Date of Award


Document Type

Thesis - ECU Access Only


Edith Cowan University

Degree Name

Doctor of Philosophy


School of Medical and Health Sciences

First Supervisor

Associate Professor Peter Roberts

Second Supervisor

Dr Peter Burton

Third Supervisor

Dr Katherine Sanders


Overall, this doctoral study aimed to contribute to the existing knowledge and clinical practice regarding male infertility, especially in relation to the potential sources and clinical impact of sperm apoptosis and reactive oxygen species (ROS) in the context of assisted reproductive technology (ART).

This thesis encompasses three independent studies, each of which addresses a single aim related to different aspects of male infertility and ART practices.

Study 1 examined the relationship between sperm apoptosis and ROS levels and ART outcomes. On the day of oocyte retrieval, the levels of sperm apoptosis (measured using Annexin V staining) and intracellular ROS (measured using dihydroethidium staining) were measured in neat and processed semen samples from a total of 170 male patients whose partners were undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatment cycles. In general, higher levels of apoptosis and ROS were associated with lower sperm motility, sperm count and advanced paternal age. No correlation was observed between sperm apoptosis and fertilisation rate, blastulation rate, day 5 embryo quality (D5EQ) and clinical pregnancy. Similarly, there was no correlation between sperm ROS levels and fertilisation rate and clinical pregnancy. However, higher levels of ROS might be associated with poorer early stages of embryonic development, as indicated by blastulation rate and D5EQ. Together, the findings suggest that although sperm apoptosis and ROS had an adverse effect on sperm parameters, the effect on clinical outcomes was minimal.

Study 2 compared the efficacy of three sperm preparation methods, namely density gradient centrifugation (DGC), the swim-up method and SeaforiaTM, in reducing the levels of sperm apoptosis and ROS. The measurement of sperm apoptosis and ROS levels were performed on a total of 17 pooled semen samples before and after sperm preparation. The main findings suggest that all three methods effectively reduced sperm apoptosis levels in the neat samples. However, only swim-up and SeaforiaTM were effective in reducing the ROS levels. Compared with DGC and swim-up, SeaforiaTM also produced samples with a higher total yield.

Study 3 investigated the effects of different time intervals during ART sperm preparation on clinical outcomes. Three time intervals including pre-wash interval (time between ejaculation and sperm preparation), post-wash interval (time between sperm preparation and insemination/sperm injection) and total interval (time between ejaculation and insemination/sperm injection) were calculated and retrospectively analysed from a total of 8,079 IVF/ICSI cycles. The results showed that prolonged pre- and post-wash and total intervals had an adverse effect on sperm motility and clinical outcomes including fertilisation rate and pregnancy outcomes. Intracellular ROS production during prolonged incubation was considered to be the most likely facilitator of this observed effect.

In summary, this thesis demonstrates that although their effect on ART outcomes were at low levels, higher levels of sperm apoptosis and ROS were associated with poorer sperm parameters, and suggestive of, a lower chance of natural conception. The cumulative findings of this thesis are indicative of the central role of ROS levels in sperm physiological functions and suggest that minimising intracellular ROS production through sperm preparation techniques and time optimisation may be beneficial to improving ART outcomes