Development of Normal Reference Values for Seminal Reactive Oxygen Species and Their Correlation With Leukocytes and Semen Parameters in a Fertile Population

Pós-Graduação Divisão de Urologia, University of Sao Paulo, Rua Dr Eneas de Carvalho Aguiar 255 7 andar, ICHC-CEP: 05422-970 Sao Paulo, SP, Brazil.
Journal of Andrology (Impact Factor: 2.47). 02/2007; 28(4):613-20. DOI: 10.2164/jandrol.106.001966
Source: PubMed


Although reactive oxygen species (ROSs) are clearly implicated in the pathogenesis of male infertility, few studies have attempted to define the basal levels of ROSs in fertile men. Levels of ROSs are highly influenced by the presence of leukocytes and are associated with decreased seminal parameters. The objective of our study was to determine the normal ROS reference values in neat and washed semen of a fertile population and to correlate the leukocyte concentrations with seminal parameters. We evaluated 114 fertile men seeking vasectomy and 47 subfertile patients as a positive control. All samples were subjected to semen analysis and Endtz testing; chemiluminescence assay was used to determine ROS levels. All seminal parameters were significantly higher in the fertile men than in the subfertile patients. In nonleukocytospermic samples, ROS levels were lower in the fertile men than in the subfertile patients in neat (0.29 [0.18, 0.54] vs 0.94 [0.38, 1.51]) (P = .001) and washed semen (5.73 [1.90, 14.71] vs 23.4 [9.46, 115.55]) (P = .001). Similarly, in samples with leukocytes (Entdz, less than 1 x 10(6)/mL), ROS levels were lower in the fertile men in neat (0.75 [0.27, 1.71] vs 2.0 [0.97, 27.41]) (P = .001) and washed semen (15.85 [4.18, 62.16] vs 239.83 [33.4, 1193.75]) (P < .0001). As expected, samples with leukocytes had significantly higher ROS values in washed and neat semen. In the fertile population, ROSs were positively correlated with leukocytes and negatively correlated with sperm count and motility. In semen samples without leukocytes, the normality cutoff of ROSs was 0.55 x 10(4) counted photons per minute with 76.4% area under the curve (AUC) in the neat samples and 10.0 x 10(4) counted photons per minute with 77% AUC in the washed samples. In semen samples with leukocytes, the cutoff for ROSs in neat samples was 1.25 with 72.7% AUC and 51.5 with 81% AUC in the washed samples. We defined the cutoff levels of ROSs in a fertile population. Seminal leukocyte levels below 1 x 10(6)/mL were associated with increased ROSs. ROS levels were positively correlated with leukocytes and negatively correlated with sperm motility and concentration. Patients with normal seminal parameters and lower seminal leukocyte levels may benefit from therapeutic interventions that improve semen quality.

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    • "A negative correlation between ROS production and percentage of normal sperm, concentration, and motility has been previously demonstrated [27,28]. We have also reported similar results in a fertile population [27]. However, the correlation between ROS and semen parameters among proven fertile donors who have fathered a child within the past 2 years and infertile patients has not been studied. "
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    ABSTRACT: Background A routine semen analysis is a first step in the laboratory evaluation of the infertile male. In addition, other tests such as measurement of reactive oxygen species can provide additional information regarding the etiology of male infertility. The objective of this study was to investigate the association of semen parameters with reactive oxygen species (ROS) in two groups: healthy donors of unproven and proven fertility and infertile men. In addition, we sought to establish an ROS cutoff value in seminal plasma at which a patient may be predicted to be infertile. Methods Seminal ejaculates from 318 infertile patients and 56 donors, including those with proven fertility were examined for semen parameters and ROS levels. Correlations were determined between traditional semen parameters and levels of ROS among the study participants. ROS levels were measured using chemiluminescence assay. Receiver operating characteristic curves were obtained to calculate a cutoff value for these tests. Results Proven Donors (n = 28) and Proven Donors within the past 2 years (n = 16) showed significantly better semen parameters than All Patients group (n = 318). Significantly lower ROS levels were seen in the two Proven Donor groups compared with All Patients. The cutoff value of ROS in Proven Donors was determined to be 91.9 RLU/s with a specificity of 68.8% and a sensitivity of 93.8%. Conclusions Infertile men, irrespective of their clinical diagnoses, have reduced semen parameters and elevated ROS levels compared to proven fertile men who have established a pregnancy recently or in the past. Reactive oxygen species are negatively correlated with traditional semen parameters such as concentration, motility and morphology. Measuring ROS levels in the seminal ejaculates provides clinically-relevant information to clinicians.
    Full-text · Article · May 2014 · Reproductive Biology and Endocrinology
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    • "(54). An earlier study (n=161) established that patients with normal seminal parameters and lower seminal leukocyte levels might benefit from this type of therapeutic intervention to lower ROS levels and improve semen quality (55). Oxidative stress is relevant for female fertility as well (56). "
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    ABSTRACT: Male factor infertility or subfertility is responsible for up to 50% of infertility cases. A considerable body of recent studies indicates that lifestyle as well as environmental and psychological factors can negatively affect male fertility, more than previously thought. These negative effects have been shown in many cases to be reversible. This review aims to provide a rationale for early clinical attention to these factors and presents a non-exhaustive evidence-based collection of primary relevant conditions and recommendations, specifically with a view to making first line diagnostics and recommendations. The presently available evidence suggests that considering the high cost,success rates, and possible side effects of assisted reproduction techniques (ART), such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), early efforts to improve male fertility appear to be an attainable and worthwhile primary goal.A series of searches was conducted of Medline, Cochrane and related databases from November 14th, 2010 to January 26th, 2012 with the following keywords: male, fertility, infertility, sperm defects, IVF, ICSI, healthy habits, and lifestyle. Subsequent follow-up searches were performed for upcoming links. The total number of studies contemplated were 1265; of these, 296 studies were reviewed with criteria of relevance; the date of study or review; study sample size and study type;and publishing journal impact status. Data were abstracted based upon probable general clinical relevancy and use. Only a selection of the references has been reflected here because of space limitations. The main results obtained were evidence-supported indications as to the other causes of male infertility, their early detection, and treatment.
    Full-text · Article · Mar 2013 · International Journal of Fertility and Sterility
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    • "Indeed, to investigate the role of OS in sperm biology and in general in male infertility many tools can be employed. These include evaluation of ROS production (Athayde et al. 2007) and/or the ability to counteract oxidative agents by enzyme and non-enzyme systems (Pasqualotto et al. 2008). In addition several studies investigated OS indirectly by revealing its noxious effects on sperm structure and function (Li et al. 2004), such as the formation of 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage. "
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    ABSTRACT: Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.
    Full-text · Article · Mar 2013 · Reproduction
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