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Exploring the electrochemical detection of 8-oxoguanine within human sperm

Shehzad, Mahaah (2017) Exploring the electrochemical detection of 8-oxoguanine within human sperm. Masters thesis (MSc), Manchester Metropolitan University.

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Abstract

The major causes of male factor infertility is poor sperm quality and DNA damage. Sperm DNA damage can impair sperm function, fertilization rates and is associated with increased miscarriage rates. Over the past decade, there has been increasing interest in the role of sperm nuclear DNA integrity in male factor infertility. Excessive production of reactive oxygen species (ROS) can cause irreversible DNA damage and form DNA lesions. The most common DNA lesion is 7,8- Dihyrdo-8-Oxoguanine (8-oxoGuanine), which derives from the oxidation of Guanine. Measuring DNA damage in sperm is becoming an important procedure to assess sperm quality. Development of methods which quantify the level of 8-oxoGuanine (HPLC, LC-MS) and detect the level of DNA fragmentation (Comet) in a biological sample have become an area of interest. Recent technology (screen-printed electrodes- SPEs), has demonstrated a simple and cost-effective alternative of measuring 8-oxoGuanine. This study aimed to investigate the use of SPEs, in the detection of 8-oxoGuanine in sperm DNA and seminal plasma, and to correlate degree of DNA fragmentation via the comet assay. These findings may enable future diagnostic techniques. Sperm cells were separated from their seminal plasma, treated with a concentration gradient of H2O2. Sperm cells underwent DNA extraction and were stored for analysis via the comet assay. This study focused inducing oxidative damage, assessing DNA fragmentation in sperm cells and measuring the level of 8-oxoguanine in extracted sperm DNA and seminal plasma. A significant difference was found in between the DNA % in tail and increasing concentrations of H2O2 in the comet assay. There was no detection of 8-oxoguanine in H2O2 treated sperm DNA, however 8-oxoguanine was detectable in seminal plasma when using the SPEs. Further work using the SPEs to detect 8-oxoguanine in a biological matrix is required.

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