Testing The Health And Therapeutic Potential Of In Vitro Derived Oocytes For The Restoration Of Female Fertility And The Treatment of Infertility

The freezing and banking of very early-staged eggs within ovarian tissue has been successfully used to preserve the fertility of girls and young women who are likely to lose their ovarian function as a result of the destructive effects of cancer treatments. Small pieces of the ovary can be surgically recovered from patients and stored at liquid nitrogen temperatures for as long as required. When the patient wishes to start her family, her stored ovarian tissue is transplanted back into her body with aim of producing fertile oocytes and a pregnancy either naturally or with the help of assisted reproduction technologies such as in vitro fertilisation (IVF). Over 150 babies have now been born worldwide using this technology. However, the transplantation method for fertility restoration is not suitable for all cancer patients. For girls and women with blood diseases such as leukaemia, and/or cancers such as breast cancer there is a risk that the cancer will be restarted from the transplanted tissues. For these patients a safer option is to grow the early staged eggs from the frozen-thawed ovarian tissue to maturity in the laboratory- a technology called the in vitro growth and maturation (IVGM) of eggs. The laboratory grown eggs can then be fertilised using IVF so that healthy embryo(s) can be transferred to the patient to produce the desired pregnancy. These technologies can also be used treat healthy but infertile women who are undergoing assisted conception using donor eggs.
The development of methods to grow eggs from their earliest stages in the laboratory is very challenging. Never-the-less advances are being made towards this goal. It is now possible to produce full sized, mature eggs in the laboratory in both humans and ruminant species such as cows and sheep. Two different culture approaches are showing significant promise these are: (i) a fast growth system over 21 days (FasGro); and (ii) a slow growth system over 40-60 days (SloGro). The FasGro and SloGro systems have been developed in parallel for both human and ruminant eggs. However, it is not yet clear which of these methods is best at producing healthy eggs that can be fertilised to produce embryos for future transfer to produce a pregnancy and baby. Before new technologies such as IVGM can be used to restore the fertility of cancer survivors or to treat infertile women it is essential that they are rigorously tested in a suitable animal model to prove they are both safe and effective.
This project aims to test the potential of IVGM eggs to used as a treatment to restore fertility in women. Sheep ovary tissues will be used as a mimic for human ovaries. Specifically, the project will grow eggs in the laboratory from frozen-thawed sheep ovary tissues using the FasGro and SloGro methods. The project will test the health of the laboratory-grown eggs and their ability to be fertilised using IVF and to produce embryos that will implant and produce a pregnancy and live offspring. The nutritional and genetic fingerprints of laboratory-derived eggs and embryos will be combined with microscope imaging and used as health screens to confirm whether the IVGM eggs are normal when compared to similar cells grown naturally in the body. An embryo transfer trial will be conducted in sheep to establish which IVGM egg production method is most effective at delivering healthy lambs. Key measurements of health and development will be made in follow-up studies of the newborn offspring from laboratory-grown eggs. Monitoring will continue as the animals grow to adulthood and will be compared to naturally conceived animals. This research is vital to enable selection of the best IVGM method and to ensure that the technology is both efficient and safe before it is used to treat patients.

Premature ovarian insufficiency (POI) or total sterility can occur in girls & young women as a result of genetic defects, surgery, abdominal trauma, or more commonly as a side effect of radiation or chemotherapy treatment for cancers or haematological disease. POI has adverse clinical & psychological effects on women's health. Ovarian cryopreservation can now be used to safeguard the fertility of girls & young women at high risk of sterility or POI. Primordial follicles, containing primordial oocytes, can be cryopreserved in ovarian cortex & stored long-term at -196C. When the patient wishes to start her family, the stored tissue is autografted back at an orthotopic or heterotopic site to restore fertility. However, for girls & women with blood-born leukaemias or cancers with a high risk of ovarian metastasis, ovarian autografting risks reseeding the cancer through the transplant. For these individuals fertility restoration can only be safely achieved in the laboratory by the complete in vitro growth & maturation (IVGM) of cryopreserved primordial oocytes into fertile metaphase II (MII) oocytes. Following in vitro fertilisation, a healthy embryo can be transferred to the patient to produce a pregnancy. The therapeutic potential of IVGM is huge but the technology is extremely challenging. Never-the-less significant advances in IVGM have recently been made by the applicants' laboratories. We now aim to: (i) complete IVGM optimization by testing the health, fertility & developmental competence of oocytes & embryos derived using slow vs. fast grow IVGM systems; (ii) conduct a pilot study to test the implantation & pregnancy potential of embryos derived from IVGM oocytes in (i) & (iii) conduct an embryo transfer trial to confirm the safety & hence therapeutic potential of IVGM oocytes for fertility restoration. These studies will be conducted in sheep as a proven, physiologically and clinically relevant model for fertility preservation & restoration in women.

Normal ovarian function underpins lifelong health and wellbeing in women. When it is disrupted there is a significant burden for women, their families and healthcare providers. Normal ovarian function can be compromised in girls and young women as a result of genetic defects, abdominal trauma, or more commonly as a side effect of radiation or chemotherapy treatment for cancers or blood diseases. If there is a reduction in the number of eggs formed or an increase in the number of eggs used there will be fewer eggs in the ovary and the woman will be at risk of premature ovarian insufficiency (POI) or total sterility that may have adverse clinical & psychological effects on health. Ovarian cryopreservation followed by the complete in vitro growth and maturation (IVGM) of eggs can now be proposed as a means to safeguard the fertility of girls and young women at high risk of sterility or POI. Significant recent advances have been made in both IVGM technology and in our abilities to test the health and normality of eggs grown in the laboratory. The stage is now set to use these advances to test the therapeutic potential of in vitro derived eggs for fertility restoration. The proposed project is vital to enable selection of the best IVGM method and to ensure that the technology is both efficient and safe before it is used to treat patients.

The research project will have a direct impact on key STAKEHOLDERS that include patients who suffer from POI. The legacy of this project will also impact on academics, reproductive and developmental scientists and clinicians as well as the wider scientific community including biomedical scientists, computational biologists, and healthcare professionals. The team of applicants have established, extensive communication networks in these areas that will be used to discuss and distribute the findings of the proposed studies to both the scientific and clinical communities and to the public.

The unique collaborative network established for this project will impact on the MRC through realization of the potential of past, publicly funded research investments, as well as additional analyses of existing research tissue and data banks.

THE UK GOVERNMENT and NHS will benefit in the medium to long-term from the proposed research through: improved health and wellbeing of cancer survivors; the development of new infertility therapies; and expansion of its research base.

HEALTH POLICY MAKERS in the UK and beyond will benefit from this research in the median term through the generation of increased understanding of the ramifications of- and treatment options for- ovarian dysfunction and its contribution to lifelong health. In the long term this will positively impact on the health and wealth of the UK in terms of better health and fertility for subsequent generations, and has the potential to lessen the burden of infertility treatment costs upon the healthcare services.

THE GENERAL PUBLIC will benefit in the medium term, as we aim to disseminate our findings beyond the academic community to increase awareness of ovarian dysfunction and the need for fertility preservation for key groups of patients. By interfacing with the clinical community the project will impact directly upon health professionals through the development of new therapeutic paradigms. We will disseminate our findings beyond the academic community to increase awareness of fertility preservation strategies and by interfacing with the clinical community this knowledge will impact directly upon health professionals.

THE COMMERCIAL SECTOR will benefit in the medium term as a key driver in this application is realization of the potential of a novel therapeutic pathway as well as core and platform culture technologies and associated intellectual property. In addition to patient treatment, the core IVGM technology underpinning this application may impact on animal breeding and conservation programmes.