LICAMM Bioengineering and evaluation of artificial ovaries for fertility preservation.

In recent years the improvement in the diagnosis, management and treatment of a range of solid and haematological malignancies has lead to a marked increase in the chances of long-term survival for a significant number of children and adolescents. Unfortunately germ cells, like the cancer cells that are their intended targets, are highly susceptible to damage by radiation and by the alkylating agents and platinum compounds that are commonly used in chemotherapy formulations. Thus the radiotherapy and chemotherapies used to cure many cancers can render patients of either sex or any age temporarily or even permanently infertile. This adverse side effect of cancer treatment is particularly relevant to young girls who, in the event of ovarian failure, are faced with the prospects of life long hormone replacement therapy- a regime that is not, it, risk free. Health care professionals are therefore faced with the challenge of not only developing and improving the technologies that will treat the cancer and protect the individual’s wellbeing, but they must also implement strategies that will conserve the fertility of these young patients.

<p>The current options available to preserve the fertility of young female cancer patients range from no medical intervention at all to the use of invasive procedures to harvest and freeze-store tissues or isolated cells. Assisted reproductive techniques can be used for the collection and storage of mature oocytes (eggs) and embryos.</p> <p>However these approaches are only suitable for patients of reproductive age and it is clearly not an option for most adolescents or for any prepubertal girls. An alternative approach that is suitable for both children and adults is the cryopreservation of ovarian tissue. Ovarian tissue cryopreservation involves freezing the earliest staged primordial oocytes in situ either through freezing small pieces of the surface of the ovary or by freezing the whole organ.</p> <p>The tissue fragments or ovary can then be transplanted back to the patient when she is in remission and wishes to start her family. Unfortunately, ovarian autografting is not suitable for patients with blood bourn diseases such as leukemia or steroid related cancers as these diseases carry a high chance of ovarian metastasis and there is therefore a high risk of reintroducing the cancer to the body through the transplanted tissue.</p> <p>Where any such risk exists a safer alternative for the restoration of fertility is to grow the immature oocytes in the ovarian tissue to maturity in the laboratory through a process known as the in vitro growth (IVG) of follicles. Once the eggs are fully grown they can then be matured by in vitro maturation (IVM) and fertilised using in vitro fertilisation (IVF)technologies so that only embryos that are free from cancer cells are transferred back to the patient to establish a pregnancy.</p> <p>The development of technologies for the in vitro derivation of fertile oocytes from the most abundant primordial follicles holds many attractions for assisted conception, in humans and for animal production technology. Any culture strategy designed to support the complete IVG of oocytes must mimic the developmental sequence of events and cellular checkpoints seen in vivo in terms of follicle and oocyte growth rates, gene expression patterns and molecular milestones and metabolic requirements.</p> <p>During their extended growth phase oocytes progressively synthesise and accumulate the payload of proteins and RNAs that are required to support both nuclear and cytoplasmic maturation of the fully-grown oocytes and early post-fertilisation development of the zygote. Finally, there are stage specific changes in DNA methylation of the genes associated with genomic imprinting during oocyte growth and early embryo development. The team at Leeds has recently developed a physiologically relevant, 3D culture strategy for follicle that supports the complete in vitro development of oocytes over extended periods of up to 3-4 months.</p> <p>While it is possible to grow oocytes from primordial stages using our multiphase 3D approach, the system is inefficient, extended open culture systems are at risk of contamination, and the health and genetic normality of the oocytes so derived remain to be tested. The complexity of the tissue and the need for a tightly controlled culture environment during IVG may be resolved using a microfluidic centred culture approach. Using Organ on chip (OoC) microfluidic devices, cells are perfused in μm-sized chambers to more closely replicate organ physiology in vivo than conventional disaggregation cultures in vitro.</p> <p>The stage is therefore set to test whether an ovary OoC microfluidic device can be developed and used to enhance the health and developmental competence of in vitro-derived oocytes from ovarian tissues.</p> <p>This Ph.D. project will therefore develop and test an OoC model of the ovary as a vehicle to support the complete in vitro production and fertilisation of oocytes. The Ph.D. will address the hypothesis that  “Organ on Chip models can be used to support the in vitro production and fertilisation of healthy oocytes from primordial follicles in vitro”.</p> <h3>Experimental Plan:</h3> <p>The Reproduction and Early Development Research Group in the LICAMM Institute and have been instrumental in establishing the technology to grow oocytes from large animals and humans to maturity in the laboratory (Prof Picton) but also to test their health and genetic normality (Dr Huntriss). Dr Pensebene has a background in nanotechnology she has developed ultrathin membranes to support in vitro cell growth and to repair fetal membrane in utero. She has developed the OoC model of the endometrium for studying the effect of environmental exposure to bacteria and dioxin on the establishment and development of pregnancy. </p> <p>This project aims to combine the recent advances in nanotechnology, microfluidics, and follicle and oocyte culture to test the health and efficiency of complete IVG, IVM and IVF of immature eggs derived from fresh ovarian tissues. The proposed experiments will ultimately test the developmental capacity of IVG/IVM oocytes derived using either: (a) an in-house, open and highly defined multi-step, physiological, follicle culture system which takes 40-60 days to complete; or (b) a novel, closed microfluidic ovary OoC co-culture strategy. A series of end point assays will be used to evaluate the health and developmental competence of oocytes derived using both approaches. Follicle and oocyte size will be measured and growth rates recorded. Follicle development and culture stress will be assessed by measuring follicle and oocyte metabolism and waste product production in samples of spent culture media. The capacity of in vitro-derived oocytes to complete meiotic maturation and produce fertile gametes will ultimately be assessed using methods for IVM and IVF. Embryo developmental potential will be quantified by culturing embryos for 7 days and recording embryo cleavage rates, blastocyst production and blastocyst cell number. Tissues will be archived throughout the cultures for later molecular analysis. All of the technologies for IVG, IVM and IVF of sheep oocytes, together with analytical techniques used to assess growth and fertility are in routine use in the Leeds laboratory. Due to the limited availability of human ovarian tissue for research, sheep ovaries and oocytes will be used as a model for human ovary/oocytes through the course of this Ph.D. The novel ovary OoC microfluidic device will be fabricated in Polydimethylsiloxane (PDMS) and biocompatible polymers using soft lithography and spin coating deposition techniques. Fabrication and assembly of the devices will be carried out at the School of Electronic and Electrical Engineering in a state of the art class 100 clean room.</p> <h3>Experiment 2</h3> <p>Molecular evaluation of the health and normality of in vitro-derived oocytes. Samples of oocytes, embryos and follicular somatic cells will be archived from Experiment 1. These samples will be used for quantification of the expression of key gene markers of oocyte (e.g. Gdf-9, Bmp15) and follicle development (e.g. Fshr, Lhr, Amh, Has2, Egfr, Amphiregulin, Epiregulin, Ptx2) and embryo health and imprinting status (e.g. Oct4, H19, Igf2r, Snrpn). All of the techniques required for single egg and embryo RNA and DNA extraction, amplification and molecular analysis are fully validated and are in routine use in the Leeds laboratory.</p> <h3>Experiment 3</h3> <p>Fabrication and testing of the novel ovary OoC microfluidic device. Cultures will be conducted to test the utility of our new microfluidic device as a vehicle to co-culture (i) ovarian stroma; (ii) ovarian cortex; (iii) preantral ovarian follicles and (iv) cumulus oocyte complexes over extended periods. Replicate cultures will be conducted for each combination of cell types. Viable cell number and parameters of follicle and oocyte growth and maturity will be recorded. Follicle and oocytes morphology will be assessed histologically at the end of culture in fixed tissues.</p> <h3>Experiment 4</h3> <p>Comparison of the efficacy of the open, multi-phase 3D culture system and the ovary OoC for the in vitro production of oocytes. This experimental series will directly compare the 2 culture strategies as vehicles for the complete IVG of follicles and oocytes. Follicle growth dynamics, the capacity for oocytes from both sources to undergo IVM, IVF and embryo development will be used to test the efficacy of both culture approaches. Oocytes and embryos will be archived for analysis using the range of molecular markers of oocyte development and normality as defined during Experiment 2.</p> <h3>Environment</h3> <p>This multidisciplinary project will predominantly be conducted in the Light laboratories. The ovary OoC will be fabricated at the School of Electronic and Electrical Engineering, assembled and characterized in the class 100 clean room. Cell loading and testing will be carried out in the LIGHT laboratories. In addition to the acquisition of generic skills in data analysis, presentation and thesis writing etc, the student will receive training in: (a) tissue culture methods (aseptic cortex harvest, follicle and oocyte isolation and culture, IVM, IVF and embryo culture; (b) metabolism assays; (c) molecular techniques (RNA isolation, real time PCR, cDNA generation, cloning and sequencing) and (d) microfluidic device fabrication and testing.</p> <h3>References:</h3> <p>Anckaert E, De Rycke M, Smitz J.(2013) Culture of oocytes and risk of imprinting defects. Hum Reprod Update 19(1):52-66.</p> <p>Chambers EL, Gosden RG, Yap C, Picton HM (2010). In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility. Hum Reprod. 2010 Oct; 25(10): 2559-68</p> <p>Gnecco JS, Pensabene V, Li DJ, Ding T, Hui EE, Bruner-Tran KL, Osteen KG (2017) Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium. Ann Biomed Eng. 45(7):1758-1769</p> <p>Huntriss J, Lu J, Hemmings K, Bayne R, Anderson R, Rutherford A, Balen A, Elder K, Picton HM (2017). Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos. J Assist Reprod Genet. 34(1):23-31</p> <p>Huntriss J, Woodfine K, Huddleston JE, Murrell A, Picton HM (2015). Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®. Methods Mol Biol. 2015;1315:259-70. doi: 10.1007/978-1-4939-2715-9_19.</p> <p>Labrecque R, Sirard MA (2014). The study of mammalian oocyte competence by transcriptome analysis: progress and challenges. Mol Hum Reprod 20(2):103-16.</p> <p>Newton HL, Glaser AW, Picton HM (2017). Fertility preservation options in prepubertal females: feasibility, safety and outcomes. Minerva Ginecol. 69(6):568-586</p> <p>Pensabene V, Costa L, Terekhov AY, Gnecco JS, Wikswo JP, Hofmeister WH (2016). Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips. ACS Appl Mater Interfaces. 31;8(34):22629-36</p> <p>Picton HM, Harris SE, Muruvi W, Chambers EL (2008). The in vitro growth and maturation of follicles. Reproduction. 136(6):703-15</p> <p>Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, Picton HM, Plancha C, Shea LD, Stouffer RL, Telfer EE, Woodruff TK, Zelinski MB (2010). Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation. Hum Reprod Update. 16(4):395-41</p> <p>Stewart KR, Veselovska L, Kelsey G (2016) Establishment and functions of DNA methylation in the germline. Epigenomics. 8(10):1399-1413</p> <p> </p>

<p>Please note these are not standalone projects and applicants must apply to the PhD academy directly.</p> <p>Applications can be made at any time. You should complete an <a href="https://medicinehealth.leeds.ac.uk/faculty-graduate-school/doc/apply-2">online application form</a> and attach the following documentation to support your application. </p> <ul> <li>a full academic CV</li> <li>degree certificate and transcripts of marks (or marks so far if still studying)</li> <li>Evidence that you meet the programme’s minimum English language requirements (if applicable, see requirement below)</li> <li>Evidence of funding to support your studies</li> </ul> <p>To help us identify that you are applying for this project please ensure you provide the following information on your application form;</p> <ul> <li>Select PhD in Medicine, Health & Human Disease as your planned programme of study</li> <li>Give the full project title and name the supervisors listed in this advert</li> </ul>

A degree in biological sciences, dentistry, medicine, midwifery, nursing, psychology or a good honours degree in a subject relevant to the research topic. A Masters degree in a relevant subject may also be required in some areas of the Faculty. For entry requirements for all other research degrees we offer, please contact us.

Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The minimum requirements for this programme in IELTS and TOEFL tests are: • British Council IELTS - score of 7.0 overall, with no element less than 6.5 • TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.

<p>For further information please contact the Faculty Graduate School<br /> e: <a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a></p>