Preventing aneuploidy in oocytes; mechanisms, markers and their potential use in clinic

Currently 15% of couples are infertile, largely as a result of an error that originates in women when the immature oocyte divides to produce an egg. Many couples now rely on IVF to have children. However, this is often a long, distressing and expensive procedure that does not always produce a live birth. We aim to relieve these pressures by developing treatment strategies that will significantly improving IVF success rates.

In a perfect division, the oocyte aligns all of its chromosomes accurately so that they may be divided equally between the future egg, and a much smaller polar body which is a waste product of this division. Unfortunately, this process fails frequently in human oocytes and eggs are often produced with an incorrect number of chromosomes, a condition known as aneuploidy. This error is the primary genetic reason for failed pregnancy, miscarriage and babies born with developmental disabilities. Indeed, nearly half of all miscarriages are aneuploid.

In complete contrast to human oocytes, the equivalent division is not error prone in young mice and the majority of their oocytes divide normally, producing perfect eggs. My previous research has identified the existence of a mechanism which acts in the mouse oocyte to prevent aneuploidy. I now wish to understand how this mechanism, and other related mechanisms act, so that we may determine why human oocytes are let down through these processes. This information will be of significant benefit to the treatment of infertility.

To achieve this, my team will uncover the molecular basis of key interactions between proteins involved in preventing aneuploidy in mouse oocytes. Following this we will use a novel, cutting edge technology to measure the levels of many proteins involved in regulating cell division in single oocytes. This will be carried out in both mouse oocytes and in human oocytes and will uncover how the balance of these proteins changes in populations of oocytes that commit frequent errors. This information has the potential to identify ways in which we may alter the protein balance in human oocytes so that they are less likely to produce aneuploid eggs, improving IVF success rates.

Using mouse oocytes, we will take this a step further, only analysing the protein content of the oocytes waste polar body while fertilising the egg. By this method we can score resulting embryos for a number of indicators of health and directly relate this score back to protein content of the polar body. This strategy will identify protein markers of oocyte viability in polar bodies and, will pave the way to developing this strategy for use in human IVF, providing a quick and cost effective way for IVF clinicians to select the embryo most likely to produce a healthy baby.

This research is important for the following reasons:

- The UK spends ~£350,000,000 per year on 70,000+ rounds of IVF, these numbers are increasing.
- Even in younger women, most will undergo at least 3 cycles of IVF before a live birth, this number is often many more.
- Clinical depression, grief, anxiety-related illness, and relationship problems and are all firmly associated with infertility and its treatment. Indeed, psychological symptom scores in female patients suffering infertility are equivalent to other chronic medical conditions such as cancer.

Taking into account the factors above, even modest improvements, either by improving success rates, or by allowing women to make earlier choices, will have enormous, global impact.

My research concerns cell cycle protein regulation in mammalian oocytes, in particular cyclin B1-CDK1 activity. In prometaphase, sufficient cyclin B1-CDK1 activity must be maintained while cells align their chromosomes. If chromosomes segregate before they are aligned, daughter cells may become aneuploid (contain incorrect chromosome numbers). Human oocytes are frequently aneuploid, this is the primary genetic cause of failed pregnancy, miscarriage and developmental disabilities in babies.

My research has uncovered a novel mechanism of cyclin B1-CDK1 activity protection that prolongs prometaphase and so prevents aneuploidy in the oocytes of young mice. We find that an excess of free cyclin B1 is destroyed in preference to CDK1-bound cyclin B1 by a previously unidentified degron. Free cyclin B1 thereby acts as a decoy, occupying the cellular destruction machinery so that cyclin B1-CDK1 activity is preserved for long enough to correctly align chromosomes, preventing cell division errors in these oocytes.

I aim to determine the molecular mechanism by which free cyclin B1 is preferentially destroyed, and to investigate whether this mechanism becomes compromised in aged mouse oocytes where aneuploidy rates increase significantly. I will then determine the contribution of this mechanism in preventing aneuploidy in human oocytes, using imaging mass cytometry (IMC) to simultaneously analyse cyclin B1 levels relative to proteins involved in chromosome alignment. This will reveal proteins which are aberrantly expressed in error prone oocytes, identifying potential points of intervention. In mouse I will take this further, analysing the waste polar body (PB) of the oocyte by IMC while fertilising the oocyte, relating subsequent embryo health to the protein content of the PB; developing a non-destructive protocol able to identify the most viable embryo for transfer following IVF.

Infertility is a substantial world health problem and its treatment requires substantial financial input. In developed countries, approximately 15% of couples suffer infertility (ref. 1). In the UK we currently spend ~£350,000,000 per year on more than 70,000 cycles of IVF. Approximately 1/3rd of this is attributed to NHS spend and 2/3rds through personal funding. Furthermore, largely due to the trend toward delaying childbirth, the number of couples seeking assisted reproductive therapies (ARTs) increases each year.

The vast majority of infertility and IVF failures are of oocyte origin and involve an event which generates an oocyte with an incorrect number of chromosomes; aneuploidy. Indeed, nearly half of all miscarriages are aneuploid. The research generated in this proposal will impact upon both our understanding of the mechanisms involved in preventing aneuploidy, and will inform on ways in which we may improve IVF success rates by grading the viability of oocytes selected for transfer in ARTs.

The overall success rate of IVF for all ages is currently ~65% after 6 cycles, however many women undergo many more cycles without success. Even in younger women, most will undergo at least 3 cycles of IVF before a live birth. Alongside this, and importantly, clinical depression, grief, anxiety-related illness, and relationship problems are all firmly associated with infertility and its treatment. Global psychological symptom scores in female patients suffering infertility are equivalent to other chronic medical conditions such as cancer (ref. 2). Any improvement which alleviates the distress and strain of infertility, either by improving success rates, or by allowing women to make earlier choices, is likely to have global impact.

Specifically, we aim to develop a protocol (using the oocytes waste polar body) able to inform on embryo viability. This will be achieved by generating a unique map of up to 60 different proteins involved in chromosome alignment and cell cycle progression in both mouse and human oocytes, identifying protein markers associated with the embryo health. In addition, our strategy will identify proteins which are aberrantly expressed in oocytes undergoing aneuploidy. This information could be used to rescue oocytes from committing errors so that they may be used for IVF.

At present, only subjective morphological criteria are used to select the single embryo for transfer in ARTs. By identifying markers of oocyte competency, we may place a numerical value on the viability of each individual oocyte, reducing the time, cost and physical health problems associated with infertility and its treatment by reducing the number of IVF cycles necessary to produce a pregnancy. Importantly, we may also identify situations that are not compatible with life, revealing errors that are likely to result in repetitive failed pregnancies (for example mutations in key proteins). Where interventions are not possible, women could be advised to engage in alternatives at an earlier stage, again limiting both physical, psychological and financial stress.

1. Sharma, R., et al., Lifestyle factors and reproductive health: taking control of your fertility. Reproductive biology and endocrinology 1477-7827-11-66 (2013).
2. Domar, A., et al., The psychological impact of infertility: a comparison with patients with other medical conditions. J Psychosom Obstet Gynaecol 14, 45-52 (1993).