Biological Significance & Mechanisms of Alkyl DNA Damage in Human Sperm and Fertility

Abstract

Infertility is a global concern affecting approximately one-third of all couples trying to conceive, with 50% of cases associated with male factor infertility, and of those diagnosed, ~33% are categorised with unexplained infertility. The population is exposed to a multitude of potentially harmful compounds, both in the work and social environment, increasing the chance for detrimental effects upon our health, including fertility. One class of such compounds are N-nitrosamines, found in tobacco, beer, drinking water and food. N-nitrosamines can be broken down by cytochrome p450 enzymes to produce alkylating agents capable of interacting with our DNA with the potential to cause DNA damage and subsequent mutations. A cohort of 248 men attending the Department of Reproductive Medicine at St Mary’s Hospital (Manchester, UK) were asked to complete a questionnaire on occupational and social exposures, including smoking & drinking habits, diet and exercise routines. Semen analysis data and assisted reproductive technology outcome data was collected for each patient, to determine associations between exposures and semen parameters or birth outcomes. The polyamines spermine and spermidine and amino acid glycine, all previously shown at high concentrations in seminal plasma, were chosen as potential endogenous sources of N-nitrosamines following nitrosation by dietary nitrites or reactive oxygen/reactive nitrogen species. The DNA model 4-(4-nitrobenzyl-pyridine) and plasmid DNA were used to determine the potential for spermine and glycine to act as alkylating agents, causing DNA damage, following nitrosation. Potential metabolite adducts of N-nitrosated spermine, spermidine and glycine, were synthesised within 23basepair oligodeoxyribonucleotides at the O6 position of guanine in order to determine whether such adducts could be recognised and repaired by the protein O6-methylguanine-DNA-methyltransferase. Statistical analysis identified several associations between exposures and semen parameters or ART outcomes including a negative association between male age and semen volume, negative correlations between men eating red meat and the number of embryos which reached transfer and the number of pregnancies. Spermine and glycine were shown to produce alkylating agents following nitrosation and induce single-strand breaks, and of the nine oligodeoxyribonucleotides synthesised all nine were recognised and repaired by O6-methylguanine-DNA-methyltransferase. This work identifies two possible sources of endogenous alkylating agents found in high concentrations within seminal plasma, with the potential to induce DNA damage, mutations or single-strand breaks within spermatozoa, which could affect male fertility. Questionnaire data supports previous associations found within other studies while identifying several new associations.

Date of Award	3 Feb 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Povey (Supervisor) & Daniel Brison (Supervisor)