Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men Jolanta Guz1, Daniel Gackowski1, Marek Foksinski1, Rafal Rozalski1, Ewelina Zarakowska1, Agnieszka Siomek1, Anna Szpila1, Marcin Kotzbach2, Roman Kotzbach3, Ryszard Olinski1* 1 Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland, 2 Gynecology practice, Specialist medical practice, Bydgoszcz, Poland, 3 Department of Obstetric Nursing, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland Abstract Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG) and 8-oxo-7,8dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods. The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/106 dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues. Citation: Guz J, Gackowski D, Foksinski M, Rozalski R, Zarakowska E, et al. (2013) Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men. PLoS ONE 8(7): e68490. doi:10.1371/journal.pone.0068490 Editor: W. Steven Ward, University of Hawaii at Manoa, John A. Burns School of Medicine, United States of America Received April 25, 2013; Accepted May 29, 2013; Published July 12, 2013 Copyright: ß 2013 Guz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Polish state funds for science for years 2010-2013, grant N N407 171439. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: DNA in sperm and that observed in surrogate tissue (leukocytes) which represents other cells in the body. When investigating a possible link between oxidative stress and abnormal spermatozoa it is important to apply an appropriate biomarker of oxidative stress. The most popular way of exploring oxidative stress includes measures of oxidative DNA damage which can be assessed by determination of 8-oxodG level in cellular DNA. It is also believed that lymphocytes are surrogate cells, which should inform about oxidative stress - measured as a certain level of 8-oxodG - in other tissues [6,7]. In addition, the whole body burden of oxidative stress may be assessed by the determination of urinary excretion of oxidatively modified bases/ nucleosides [8,9]. Therefore, in the present study, for the first time the broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the groups of fertile and infertile subjects. These parameters include: (i) 8oxodG level in DNA isolated from leukocytes and spermatozoa; (ii) 8-oxodG and 8-oxoGua levels in urine; (iii) antioxidant vitamins (A, C and E) and uric acid. Low molecular weight antioxidants are regarded as effective free radical scavengers, therefore they can protect biomolecules such as DNA either directly or indirectly. Introduction One of the major factors which define fertility is the quality of spermatozoa and it has been recently estimated that one in twenty men exhibits some form of defect in sperm quality, which in turn may be involved in male factor infertility which is responsible for half of all infertility cases [1]. There is considerable evidence that oxidative stress plays a major role in the etiology of male infertility. Thus, abnormal spermatozoa frequently display typical features of oxidative stress i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity [2–4]. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility [5]. Despite significant progress in the last decade regarding a possible role of oxidative stress in the etiology of defective sperm function and male infertility some important issues, outlined below, have not yet been addressed: (i) whether oxidative stress in patients with abnormal spermatozoa is restricted to semen only, or whether the whole organism is oxidatively stressed as well; (ii) whether there is any association between oxidatively damaged PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e68490 PLOS ONE | www.plosone.org 2 60.0 (52.5–64.5) 20.5 (14.0–30.0) 0.56 (0.26–1.10) Total motility (%) Sperm morphology/ normal forms (%) Round cells concentration (106/ml) Values are expressed as median and interquartile range. doi:10.1371/journal.pone.0068490.t001 41.0 (35.5–49.5) Sperm concentration (106/ml) Progressive motility (%) 37.09 (23.99–63.85) Age 113.90 (74.59–181.40) 33.0 (28.0–35.5) Parameter Total sperm number (106/ejaculate) Control group/ normozoospermia n = 32 0.42 (0.16–0.80) 18.0 (12.0–22.0) 44.0 (29.0–58.0) 10.0 (3.0–19.0) 42.25 (16.72–118.45) 15.94 (8.02–41.85) 33.0 (28.0–36.0) All n = 91 Patients 0.54 (0.29–0.97) 19.0 (13.0–22.0) 44.0 (30.0–59.0) 11.0 (5.0–17.0) 110.36 (58.5–188.80) 40.00 (21.12–61.70) 31.0 (28.0–36.0) Asthenozoospermia n = 47 Table 1. Characteristic of subjects’ age and semen qu (...truncated)