Oxidative stress, sperm DNA fragmentation and outcome of assisted reproduction

Abstract

The aim of this thesis was to determine whether the addition of dithiothreitol (DTT) treatment and LIVE/DEAD cell differential staining in the current protocols used for assessment of sperm DNA fragmentation and oxidative DNA damage, will improve the detection of sperm DNA damage. Investigations were also undertaken to determine the levels of oxidised nucleic acids in seminal plasma and blood serum as indicators of local and systemic oxidative stress; and to explore the relationships between these parameters and the outcome of assisted reproduction. A total of 92 patients undertaking their third cycle or less for either an Intrauterine Insemination (IUI) or an In vitro Fertilisation/Intra-Cytoplasmic Sperm Injection (IVF/ICSI) treatment were recruited for the study. Assessment of percentage sperm DNA fragmentation was performed using the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate (dUTP) nick-end labeling (TUNEL) assay in all cases, using an existing protocol that was implemented in a clinical setting by Dr Laura Surmon. The assessment of oxidative DNA damage was determined using the percentage 8-oxo-7, 8- dihydro-2' deoxyguanosine (8OHdG) as the biomarker, a fluorescence-labeled 8OHdG- specific protein and a protocol designed by Dr Laura Surmon. The method involved taking approximately 200I¼l of spermatozoa prepared for treatment that had a concentration of at least 10 million/ml. Both assays were performed simultaneously using the current protocol assay with the updated protocol assay i.e. the addition of DTT and LIVE/DEAD stain for sperm DNA fragmentation and oxidative sperm DNA damage. Seminal plasma was also collected and used for mass spectrometry (MS) analysis as an indicator of the levels of oxidative stress local to the male reproductive tract. Although seminal plasma contains very low levels of DNA when compared to spermatozoa itself, isolating DNA and RNA in sperm cells was not necessary since the TUNEL and 8OHdG assays already assess this. The study was focusing on isolating the fluid component in particular, to localise the source of oxidative stress. Furthermore, a sample of blood was also collected from the male on the day of treatment and the serum isolated and used for MS to determine levels of systemic oxidative stress. Of the 92 patients recruited for the study only 78 were included. Sample size was restricted due to a number of male patients not consenting to providing a blood sample on the day of treatment and some samples containing RNA and/or DNA at levels below that detectable by MS. Of the 78 patients however, 25 patients had an IUI cycle and 53 patients underwent an IVF/ICSI cycle. There were no significant differences between the IUI and IVF/ICSI treatment groups in terms of male or female age, or the presence of a significant female infertility factor or abnormal semen parameters on the day of treatment. There were no significant differences between the current and updated assay protocols which included DTT and LIVE/DEAD stain in terms of percentage sperm DNA fragmentation (TUNEL) or oxidative sperm DNA damage (8OHdG). However there was a noticeable effect when DTT was applied in the updated assay, which seemed to have made the sperm heads swell and therefore identifying each sperm cell became more complex. Further work needs to be done to experimentally determine how DTT may best be incorporated into the assays to increase access to DNA damage while also limiting the disruption to the spermatic cell membrane and thus maintaining overall cell and assay, integrity. The percentage of sperm DNA damage as determined using the TUNEL assay was found to be significantly lower on the day of treatment as compared to the time of initial semen analysis. It is possible that the course of antioxidants prescribed by the medical director, has led to a reduction in the degree of DNA damage in the form of DNA strand breaks by the time of treatment. Such reductions may therefore mean that the less invasive IUI cycle may become an option for previously unsuitable patients. This study has also demonstrated a significant correlation between seminal plasma levels of oxidised nucleic acids and sperm DNA fragmentation (r = 0.457, p < 0.05), which suggests that sperm DNA damage may be due to oxidative stress in the male reproductive tract rather than systemic oxidative stress as addressed in results. Ongoing investigations need to be done to confirm these findings and further explore the role of 8OHdG and sperm DNA fragmentation in fertility and their impact on the outcome of assisted reproduction. In terms of the outcome of assisted reproduction following IUI (n = 25), the mean percentage sperm DNA fragmentation as determined using the current TUNEL assay protocol was significantly lower for those couples achieving a live birth (2.5% vs 10.3%, p < 0.001). Through receiver operating characteristic (ROC) analysis, a threshold of 3.5% sperm DNA fragmentation was shown to have a good sensitivity and specificity for predicting an outcome of a live birth following an IUI in this subset of patients. A threshold

Date of Award	2016
Original language	English