David Miller

Profile

Having completed my PhD in dental biochemistry in 1982, I went on to study cell cycle proteins and microtubule dynamics in the slime mould, Physarum before moving to Liverpool University. There, I began a career-long interest in heat shock proteins and their ability to protect cells against environmental stress. Initially, this work used the eggs and larvae of the brine shrimp Artermia salina (the eggs are very tolerant to desiccation), which I continued following my appointment as senior lecturer in biochemistry in Liverpool John Moores University 

Research interests

I am interested in the molecular and genetic aspects of male infertility and reproductive function and the underlying causes of male factor infertility and reproductive dysfunction, the great majority of which, has unknown causes. Using wholly non-invasive methods that make use of ejaculate spermatozoa, we are compiling an RNA database that describes the fertile male and comparing this with RNA profiles obtained from the semen of infertile men. In this way, we aim to identify gene expression pathways that are affected in the infertile male. At the same time, if we know what RNAs are present in the spermatozoon, we can identify, by comparison with the RNAs that are present in the egg, those that are specific to and delivered by the sperm. Once we have identified these 'male' specific messages, we can begin assessing whether they have any function or influence on the developing embryo. In a similar vein, we are looking more closely at how chromatin is packaged in the spermatozoon. So far we have found that DNA in human and murine spermatozoa is packaged by a mixture of histones (as is the case with all somatic cells) and protamines (peculiar to spermatozoa). More interestingly, we have important evidence that different regions of the DNA are differentially packaged into these two compartments such that genic regions are enriched in the histone bound regions. This has important implications for male fertility because it suggests that some important stretches of DNA in sperm chromatin, containing particular gene sequences may be more susceptible to intrinsic or externally regulated damage, leading to sperm dysfunction. See Saida et al, 2011 and Arapanhi et al, 2009. This has extended into a major translational study looking at sperm selection for Intra-Cytoplasmic Sperm Injection (ICSI). 

The male contribution to human infertility is not fully understood and estimates of its prevalence vary. However, concern over the recent decline in sperm counts at least in the developed world, alongside the increasing age at which couples come forward for treatment, has led to calls for improvements in the care of the male partner. Such improvements include a better understanding of the causes of male infertility and how best to ameliorate the condition sufficiently to boost treatment success rates (hitherto focused mainly on boosting female fertility). With the advent, rapid uptake and expansion of interventional intracytoplasmic sperm injection (ICSI) treatment, the practitioner has only one chance per egg to pick the right sperm with the greatest potential for live birth for injection and methods aimed at increasing the likelihood of doing so are in development. One such method involves the selection of sperm based on their innate ability to bind hyaluronic acid (HA), which occurs naturally in the cumulus–oophorous complex. Such sperm appear to have better indicators of genomic integrity, including lower levels of deoxyribonucleic acid (DNA) fragmentation, chromosomal aneuploidy and cytoplasmic retention and, hence, increased maturity relating to these measures. A number of clinical trials have tested the claim that ICSI with hyaluronan selected sperm improves clinical outcomes, but with the exception of a reduction in miscarriage following the use of selected sperm, results for other outcomes, including live births have remained equivocal at best. Hyaluronic Acid Binding sperm selection (HABSelect) was designed to detect a minimum 5% difference (per cycle started) in full-term live birth outcomes, which was also sufficient to detect significant changes in other (secondary) outcomes including miscarriage rates.HABSelect was a parallel-arm, randomised clinical trial with associated laboratory-based studies investigating sperm DNA integrity (fragmentation and compaction). The intervention was based on sperm binding to the HA substrate in the Conformité Européenne (CE) and UK Medicines and Healthcare products Regulatory Agency- approved PICSITM dish (Origio, Måløv, Denmark). This substrate binds and immobilises sperm for ICSI. The study was as inclusive as possible with regard to both partners’ eligibility to participate. Approximately 6700 couples were assessed for eligibility and 2772 were randomised into either the selection (PICSI, n = 1387) or the control (standard ICSI, n = 1385) arm of the trial, although six couples were excluded post-randomisation. as they were subsequently found not to have met eligibility criteria. Following post-randomisation withdrawals, the number of couples included in the primary analysis was 2752. Following treatment, residual sperm samples were frozen and stored for retrospective analysis of DNA integrity using a number of complementary assays measuring variables for DNA fragmentation [acridine orange, comet and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)] and compaction [aniline blue (AB)] with HALO assays linking both variables.

Approximately 6700 couples were assessed for eligibility in HABSelect and 2772 were finally randomised into either the selection (PICSI) or control (standard ICSI) arms of the trial. Outcome data were available for 2752 couples. For the primary outcome, 379 out of 1381 (PICSI 27.4%) and 346 out of 1371 (ICSI 25.2%) of eligible couples randomised achieved a full-term live birth (≥ 37 weeks). This corresponds to an odds ratio for all treatment cycles of 1.12 [95% confidence interval (CI) 0.95 to 1.34], which was not statistically significant (p = 0.18). Of the secondary outcomes, miscarriage rates per couple treated were significantly reduced in the PICSI arm, with 60 out of 1381 (4.3%) clinical pregnancies lost per couple treated, compared with ICSI at 96 out of 1371 (7.0%), corresponding to an odds ratio for all treatment cycles of 0.61 (95% CI 0.43 to 0.84; p = 0.003). Clinical pregnancy rates (CPRs) per couple treated were not significantly different and subgroup analyses of both primary and miscarriage outcomes across hyaluronic acid binding score (HBS), female age, anti-Müllerian hormone or follicle stimulating hormone subgroups did not find a difference in treatment effect. DNA fragmentation in the sperm prepared for ICSI/PICSI was not discriminatory of clinical outcomes, although DNA compaction may have influenced establishment of CP. The mechanistic analysis, which explored the relationship between mechanistic and clinical data for the purposes of hypothesis generation, found statistically significant relationships between HBS, sperm motility, sperm concentration and sperm DNA integrity. Since the trial results were published (see Kirkman-Brown et al, 2019 and Miller et al, 2019), we have uncovered evidence that sperm DNA integrity is integral to the chances of successful treatment and that sperm selection with hyaluronan can eliminate almost 40% of miscarriages among older women. We believe our evidence links hyaluronan sperm selection with better paternal DNA quality and integrity. 

I was closely involved in the REPROTRAIN International Training Network, coordinated by Rafael Oliva in Barcelona with three projects based in Leeds. Two projects provided training for Early Stage Researchers (ESR) to undertake research in the composition and packaging of human and bovine sperm chromatin. With a focus on sperm quality based on both differential density gradient centrifugation and the ability to bind hyaluronic acid, one of these projects (ESR1) examined the RNA composition of 'competent' and 'incompetent' sperm using a combination of microarray and second generation RNA sequencing. Both mRNAs and miRNAs were investigated as part of this project and aspects of sperm RNA and protein turnover and synthesis on mitoribosomes were explored. A second ESR post looked at differences in DNA composition of histone packaged DNA in 'competent' and 'incompetent' sperm again focusing on their sedimentation characteristics and hyaluronan binding properties. We were particularly interested in exploring the relationship between sperm quality and histone retention, with regard to differential DNA packaging (into histone and protamine binding domains) and the work will also involve a combination of microarray and second generation DNA (exome) sequencing. Localising chromatin domains in sperm nuclei. A third post for a more experienced researcher (ER) explored the use of seminal plasma as a surrogate for assessing prostate health (ranging postatic intra-epithelian neoplasmia or PIN to full Gleason 7 prostate cancer and we are developing an RNA-based diagnostic for this purpose. 

Qualifications

• BSc (Hons)
• PhD

Professional memberships

• Treasurer and Company Secretary, Society for Reproduction and Fertility
• Treasurer, British Andrology Society.

Student education

I have been actively involved in student education throughout my career, most recently, on the twin Masters programmes in Clinical Embryology, including our one year, full-time residential and two year, part-time distance-learning programmes. I teach mainly on Andrology-related subjects (spermatogenesis and infertility) but I also teach sex determination and differentiation, maternal serum screening in prenatal diagnosis and the molecular genetics of early development. 

Research groups and institutes

• Leeds Institute of Cardiovascular and Metabolic Medicine
• Rare Diseases & Genetics