Robert John Aitken

DNA damage to spermatozoa has impacts on fertilization and pregnancy.

DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.

Redox Regulation of Human Sperm Function: From the Physiological Control of Sperm Capacitation to the Etiology of Infertility and DNA Damage in the Germ Line

Defective sperm function is the largest single defined cause of human infertility and one of the major reasons we are witnessing an exponential increase in the uptake of assisted conception therapy in the developed world. A major characteristic of defective human spermatozoa is the presence of large amounts of DNA damage, which is, in turn, associated with reduced fertility, increased rates of miscarriage, and an enhanced risk of disease in the offspring. This DNA damage is largely oxidative and is closely associated with defects in spermiogenesis. To explain the origins of this DNA damage, we postulate that spermiogenesis is disrupted by oxidative stress, leading to the creation of defective gametes with poorly remodeled chromatin that are particularly susceptible to free radical attack. To compound the problem, these defective cells have a tendency to undergo an unusual truncated form of apoptosis associated with high amounts of superoxide generation by the sperm mitochondria. This leads to significant oxidative DNA damage that eventually culminates in the DNA fragmentation we see in infertile patients. In light of the significance of oxidative stress in the etiology of defective sperm function, a variety of antioxidant therapies are now being assessed for their therapeutic potential.

Oxidative stress and male reproductive health

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair (BER) pathway, 8-oxoguanine glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.

Stimulation of mitochondrial reactive oxygen species production by unesterified, unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P<0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.

Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion.

The First International Conference on Male-Mediated Developmental Toxicity, held in September 1992, reported that the spermatozoon can bring genetic damage into the oocyte at fertilization and thereby contribute to subsequent abnormal pregnancy outcomes (Olshan and Mattison, 1994). At that time, laboratory tests for genetic defects in sperm were at an early stage of development and were relatively untested in the clinic and the field. A breakout group at that meeting discussed the need for improved sperm biomarkers of adverse reproductive effects and concluded that sensitive, reliable, and practical methods

New insights into the molecular mechanisms of sperm-egg interaction

Abstract.At the moment of insemination millions of mammalian sperm cells are released into the female reproductive tract in order to find a single cell – the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilisation, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, that surrounds the oocyte and initiate the chain of cellular interactions that will culminate in fertilization. These exquisitely cell- and species-specific recognition events are among the most strategically important cellular interactions in biology. Understanding the cellular and molecular mechanisms that underpin them has implications for diagnosis of the aetiology of human infertility and the development of novel targets for fertility regulation. Herein, we describe two models indicating the plethora of highly orchestrated molecular interactions underlying successful sperm zona binding and sperm oocyte fusion.

Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.

Outcomes for Patients with Ischaemic Stroke and Atrial Fibrillation: The PRISM Study (A Program of Research Informing Stroke Management)

Background: In the past decade the prevalence of atrial fibrillation (AF) has been increasing in ageing populations while stroke prevention and management have advanced. To inform clinician practice, health service planning and further research, it is timely to reassess the burden of AF-related ischaemic stroke. Methods: We identified patients aged 18+ years with a primary or stay diagnosis of ischaemic stroke (ICD-10-AM I63.x), from July 1, 2000 to June 30, 2006, using an administrative health dataset of all hospitalisations in New South Wales (population ∼7 million). Fact of death was determined to December 2007. Results: Of the 26,960 index cases of ischaemic stroke, 25.4% had AF recorded during admission. Median age for AF and non-AF patients was 80.4 and 75.2 years, respectively (p < 0.001). Mortality was significantly higher in patients with AF at 30 days (19.4 vs. 11.5%), 90 days (27.7 vs. 15.8%) and 365 days (38.5 vs. 22.6%) (p values <0.0001). Adjusting for age and co-morbidities reduced these differences, with 90-day mortality of 20.9% in AF patients versus 14.7% in non-AF patients (p value <0.0001). The effect of AF on outcomes appears stronger in younger stroke patients relative to patients without AF (p valueinteraction <0.0001). At 30 days, the relative risk of mortality due to AF was 3.16 (95% CI 1.92–5.25) amongst those younger than 50, 1.71 (95% CI 1.32–2.22) in patients aged 50–64 years, 1.39 (95% CI 1.16–1.66) in patients aged 65–74 years, 1.29 (95% CI 1.17–1.43) in those aged 75–84 years, and 1.23 (95% CI 1.13–1.33) in those aged 85+ years. AF patients, surviving admission, spent a median of 19.2 days (95% CI 18.4–20.1) in hospital compared with 14.5 days (95% CI 13.9–15.1) for patients without AF (p < 0.001), with differences in length of stay greatest in younger patients (p valueinteraction <0.0001). 90-Day stroke survivors with AF spent an average of 21.5 days (95% CI 20.6–22.4) in hospital versus 16.6 days (95% CI 15.9–17.2) in those without AF. AF patients accessed more in-hospital rehabilitation (36.6%; 95% CI 35.0–38.2) than patients without AF (31.8%; 95% CI 31.0–32.7) (p value <0.0001), and differences in the proportion of AF versus non-AF patients accessing rehabilitation was greatest in younger patients (p valueinteraction <0.0006). Conclusions: Ischaemicstroke patients with AF have substantially worse outcomes than patients without AF, which can be partly explained by older age and greater co-morbidities. We have quantified the large effect of AF in younger patients and our results strongly argue for new antithrombotic research in young AF patients.

The presence of a truncated base excision repair pathway in human spermatozoa that is mediated by OGG1

Summary DNA repair has long been considered impossible in human spermatozoa due to the high level of DNA compaction observed in these cells. However, detailed examination of the base excision repair pathway in human spermatozoa has revealed the presence of an enzyme critical to this pathway, 8-oxoguanine DNA glycosylase 1 (OGG1). This glycosylase was associated with the sperm nucleus and mitochondria and could actively excise 8-hydroxy-2′-deoxyguanosine (8OHdG), releasing this adduct into the extracellular space. This activity was significantly reduced in the presence of cadmium (II), a recognized inhibitor of OGG1, in a time- and dose-dependent manner (P<0.001). Remarkably, spermatozoa do not possess the downstream components of the base excision repair pathway, apurinic endonuclease 1 (APE1) and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1). The absence of these proteins was particularly significant, as APE1 is required to create a 3′-hydroxyl (3′-OH) terminus at the apurinic site created by OGG1, which would be recognized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. As a result, TUNEL was unable to detect oxidatively induced DNA damage in spermatozoa following exposure to hydrogen peroxide. In the same cells, intracellular and extracellular 8OHdG could be clearly detected in a manner that was highly correlated with the outcome of the sperm chromatin structure assay (SCSA). However, incubation of these cells for 48 hours revealed a time-dependent increase in TUNEL positivity, suggesting the perimortem activation of a nuclease. These results emphasize the limited capacity of mature spermatozoa to mount a DNA repair response to oxidative stress, and highlight the importance of such mechanisms in the oocyte in order to protect the embryo from paternally mediated genetic damage.

The Paradoxical Relationship Between Stallion Fertility and Oxidative Stress

ABSTRACT The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using “dismount” sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2′-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 69.6%) and acrosome integrity (40.2% vs. 50.1%) than those that did not. We hypothesized that the most fertile spermatozoa exhibited the highest levels of oxidative phosphorylation (OXPHOS), with oxidative stress simply being a by-product of intense mitochondrial activity. Accordingly, an experiment to investigate the relationship between oxidative stress and motility was conducted and revealed positive correlations between mitochondrial ROS and total motility (R2 = 0.90), rapid motility (R2 = 0.89), average path velocity (VAP; R2 = 0.59), and curvilinear velocity (VCL; R2 = 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R2 = 0.46), rapid motility (R2 = 0.51), average path velocity (R2 = 0.62), and VCL (R2 = 0.56), supporting the aforementioned hypothesis. The relative importance of OXPHOS in supporting the motility of equine spermatozoa was contrasted with human spermatozoa, which primarily utilize glycolysis. In this study, mitochondrial inhibition significantly reduced the velocity (P < 0.01) and ATP (P < 0.05) content of equine, but not human, spermatozoa, emphasizing the formers relative dependence on OXPHOS. The equine is the first mammal in which such a positive relationship between oxidative stress and functionality has been observed, with implications for the management of stallion fertility in vitro and in vivo.