Measuring Reactive Oxygen Species in Semen for Male Preconception Care: A Scientist Perspective

Abstract

Oxidative stress and elevated levels of seminal and sperm reactive oxygen species (ROS) may contribute to up to 80% of male infertility diagnosis, with sperm ROS concentrations at fertilization important in the development of a healthy fetus and child. The evaluation of ROS in semen seems promising as a potential diagnostic tool for male infertility and male preconception care with a number of clinically available tests on the market (MiOXSYS, luminol chemiluminescence and OxiSperm). While some of these tests show promise for clinical use, discrepancies in documented decision limits and lack of cohort studies/clinical trials assessing their benefits on fertilization rates, embryo development, pregnancy and live birth rates limit their current clinical utility. In this review, we provide an update on the current techniques used for analyzing semen ROS concentrations clinically, the potential to use of ROS research tools for improving clinical ROS detection in sperm and describe why we believe we are likely still a long way away before semen ROS concentrations might become a mainstream preconception diagnostic test in men.

Keywords: ART; fertility; pregnancy; seminal plasma; sperm.

Figure 1

Sources of ROS generated exogenously and endogenously in sperm. At low levels, ROS contributes to successful fertilization by increasing membrane fluidity through cholesterol exudation and through tyrosine phosphorylation of target proteins required for fertilization. Certain ROS species directly inhibit tyrosine phosphatases and others contribute to the activation of cyclic adenosine monophosphate (cAMP), leading to protein kinase A activation and phosphorylation of target proteins.

Figure 2

(a) ROS formed within the cell starts as superoxide (O₂⁻) created from ATP generation in mitochondria and from NADPH oxidase (NOX), and superoxide dismutates, facilitated by the protein superoxide dismutase (SOD) into hydrogen peroxide (H₂O₂). Hydrogen peroxide can then be neutralized by catalase (CAT) to create water (H₂O) and singlet oxygen (O₂). However, in the presence of ferrous iron it will react, creating hydroxyl radical (OH⁻). (b) If there is nitric oxide (NO⁻) present, then it will react to create peroxynitrite (ONOO⁻), a potent but unstable oxidant. (c) These ROS contribute to sperm damage through oxidation of the guanine base in DNA, creating single strand breaks, double strand breaks and DNA fragmentation, or through its oxidation of polyunsaturated fatty acids, creating lipid radicals that self-propagate, resulting lipid peroxidation.

Figure 3

Reducing sperm mitochondrial superoxide concentrations with 100 µM MnTBAP in a mouse model of in vitro H₂O₂ exposure significantly impairs 2-cell embryo development. (a) Proportion of sperm with progressive motility; (b) Sperm mitochondrial superoxide mean fluorescence intensity (MFI) (MitoSox Red, (MSR) a specific mitochondrial superoxide indicator); (c) Proportion of 2-cells 24 h post insemination with 10,000 sperm and (d) Linear regression of sperm superoxide concentrations and 2-cell cleavage rates (grey squares: 100 µM MnTBAP, white circles: 3000 µM H₂O₂). Data is representative of 4 CBAF1 males (represented by different symbols on graphs) and 24 super ovulated 3–4 weeks old CBAF1 females. Female mice were super ovulated with 5IU of PMSG followed 48 h with 5IU of HCG. Sperm was collected from male mice 15 h post HCG and incubated in 3000 µM of H₂O₂ in G-IVF PLUS (Vitrolife) for 1 h at 37 °C, 5% O₂ and 6% CO₂. Sperm samples were split into two groups and either incubated in (1) Control: 3000 µM of H₂O₂ in G-IVF PLUS or (2) 100 µM of MnTBAP: 3000 µM of H₂O₂ in G-IVF PLUS for a further 1 h at 37 °C, 5% O₂ and 6% CO₂. Cumulus oocyte complexes (COC) were collected from female mice 15.5 h post HCG. Sperm were washed of their treatments after backfilling with 3 mL of G-IVF PLUS by centrifugation 400× g for 5 min. COCs were inseminated with 10,000 sperm 16.5 h post HCG. Following a 4 h fertilization, zygotes were washed and cultured in G1 PLUS (Vitrolife) for 24 h at 37 °C, 5% O₂ and 6% CO₂ at which 2-cell embryo cleavage rates were assessed. Prior to insemination, sperm progressive motility was assessed in 200 sperm per sample and superoxide concentrations assessed after 30 min incubation at 37 °C of 5µM of MSR and 10,000 sperm assessed on a FACS Canto II. Data was analysed by a paired t-test or a simple linear regression showing the slope and 95% confidence intervals.