Spectroscopic Probes of Energy Regulation & Metabolism (SPERM): Using High Resolution MR spectroscopy to identify biomarkers of male fertility

Approximately 20% of young men have poor quality semen and in 50% of couples undergoing in vitro fertilisation (IVF) there is a problem with the sperm from the male partner. Whilst often the problem is because the male has a low sperm count, sometimes it is because his sperm don't swim very well. In such cases, sperm can be injected into his partner's eggs to help fertilisation happen (a process called Intra-cytoplasmic Sperm Injection or ICSI). But this adds to the cost of infertility treatment and is not always funded by the NHS. Currently there are no effective therapies to improve poor sperm motility, but to be able to give sperm a 'boost' might avoid the need for high-tech procedures such as ICSI in preference to less technical ones, or could avoid the need for assisted conception altogether if the therapy was very successful.

Unfortunately we know very little about the way that sperm generate the energy necessary for their tail to beat and power them forwards. There are a number of ways they could do this and a range of different 'fuels' they could burn. Therefore, in this project, we propose to use some of the techniques developed by radiologists to examine the energy pathways used by cancer cells to develop new chemotherapy treatments. These techniques rely on detecting the molecular signature of atoms when they are placed in a magnetic field and are very similar to the technology used by doctors to scan a patient to see inside them (MRI). We have already developed a method to prepare sperm and place them inside the scanner and pilot data suggests that we can see key molecules involved in the production of sperm energy. In addition, we have also added some labelled 'fuels' to the sperm to watch how they use them. This suggests our techniques work and now we plan to undertake a more comprehensive analysis of sperm from different men.

We will obtain semen from men with different fertilities and prepare their sperm to be scanned under different conditions. We will examine whether we can see differences in the molecules used to make and provide sperm energy between men whose sperm swim well and those whose sperm swim badly. In addition, we will also attempt to separate good swimming sperm from poor swimming sperm in the same man, to see if we can detect differences. If we can, then this may lead to two clinical developments.

First, it may allow us to develop a better diagnostic test of sperm motility that is easier and cheaper to perform than the current method. At the moment, in order to diagnose male infertility related to poor sperm motility, a scientist has to observe the motility of sperm using a light microscope. This is labour intensive, prone to error and has developed little since the 1950s. A new test based on the presence or absence of one of more metabolite signals (or combination of signals) could improve this and lead to cost savings for the NHS.

Second, if our techniques are able to identify deficiencies in the sperm energy molecules, this opens up the possibility of undertaking further research to devise potential cures. We suggest that this might be possible through one or more of the following four ways: (a) developing drugs that could be taken by the man or woman in the weeks and months prior to conception; (b) advice about specific foods or nutritional supplements to enhance sperm motility; (c) the development of sperm-motility stimulating lubricants or pessaries that could be used by the couple during sex; or (d) sperm-motility stimulating culture media that could be used in IVF and other assisted conception technologies.

In conclusion, we propose that our research has the potential to provide a better understanding of how sperm make and use energy to swim. By filling that gap in knowledge, and by developing a new collaboration between fertility specialists and radiologists, we think we have the potential to radically change the diagnosis and treatment of male infertility.

This project aims to use the techniques of high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy to examine human sperm energy metabolism. The aim is to shed new light on an age-old debate of how sperm are appropriately fuelled and whether dysfunctions of sperm metabolism are contributing to the male infertility related to poor sperm motility (asthenozoospermia). We propose using the following techniques:

(i) High Resolution Magic Angle Spinning (HR-MAS) 1H NMR spectroscopy at 9.4T of sperm pellet in a zirconium rotor and spun at a frequency of 3-6 KHz at an angle thetam = 54.74 degrees to the magnetic field direction;

(ii) High Resolution NMR spectroscopy at 9.4T of sperm incubated with 13C labelled substrates including 13C1-pyruvic acid, 13C2-pyruvic acid, 13C1-lactate, 13C5-glutamine, 13C1-6,d7-glucose, 13C2-fructose, and 13C1-butyric acid (fatty acid analogue); and

(iii) Dissolution Dynamic Nuclear Polarisation enhanced 13C NMR spectroscopy using a HyperSense DNP polariser linked to a 7T Bruker MRI/S system to examine relatively short-term metabolic changes in sperm.

For each technique, sperm will be obtained from healthy male volunteers (aged 18 to 65) and their motility assessed by computer to quantify the proportion and speed of motile sperm (and define asthenozoospermia in a robust way). Sperm will then be isolated from semen using a two-step 45/95% (v/v) Percoll gradient suspended in phosphate buffered saline and then removing any residual leukocytes by subjecting sperm found both in the pellet and at the 45/95 interface to magnetic cell sorting with CD45 Dynabeads.

For each of our objectives, we will scan sperm from at least 6 different males to compare both between and within male differences in metabolite concentrations, metabolism of labelled substrates and real time metabolic kinetics. Metabolites will be identified from spectra using the dedicated NMR processing packages (Bruker Topspin) and custom software (Matlab).

Our proposed research into human sperm metabolism has the possibility to impact on six potential beneficiaries:

(i) The wider public (including but not limited to couples attempting conception or those where infertility has been diagnosed);

(ii) Quangos and government agencies involved in the regulation (e.g. the Human Fertilisation and Embryology) or delivery (e.g. the National Health Service) of fertility treatments, or the development of clinical guidelines (e.g. the National Institute for Health and Clinical Excellence or the many Clinical Commissioning Groups in England or their equivalents in Wales, Scotland and Northern Ireland) or around the world.

(iii) Non-governmental organisations concerned with fertility and reproductive medicine such as the "United Nations Development Programme / United Nations Population Fund / United Nations Children's Emergency Fund / World Health Organisation / World Bank Special Programme of Research, Development and Research Training in Human Reproduction" (otherwise known as the WHO Human Reproduction Programme).

(iv) Voluntary Organisations and Charities involved in fertility (e.g. Infertility Network UK, British Fertility Society, European Society of Human Reproduction and Embryology, International Federation of Fertility Societies);

(v) The media (e.g. the news media but also those involved in the production of science programmes or radio and television documentaries and lifestyle magazines); and

(vi) Businesses and industries with diagnostic products for the fertility market (e.g. Microm Ltd) or marketing pharmacological (e.g. Merck Serono Ltd) or putative nutritional (e.g. Sigma Tau, the makers of Proxeed Plus) agents designed to enhance fertility, or the development of culture media for use in human IVF (e.g. Cook Medical Inc).

The examples given in each case are used to highlight the type of organisation that may benefit from the research but is not designed to be exhaustive and a more definitive list will be generated once the results of the project become available. However, it is anticipated that the above might benefit from the proposed research in the following three ways:

(a) The results may allow us to develop new methods to diagnose male infertility. Current methods rely upon a scientist observing the motility of sperm using a light microscope. This is labour intensive, prone to error and has developed little since the 1950's. A new test based on the presence or absence of one of more metabolite signals (or combination of signals) could improve this and be marketed to diagnostic laboratories or IVF clinics. This could generate global revenues as well as lead to cost savings in current laboratory services and efficiencies to clinical pathways.

(b) If it is established that deficiencies in sperm metabolic pathways are a cause of asthenozoospermia, this opens up the possibility of undertaking further research to devise potential therapies. We envisage four possible routes of therapy including the development of: (a) sperm-specific pharmacological agents to be taken by the man (or woman) in the weeks and months prior to conception; (b) combinations of specific foods or nutritional supplements to enhance sperm motility; (c) sperm-motility enhancing lubricants or pessaries to be used during coitus; or (d) sperm-motility enhancing culture media for use in IVF and other assisted conception technologies. Again, these could generate global revenues to companies in the fertility sector as well as cost savings to healthcare budgets if fertility could be enhanced and the need for assisted conception avoided.

Finally, there is the potential for broader societal and public benefits to this research given that the media and public appear to have a thirst for new research findings in reproductive medicine. Therefore, interest in our project could be used as a platform to deliver important general health messages about male and female fertility.