Title: Impact of cryopreservation on oocyte and embryo health and developmental competence in IVF and oncology patients.
Funder: Health Education England

Abstract: BACKGROUND More than 35 years since the world's first in-vitro Fertilisation (IVF) baby was born, an estimated 5 million babies have now been born worldwide using Assisted Reproductive Technology (ART), with the Nobel Prize in medicine awarded to Roberts Edwards in 2010 for his pioneering work.One in 7 couples will require IVF treatment, which now accounts for 2% of all births in the UK. Significant technological advances in ART such as gamete cryopreservation, improved and extended embryo culture and the use of time-lapse incubators have resulted in major improvements in clinical pregnancy and live-birth rates. However, a number of studies show that these ART procedures may influence gene expression during the pre-implantation stages of development, which may provide early evidence of health implications for patients and their babies. This is a major issue for UK society as the care of ART children falls entirely on the NHS.Unfortunately, existing and new technologies in ART are often not properly investigated at the molecular level before clinical application. Long-term follow-up studies on children conceived in ART are difficult and expensive to carry out, cannot be comprehensive, and take many years to report data. Therefore, as newer technologies continue to emerge, it is important to ensure their safe use as early as possible. AIMSTo investigate the impact of a specific ART procedure such as cryopreservation of human MII oocytes by slow freezing and vitrification, on subsequent embryo development using known markers of oocyte and embryo developmental competence. To establish guidelines on vitrification and safety of such procedures, based on research outputs and extensive Cochrane systemic review that will evaluate pregnancy and live birth rates, child health and growth outcomes, birthweight and physiological studies such as blood pressure, developmental and behavioural studies. QUESTIONS TO BE ADDRESSED Does oocyte vitrification affect: known oocyte molecular markers (Workpackage 1; WP1) and oocyte activation and known markers of embryo development (WP2)? Is oocyte vitrification safe and effective for routine clinical use in fertility preservation and ART (WP3 and 4)? PROPOSED PLAN OF INVESTIGATION This study involving research consented gametes will be sub-divided into 4 different overlapping work packages for strategic execution and effective evaluation. WP1: Vitrification and warming of research consented oocytes using closed and open vitrification methods; subsequently cDNA will be obtained for gene expression analysis (see WP2). WP2: Using the best method of vitrification as evaluated in WP1, fresh oocytes will be vitrified-warmed and activated to generate embryos and cDNA obtained from these embryos and a control group for gene expression analysis, using poly APCR and analysis by qPCR and micro-array; following culture and timelapse microscopy. WP3 and 4: Generation of a protocol and scientific guidelines on vitrification and a high quality evidenced based Cochrane systemic review. This review is a critical foundation for evidence-based practice and STP workforce training. POTENTIAL BENEFIT TO PATIENTS AND NHS This study will facilitate our understanding of the impact of specific ART procedures on the oocyte and embryo. This is entirely in line with the NHS Constitution, which states that It works at the limits of science bringing the highest levels of human knowledge and skill to save lives and improve health. The project will help provide safe treatments and ensure long-term health of children conceived from ART. This should provide better cryopreservation outcomes for infertility and oncology patients with efficient cost-savings for the NHS and society through improved peri-natal outcomes. Our findings will provide a technical and scientific frame-work for establishing the first Altruistic Egg Donor Cryobank for NHS patients, to meet the growing demand for donor gametes in the UK.

Lay abstract: Aims To investigate the impact of cryopreservation (freezing) of eggs and embryos and how the procedure affects subsequent embryo development, egg/ embryo development and long-term health of babies arising from freezing procedures.A secondary aim will be to draw up scientific protocols and guidelines for establishing safe oocyte cryopreservation and adonor egg bank.Our understanding of the impact of freezing will enhance our ability to fully unravel how thisessential procedure can be optimised and used safely, in-order to ensure the well-being of all patients/ parents and children born from the application of this procedure. Background: managing IVF treatment Following the birth of the world's first IVF (test tube) baby (1978) in Oldham, Manchester, 5 million babies have now been born from various IVF treatment procedures. IVF is increasingly popular and continues to offer hope of pregnancy to countless numbers of couples world-wide. IVF babies now account for 2% of all births in the UK. However, IVF involves exposing sperm, eggs and early embryos to an artificial laboratory environment, including new techniques such as egg injection, embryo manipulation and egg and embryo freezing, just at the critical moment of conception. Hence, it is important for scientists to monitor the safety of IVF and the long-term impact of IVF procedures specifically freezing (cryopreservation) of eggs and embryos, on the wellbeing of IVF babies. Cryopreservation is used in IVF as it allows surplus eggs and good quality embryos to be frozen and stored for future patients use. This is also important for women diagnosed with premature ovarian failure, cancer, and women who may require donated eggs. Egg donation is increasingly required for women who delay childbearing or are not able to conceive their own genetic child for other reasons, and establishing a bank of donated cryopreserved oocytes similar to well established sperm banks would greatly improve their treatment options. Despite these routine clinical uses, safety issues and the general efficacy of freezing human eggs and embryos remains an on-going debate among fertility treatment providers and regulatory bodies such as the Human Fertilisation and Embryology Authority (HFEA) and the National Institute for Health and Care Excellence (NICE). Background: managing cancer treatments Unfortunately cancer treatment often requires heavy doses of radiation and/or chemotherapy, which results in loss of fertility in women. In view of these implications, preservation of eggs or embryos offers these women the only hope of having a family later in life. A five year survival outcome of women diagnosed in England and treated for breast cancer, hodgkin lymphoma and skin cancer is now over 80%, a major and significant improvements compared with outcomes obtained 10 years ago. Therefore as life expectancy after cancer treatment continues to improve, it becomes not just essential to maximize success rates following fertility preservation; but to also ensure that current and new methods of egg and embryo freezing remains safe and offers the best chances of pregnancy with a healthy life-birth. Design and MethodsAppropriate consent to donate eggs that are not suitable for treatment will be obtained from IVF patients in accordance with our existing HFEA research licences. Sibling eggs will be preserved using two different methods of rapid oocyte freezing (contact with liquid nitrogen and without contact) compared to an untreated control egg. The abilities of these eggs to develop and become embryos will be evaluated and information obtained on molecules which indicate oocyte and embryo health. Patient and Public Involvement We involve patients and general public extensively in our work via IVF patient open evenings, support groups, interaction with the national group Infertility Network UK (INUK), and established groups in our hospital. With funding from our local research service, I was able to hold a focus group meeting with patients to prioritise project aims and objectives for maximum patient benefit. DisseminationFindings will inform changes to routine standard operating procedures for egg and embryo freezing and will be utilised in providing a scientific framework for the establishment of the first altruistic donor egg bank in the UK for cancer and IVF patients. Findings will be disseminated at our focus groups and amongst staff and patients at the Christie Hospital, Manchester for better awareness, via charities such as CRUK, patient groups such as INUK and via the MSc in Reproductive Sciences based in Manchester and at national and international meetings; which my supervisors attend and lecture at regularly.

Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: REPRODUCTIVE HEALTH AND CHILDBIRTH