol | ROS cut off of 102.2 RLU/sec/106 sperm (sensitivity 76.4% and specificity 53.3%) |
| [40] Agarwal, A.; Sharma, R.K.; Sharma, R.; Assidi, M.; Abuzenadah, A.M.; Alshahrani, S.; Durairajanayagam, D.; Sabanegh, E. Characterizing semen parameters and their association with reactive oxygen species in infertile men. Reprod. Biol. Endocrinol. 2014, 12, 33. |
n = 56 fertile donors and n = 318 infertile men |
Luminol | ROS cut off of 91.9 RLU/sec/106 sperm (sensitivity 93.8%, specificity 68.8%) |
| [41] Vessey, W.; Perez-Miranda, A.; Macfarquhar, R.; Agarwal, A.; Homa, S. Reactive oxygen species in human semen: Validation and qualification of a chemiluminescence assay. Fertility and sterility 2014, 102, 1576–1583.e1574. | n = 23 semen samples from 19 men attending semen analysis | Luminol | No significant intra-or inter assay variation Working reagents stable for 3 months ROS measurements in samples are not stable and decline immediately after ejaculation |
| [42] Zorn, B.; Vidmar, G.; Meden-Vrtovec, H. Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection. Int. J. Androl. 2003, 26, 279–285. | n = 147 male partners of infertile couples (41 IVF and 106 ICSI) | Luminol | High ROS classified as 10 mV/sec/109 sperm observed in 43% of men Log ROS negatively correlated with fertilization above 25%; however, this was lost after adjusting for female and cycle characteristics Log ROS negatively correlated with embryo morphology after day 4 after multiple regression analysis Negative effect of ROS on pregnancy rates after IVF but not with ICSI |
| [43] Majzoub, A.; Arafa, M.; Mahdi, M.; Agarwal, A.; Al Said, S.; Al-Emadi, I.; El Ansari, W.; Alattar, A.; Al Rumaihi, K.; Elbardisi, H. Oxidation-reduction potential and sperm DNA fragmentation, and their associations with sperm morphological anomalies amongst fertile and infertile men. Arab journal of urology 2018, 16, 87–95. | n = 1168 infertile men and n = 100 fertile from general population and infertility clinics | MiOXSYS | ORP = 1.73 mV/106/mL (sensitivity 76% and 56% specificity) |
| [44] Agarwal, A.; Roychoudhury, S.; Sharma, R.; Gupta, S.; Majzoub, A.; Sabanegh, E. Diagnostic application of oxidation-reduction potential assay for measurement of oxidative stress: Clinical utility in male factor infertility. Reproductive biomedicine online 2017, 34, 48–57. | n = 106 infertile men and n = 51 fertile men | MiOXSYS | Cut-off value of 1.39 mV/106/mL (sensitivity 69.6% and specificity 83.1%) |
| [45] Agarwal, A.; Sharma, R.; Roychoudhury, S.; Du Plessis, S.; Sabanegh, E. Mioxsys: A novel method of measuring oxidation reduction potential in semen and seminal plasma. Fertil. Steril. 2016, 106, 566–573.e10. | n = 33 infertile men and n = 26 fertile men from the general population | MiOXSYS | Cut-off value of 1.48 mV/106/mL in semen (sensitivity 60% and specificity 75%) and 2.09 mV in seminal plasma (sensitivity 46.7% and specificity 81.8%) |
| [46] Agarwal, A.; Wang, S.M. Clinical relevance of oxidation-reduction potential in the evaluation of male infertility. Urology 2017, 104, 84–89. |
n = 194 infertile men and n = 29 men with repeat semen analysis |
MiOXSYS | Cut-off value of 1.57 mV/106/mL to detect one semen defect (sensitivity 70.4%, specificity 88.1) Cut-off value of 2.59 mV/106/mL for detecting oligozoospermia (sensitivity 88%, specificity 91.2%) |
| [47] Vassiliou, A.; Martin, C.H.; Homa, S.T.; Stone, J.; Dawkins, A.; Genkova, M.N.; Skyla Dela Roca, H.; Parikh, S.; Patel, J.; Yap, T.; et al. Redox potential in human semen: Validation and qualification of the miox(sys) assay. Andrologia 2021, 53, e13938. | n = 286 men undergoing routine semen analysis, and n= 854 samples for luminol validation | MiOXSYS and Luminol | No relationship between luminol RLU sec/106 and sORP mV/106/mL. A number of samples classified as low for MiOXSYS (<1.34 mV/106/mL) were classified as high ROS by luminol (cut off value of 13.8 RLU/sec/106, 86% sensitivity and 86% specificity). MiOXSYS was reproducible across operators, analyzers and days. |
| [48] Agarwal, A.; Du Plessis. SS.; Sharma, R.; Samanta, L.; Harlev, A.; Ahmad, G.; Gupta, S & Sabanegh, ES. Establishing the oxidation-reduction potential in semen and seminal plasma. Fertil Steril 2015, 104, e146. | n = 18 fertile men | MiOXSYS | Cut-off = 4.73 mV/106/mL (sensitivity = 100%, specificity = 89.5%) in sperm and 4.65 mV/mL (sensitivity = 100%, specificity = 93.8%) in seminal plasma |
| [49] Agarwal, A.; Panner Selvam, M.K.; Arafa, M.; Okada, H.; Homa, S.; Killeen, A.; Balaban, B.; Saleh, R.; Armagan, A.; Roychoudhury, S., et al. Multi-center evaluation of oxidation-reduction potential by the mioxsys in males with abnormal semen. Asian journal of andrology 2019, 21, 565–569. | n = 2092 men attending for semen analysis from 9 countries | MiOXSYS | Cut-off 1.34 mV/106/mL (sensitivity 98.1% and specificity 40.6%) |
| [50] Panner Selvam, M.K.; Henkel, R.; Sharma, R.; Agarwal, A. Calibration of redox potential in sperm wash media and evaluation of oxidation-reduction potential values in various assisted reproductive technology culture media using mioxsys system. Andrology 2018, 6, 293–300. | (i) ENHANCE WG (Vitrolife, San Diego, CA, USA); (ii) Quinn’s™ Sperm Washing Medium (SAGE, In-Vitro Fertilization, Inc., Trumbull, CT, USA); and (iii) one sperm cryopreservation medium (Freezing Medium; Test Yolk buffer, Irvine Scientific, CA, USA). | MiOXSYS | ORP (mV) in sperm prep media = 267.3 mV |
| [51] Panner Selvam, M.K.; Agarwal, A.; Henkel, R.; Finelli, R.; Robert, K.A.; Iovine, C.; Baskaran, S. The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic. Biol. Med. 2020, 152, 375–385. | n = 66 fertile men | MiOXSYS | sORPmV > 1.48 mV/106/mL or < 9.76 mV/106 |
| [52] Sallam, N.; Hegab, M.; Mohamed, F.; El-Kaffash, D. Effect of oxidative stress in semen, follicular fluid and embryo culture medium on the outcome of assisted reproduction. AIMJ 2017, 2, 59–65. | n = 50 couples with unexplained infertility undergoing IVF and ICSI | MiOXSYS | Cut off sORPmV < 1.57 mV/106/mL for fertilization and < 0.75 mV/106/mL for clinical pregnancy |
| [53] Iommiello, V.M.; Albani, E.; Di Rosa, A.; Marras, A.; Menduni, F.; Morreale, G.; Levi, S.L.; Pisano, B.; Levi-Setti, P.E. Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells. Int. J. Endocrinol. 2015, 2015, 321901. | n = 56 infertile men | OxiSperm in relation to DFI | L3 or L4 of semen oxidative stress correlated with DFI ≥ 30% |
| [54] Gosálvez, J.; Coppola, L.; Fernández, J.L.; López-Fernández, C.; Góngora, A.; Faundez, R.; Kim, J.; Sayme, N.; de la Casa, M.; Santiso, R., et al. Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress. Reproductive biomedicine online 2017, 34, 513–521. | n = 707 infertile men | OxiSperm | 76% participants categorised as L2 (medium), only 4% L3 (high) and 20% L1 (low) |
| [55] Tunc, O.; Thompson, J.; Tremellen, K. Development of the nbt assay as a marker of sperm oxidative stress. International journal of andrology 2010, 33, 13–21. |
n = 21 fertile and n = 36 infertile men |
NBT-reactivity | Cut-off = 24 μg formazan/107 sperm (sensitivity 91.7%, specificity 81.0%) |
| [56] Esfandiari, N.; Sharma, R.K.; Saleh, R.A.; Thomas, A.J., Jr.; Agarwal, A. Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa. Journal of andrology 2003, 24, 862–870. |
n = 21 infertile men and n = 9 healthy donors |
NBT-reactivity | NBT positive sperm increased in samples with leucocytes present. Cut-off = 19% (sensitivity of 100% and specificity 86.4%) |
| [57] Amarasekara, D.S.; Wijerathna, S.; Fernando, C.; Udagama, P.V. Cost-effective diagnosis of male oxidative stress using the nitroblue tetrazolium test: Useful application for the developing world. Andrologia 2014, 46, 73–79. | n = 102 subfertile and n = 30 proven fertile men | NBT-reactivity | Cut-off = 42.02 μg formazan/107 sperm (sensitivity 71.4% and specificity 70%) |
| [58] Pujol, A.; Obradors, A.; Esteo, E.; Costilla, B.; García, D.; Vernaeve, V.; Vassena, R. Oxidative stress level in fresh ejaculate is not related to semen parameters or to pregnancy rates in cycles with donor oocytes. Journal of assisted reproduction and genetics 2016, 33, 529–534. | n = 132 infertile men | OxiSperm | 43.2% were in high oxidative stress (L3) and 30.3% were low (L2) and 25.0% very low (L1) No association between oxidative stress and fertilization rate, embryo morphology or pregnancy rates |
| [59] Degirmenci, Y.; Demirdag, E.; Guler, I.; Yildiz, S.; Erdem, M.; Erdem, A. Impact of the sexual abstinence period on the production of seminal reactive oxygen species in patients undergoing intrauterine insemination: A randomized trial. The journal of obstetrics and gynaecology research 2020, 46, 1133–1139. | n = 90 infertile men | OxiSperm | Increased pigment staining related to higher ROS levels in 70% of samples with ejaculation length >4 days vs. 50% for 3–4 days abstinence and 43.3% for 0–2 days abstinence. |
| [60] Javed, A.; Talkad, M.S.; Ramaiah, M.K. Evaluation of sperm DNA fragmentation using multiple methods: A comparison of their predictive power for male infertility. Clinical and experimental reproductive medicine 2019, 46, 14–21. |
n = 50 infertile and n = 50 fertile men |
OxiSperm | Fertile group L1 (low), 39%; L2 (low–medium); 24%; L3 (medium), 11%; and L4 (high), 36%. Infertile group L1 (low), 16%; L2 (low-medium), 11%; L3 (medium), 31%; and L4 (high), 42%. No correlations with male infertility (specificity 0.32, sensitivity 0.99) |