Induced IVM: A New Approach to Oocyte Maturation in vitro · 2011. 11. 25. · 2.3.4 Oocyte nuclear...

Induced IVM: A New Approach to Oocyte Maturation in vitro

Firas Albuz, MSc.

The Robinson Institute The Research Centre for Reproductive Health,

Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health,

University of Adelaide, Australia. A thesis submitted to the University of Adelaide in total fulfilment of the requirements for the

degree of Doctor of Philosophy in Medicine.

October 2009

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Table of Contents

Abstract vii

Declaration ix

Acknowledgements x

Glossary/Abbreviations xii

Publications and Conference Proceedings xiii

Provisional Patent xvi

Visits to Overseas Laboratories xvi

Invited Guest Seminar - International xvi

Awards, Scholarship & Prizes xvii

CHAPTER 1

LITERATURE REVIEW 18

1.1 Introduction 19

1.2 Infertility Treatment and Current Limitation 21

1.3 In vitro Maturation of Oocytes 21

1.3.1 Rationale for the Clinical Use of IVM 22

1.3.2 Potential Applications of IVM (Patient Groups) 23

1.3.3 Rescue IVM (GV Oocytes from Stimulated Cycles) 25

1.3.4 IVM Limitations 25

1.3.4.1 Fertilization Rates 26

1.3.4.2 Embryo Development Rates 26

1.3.4.3 Embryo Transfer Outcomes 28

1.3.4.4 Long-Term Health (Offspring) Outcomes 28

1.3.5 Conclusion 30

1.4 Biological Aspects 31

1.4.1 Oocyte Development 31

1.4.2 Oocyte Meiotic Arrest 34

1.4.3 Cumulus Cell-Oocyte Gap Junctional Communication 34

1.4.4 cAMP 35

1.4.5 Oocyte Maturation (in vivo) 39

1.4.6 Prophase I & Chromatin Configurations 39

1.4.7 Cytoplasmic Maturation 40

1.4.8 Meiotic Resumption 42

1.4.8.1 Nuclear Maturation 42

1.4.8.2 Role of Gonadotrophins/EGF on Meiotic Resumption 43

1.4.8.3 cAMP/PDEs 43

1.4.8.4 Loss of Gap Junctional Communication (GJC) 45

1.4.8.5 MPF 48

1.4.8.6 FF-MAS 49

1.5 Oocyte Maturation (in vitro) 49

1.6 Prospective Concepts for Improving IVM Culture System 50

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1.6.1 IVM Collection and Handling Media 52

1.6.2 IVM Culture Media 53

1.6.3 Prematuration Culture (PMC) 54

1.6.3.1 Physiological Conditions & Meiotic Arrest in vitro 54

1.6.3.2 Inorganic Additives & Meiotic Arrest in vitro 55 1.6.3.2.1 Purines 55 1.6.3.2.2 Protein Kinase Inhibitors 55 1. 6.3.2.3 cAMP Modulators (AC, PDEs) 57 1.6.3.2.4 AMPK Activators 60

1.7 Conclusion 63

1.8 Hypothesis and Aims for PhD Project 64

1.8.1 Hypothesis 64

1.8.2 Aims 64

CHAPTER 2

EFFECT OF INHIBITION OF PHOSPHODIESTERASE TYPE 8 DURING IN

VITRO MATURATION OF BOVINE OOCYTE ON CAMP LEVELS, MEIOTIC

AND DEVELOPMENTAL CAPACITY 65

2.1 Abstract 66

2.2 Introduction 67

2.3 Materials & methods 69

2.3.1 Oocytes recovery and culture 69

2.3.2 cAMP modulators 69

2.3.3 Measurement of intracellular cAMP 70

2.3.4 Oocyte nuclear maturation assessment 70

2.3.5 In vitro maturation, fertilization and embryo culture 71

2.3.6 Statistical analyses 72

2.4 Results 73

2.4.1 The role of PDE8 on cAMP degradation in the cumulus-oocyte complex 73

2.4.2 The effect of PDE8 inhibition on oocyte meiotic maturation 73

2.4.3 The effect of PDE8 inhibition on cleavage and development to the blastocyst stage 74

2.5 Discussion 75

2.6 Figures 79

2.7 References 83

CHAPTER 3

SUBSTANTIAL IMPROVEMENTS IN BOVINE EMBRYO YIELD USING A

NOVEL SYSTEM OF INDUCED IVM BY EXPLOITING CAMP MODULATORS

IN PRE-IVM AND IVM 86

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3.1 Abstract 87

3.2 Introduction 89

3.3 Materials and Methods 91

3.3.1 Oocyte collection and selection (pre-IVM phase) 91

3.3.2 Oocyte in vitro maturation (IVM phase) 91

3.3.3 In vitro fertilization and embryo culture 92

3.3.4 Blastocyst differential staining 93


3.3.6 Oocyte nuclear morphology assessment 94

3.3.7 Oocyte-cumulus cells gap junctional communication assay 95

3.3.8 Statistical analysis 96

3.4 Results 97

3.4.1 cAMP content of COC and DO during pre-IVM phase 97

3.4.2 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on spontaneous oocyte maturation (GV/GVBD) 98

3.4.3 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on oocyte germinal vesicle configurations 98

3.4.4 Effect of cAMP modulators during pre-IVM and IVM on oocyte-cumulus cell gap junctional communication 100

3.4.5 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on oocyte meiotic progression to MII stage 100

3.4.6 Intracellular concentration of cAMP in oocyte after pre-IVM and IVM phase 101

3.4.7 Effect of (EGFR) kinase inhibitor on FSH-induced oocyte maturation in the presence of the type-3 PDE inhibitor (Cilostamide) 101

3.4.8 Effect of cAMP modulators during pre-IVM and IVM phase on oocyte developmental competence 102

3.4.9 Effect of cAMP modulators during pre-IVM and IVM on blastocyst cell numbers 103

3.5 Discussion 104

3.6 Figures 109

3.7 Tables 118

3.8 References 120

CHAPTER 4

SPECIFIC PRE-IVM CONDITIONS THAT IMPROVE BOVINE OOCYTE

DEVELOPMENTAL COMPETENCE 123

4.1 Abstract 124

4.2 Introduction 126

4.3 Materials & methods 130

4.3.1 Oocyte recovery, selection and incubation (pre-IVM phase) 130

4.3.2 In vitro oocyte maturation, fertilization and embryo culture 130


4.3.4 Experimental design 132


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4.4 Results 135

4.4.1 Experiment 1. The effect of increasing concentrations of forskolin and the presence/absence of IBMX in collection medium during the pre-IVM phase on COC cAMP levels 135

4.4.2 Experiment 2. The effect of pre-IVM duration on COC cAMP when incubated with increasing concentrations of forskolin and IBMX 135

4.4.3 Experiment 3. The effect of pre-IVM duration on intra-oocyte cAMP when incubated with increasing concentrations of forskolin and IBMX 136

4.4.4 Expeirment 4. The effect of increasing cAMP levels prior to IVM on oocyte developmental competence 137

4.5 Discussion 138

4.6 Figures 142

4.7 References 146

CHAPTER 5

INDUCED OOCYTE IVM SUBSTANTIALLY IMPROVES MOUSE EMBRYO

YIELD AND PREGNANCY OUTCOMES 150

5.1 Abstract 151

5.2 Introduction 153

5.3 Materials and Methods 156

5.3.1 Source of oocytes 156

5.3.2 Oocyte collection (pre-IVM phase) 156

5.3.3 Oocytes in vitro maturation (IVM phase) 157

5.3.4 In vitro fertilization 157

5.3.5 Differential nuclear staining 158

5.3.6 Embryo transfer 158


5.4 Experimental Design 159

5.4.1 COC cAMP levels and initiation of maturation 159

5.4.2 The effect of increasing doses of FSH to induce meiotic maturation of COCs matured in the presence of the type 3 PDE inhibitor (cilostamide) 159

5.4.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by induced IVM (22 hours IVM) 160

5.4.4 The effect of standard or induced IVM on oocyte meiotic maturation 160

5.4.5 The effect of time of maturation (18 or 22 hours) on oocytes matured by standard or induced IVM on oocyte developmental capacity 161

5.4.6 The effect of different cAMP modulators during the pre-IVM & IVM phases on oocyte developmental capacity 161

5.4.7 Embryo developmental capacity, pregnancy outcomes & fetal parameters of oocytes derived from induced and standard IVM and from in vivo matured oocytes 161


5.5 Results 163

5.5.1 COC cAMP measurements in vitro versus in vivo 163

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5.5.2 Increasing doses of FSH to induce meiotic maturation of COCs matured in the presence of the type 3 PDE inhibitor (cilostamide) 163

5.5.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by induced IVM 163

5.5.4 The effect of Induced IVM on GV/GVBD (0-14 hours of IVM) 164

5.5.5 The effect of Induced IVM on oocyte maturation (18 and 22 hours of IVM) 164

5.5.6 The effect of induced IVM vs. standard IVM on oocyte developmental capacity & blastocyst quality 164

5.5.7 The effect of maturing oocytes by induced or standard IVM compared to in vivo matured oocytes on developmental capacity, pregnancy outcomes & fetal parameters 165

5.6 Discussion 167

5.7 Figures 172

5.8 References 183

CHAPTER 6

FINAL DISCUSSION 187

FUTURE DIRECTIONS 198

THESIS REFERENCES 220

CHAPTER 7

APPENDICES 220

Appendix 1: Additional Experiments 221

Appendix 1: Figures 226

Appendix 2: Culture Media 233

Appendix 3: Reagents 239

Appendix 4: Bovine Blastocyst Scoring System 241

Appendix 5: Published Version of Chapter II 243

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Abstract Oocyte in vitro maturation (IVM) is a technique that would alter the management of human

infertility if success rates were notably higher. Oocyte maturation in vivo is a highly

orchestrated, induced process, whereby 3'-5'-cyclic adenosine monophosphate (cAMP)-

mediated meiotic arrest is overridden by the gonadotrophin surge. However, using standard

IVM, oocytes resume maturation spontaneously hence compromising developmental

competence. The aim of this thesis was to establish an improved system for mammalian

oocyte IVM by studying the inclusion of various forms of cAMP modulators during IVM and

examine oocyte quality and developmental capacity.

Firstly, this thesis includes a series of experiments designed to examine the effect of specific

inhibition of phosphodiesterase type 8 (PDE8) during IVM of bovine oocytes on cAMP

levels, meiotic and developmental capacity. The inhibition of PDE8 degradation resulted in a

dose-dependent increase in cAMP levels and delayed oocyte meiotic resumption. However,

the inhibition of PDE8 degradation failed to enhance oocyte developmental competence.

This thesis includes an extensive series of studies designed to establish a novel induced-IVM

system. Firstly, a pre-IVM phase was developed where immature bovine or mouse oocytes

were briefly treated with the adenylate cyclase activator, forskolin and a non-specific PDE

inhibitor, IBMX, which substantially increased intra-oocyte cAMP to in vivo physiological

levels. Secondly, to maintain oocyte cAMP levels and prevent precocious oocyte maturation,

oocytes were then matured with an oocyte-specific PDE 3 inhibitor, cilostamide and

simultaneously induced to mature by FSH. The net effect of this system was an increase in

oocyte-somatic cell gap-junctional communication and a delay in meiotic progression through

prophase I to metaphase II, extending the standard IVM interval. Moreover FSH-induced

maturation was prevented by an epidermal growth factor receptor inhibitor, AG1478,

demonstrating that induced oocyte maturation functions via secondary autocrine signalling

within the cumulus cell compartment.

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Results from the present thesis also demonstrated that induced-IVM leads to a substantial

improvement in oocyte quality, which in turn had long-term developmental consequences

improving embryo/fetal yield and pregnancy outcomes. The work presented in this thesis

validates this technology using two mammalian models. In the bovine, induced-IVM more

than doubled embryo yield (27% to 69%), relative to standard-IVM. Similarly in the mouse,

induced-IVM substantially increased fertilization rate (55% vs. 82%), embryo yield (55% vs.

86%), embryo quality, implantation rate (28% vs. 53%), fetal yield (8% vs. 26%) and fetal

weights (0.5g vs. 0.9g). All these embryo and fetal readouts using induced-IVM in mice were

equivalent to those using in vivo matured oocytes (conventional IVF).

In conclusion, induced-IVM mimics some of the characteristics of oocyte maturation in vivo

and substantially improves oocyte developmental outcomes in two disparate mammalian

species. The outcomes of the research presented in this thesis have provided a new

perspective to our understanding of the mechanisms regulating oocyte maturation and the

acquisition of developmental competence. The novel IVM system will provide new options

for a wide range of reproductive biotechnologies including livestock breeding and

conservation applications. Application of induced-IVM to human infertility will bring

substantial cost and health benefits by simplifying ART protocols.

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Declaration This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution to Firas Albuz and, to the best of my

knowledge and belief, contains no material previously published or written by another person,

except where due reference is made in the text.

I give consent to this copy of my thesis when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis (as

listed below*) resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be available on the web, via the

university`s digital research repository, the Library catalogue, the Australasian Digital Theses

Program (ADTP) and also through web search engines, unless permission has been granted

by the University to restrict access for a period of time.

* List of Publications

1. Maxime Sasseville, Firas K Albuz, Nancy Côté N, Christine Guillemette, Robert B Gilchrist and Francois J Richard. (2009). Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle. Biology of Reproduction.81: 415-425.

2. Firas K Albuz, Maxime Sasseville, Michelle Lane, David T Armstrong, Jeremy G Thompson

and Robert B Gilchrist. (2009). Induced oocyte IVM substantially improves embryo yield and pregnancy outcomes. Nature Biotechnology. Manuscript No. NBT-TR21146A (submitted 01/06/2009).

October 2009

Firas Albuz

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Acknowledgements

Achieving my infinite dream of pursuing a PhD degree was a painful and enjoyable

experience. It was just like climbing a high peak, step by step, accompanied by bitterness,

hardship, frustration, encouragement, trust and with so many people‟s kind help. When I

found myself at the top enjoying the beautiful scenery, I realized that it was, in fact,

teamwork that got me there and a group of awesome people to whom I owe a huge debt of

gratitude.

I would like to express my most sincere gratitude to my two supervisors, Dr Robert Gilchrist

and A/Prof. Jeremy Thompson, for allowing me the opportunity to undertake a PhD in the

field of research that I was always dreaming to achieve immensely. Many thanks for the

continual support, advice and encouragement throughout my project. Your enthusiasm for the

subject and never ending expertise in this area of research has been of tremendous help.

My PhD project could not have occurred without the support of Cook Australia, in particular

Kim Giliam and Aidan McMahon. Thank you for having faith in my project and work.

My very special thanks go to Prof. David Armstrong & Dr. Maxime Sasseville who have

helped me in every aspect of my study and who have provided numerous valuable comments.

Exceptional thanks goes to Dr. Michelle Lane, who helped me with embryo transfer

experiments and gave me the great opportunity to work in the clinical embryology field at

Repromed.

An enormous thanks goes out to past and present staff and students of the Robinson Institute/

The Research Centre for Reproductive Health. In particular, I would like to thank Lesley

Ritter for too many things to name so I will not even try. I would also like to thank Megan

Mitchell, Melanie McDowall, Deanne Feil & Annie Whitty for all the support over the years.

Fellow students: Kim, Sarah, Laura, Kathryn and Hannah over the past 3 years please know

that all your friendship and support have been valuable to my sanity.

I must also give gargantuan thanks to Dave Froiland and Ashley Gauld. I will miss the

Thursday lunch sessions with you guys, thanks for the great support throughout my residency

in Adelaide. I will miss our chats about almost everything and working with you in the lab.

To me, you have been more than just work colleagues; you are great friends.

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Friends sustain you on life`s journey and without them this PhD would not have been

possible. I must thank my incredible network of friends who are members of the “Olive

circle” for all the love and support they have provided throughout the course of my PhD,

Raeleen, George, Raik, Dimi, Tamer & Sarah. Moreover I would like to thank my real friend

and flatmate “the famous wine maker” Marcos for our long chats and real friendship. I will

always remember these beautiful days and your neighbourhood in Henley beach, we were

living in such a beautiful unforgettable place, I will remember this place always and forever.

Raeleen thanks for the food and the hospitality, you guys were my family in Adelaide. I am

so fortunate to have such wonderful, supportive friends and I appreciate everything that you

have done to help me.

My deep love and appreciation goes to my family with whom I shared my childhood and

whose love and support still sustain me today. My sisters and brother: Einas, Eiman and

Mohm`d who are all so much part of me and my memories of childhood. My beloved parents

have given me more than I can even realize or mention here. My mother: Samiha whose

ambition, love and sacrifice is boundless, you are always a constant source of pride, support

and encouragement and of course not to mention the untold number of sacrifices for the entire

family, and specifically for me to continue my schooling. I know that you are always proud of

me. My father: Kamal who is my role model in tolerance, patience and sacrifice; he is a man

who dedicated his whole life to see his children happy and successful. Last but not least, in

this world there are always facts, proved and unproved hypotheses. However, there is only

one hypothesis which will never ever fail: the hypothesis of love. I am finishing up my degree

and writing this thesis because of you my lovely wife Reem. You are the first, the last and

everything in my life, I love you, you put up with a lot of things, but I will never forget your

support, patience and love. Thanks for your understanding and your smile which keep me

persistent day by day. I would therefore like to dedicate this thesis to you and to my family

and to those whose names are not included, but who assisted me in one form or another. I

sincerely thank them all.

These studies were financially supported by a linkage grant (LP0562536) awarded by the

Australian Research Council (ARC) in partnership with COOK Australia and through

Australian Postgraduate Award/Industry.

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Glossary/Abbreviations AC adenylate cyclase AM acetoxy-methyl ester 5‟-AMP adenosine 5‟-monophosphate AMPK AMP-activated protein kinase ATP adenosine triphosphate BSA bovine serum albumin B-TCM bicarbonate-buffered tissue culture medium CAM calcein acetoxy-methyl ester cAMP cyclic adenosine monophosphate cAMP-PKA cAMP-dependant protein kinase A cGMP cyclic guanosine monophosphate CM cilostamide COC cumulus-oocyte complex dbcAMP dibutyryl cyclic adenosine monophosphate DMSO dimethyl-sulphoxide DO cumulus-oocyte complex derived oocyte ET embryo transfer FAF fatty acid-free FF-MAS follicular fluid meiosis activating sterol FSK forskolin FSH follicle stimulating hormone GC granulosa cell GJC gap junctional communication GV germinal vesicle GVBD germinal vesicle breakdown hCG human chorionic gonadotrophin H-TCM hepes-buffered tissue culture medium iAC invasive adenylate cyclase IBMX 3-isobutyl-1-methylxanthine ICM inner cell mass IVF in vitro fertilization IVM in vitro maturation IVP in vitro production (of embryos) LH luteinising hormone M meiosis phase of the cell cycle MGC mural granulosa cell MI metaphase I MII metaphase II MR milrinone PDE phosphodiesterase PDE3 phosphodiesterase subtype 3 PDE4 phosphodiesterase subtype 4 PDE8 phosphodiesterase subtype 8 PVA polyvinyl alcohol rhFSH recombinant human FSH RIA radioimmunoassay TE trophoectoderm

xiii

Publications and Conference Proceedings Scientific Publications

1. Maxime Sasseville, Firas K Albuz, Nancy Côté N, Christine Guillemette, Robert B Gilchrist

and Francois J Richard. (2009). Characterization of Novel Phosphodiesterases in the Bovine

Ovarian Follicle. Biology of Reproduction.81: 415-425.

[Impact Factor: 3.6]

2. Firas K Albuz, Maxime Sasseville, Michelle Lane, David T Armstrong, Jeremy G

Thompson and Robert B Gilchrist. (2009). Induced oocyte IVM substantially improves

embryo yield and pregnancy outcomes. Nature Biotechnology. Manuscript No. NBT-

TR21146A (submitted 01/06/2009).

[Impact Factor: 22.3]

Other Journal Contributions

1. Firas K Albuz, Maxime Sasseville, David T Armstrong, Jeremy G Thompson and Robert B

Gilchrist. (2008). Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on

bovine cumulus and oocyte functions. Reproduction, Fertility and Development; 20 (Suppl);

Abstract 260.

2. Maxime Sasseville, Firas K Albuz, Francois J Richard, Robert B Gilchrist. (2008). Evidence

for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle. Reproduction,

Fertility and Development; 20 (Suppl); Abstract 254.

3. Firas K Albuz, Maxime Sasseville, David T Armstrong, Michelle Lane, Jeremy G

Thompson and Robert B Gilchrist. (2009). Induced oocyte in vitro maturation (IVM)

substantially improves embryo yield and pregnancy outcomes. Reproduction, Fertility and

Development; 21 (Suppl); Abstract 131.

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Conference Proceedings

1. International


Gilchrist. (2009). Substantial improvements in embryo yield using a novel system of induced

IVM by exploiting cAMP modulators in pre-IVM and IVM. Proceedings of the twenty-fifth

annual conference of the European Society of Human Reproduction and Embryology,

Amsterdam, The Netherlands. Abstract 167. (Oral presentation).

2. Robert B Gilchrist, Firas K Albuz and Jeremy G Thompson. (2010). A new approach to

IVM and embryo IVP: Induced-IVM substantially improves embryo yield and pregnancy

outcomes. Proceedings of the thirty-sixth annual conference of International Embryo Transfer

Society Conference, Cordoba, Argentina.

2. National


Gilchrist. (2008). Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on

bovine cumulus and oocyte functions. Proceedings of the thirty-ninth annual conference of

The Society for Reproductive Biology, Melbourne, Australia. (Abstract 260)

2. Maxime Sasseville, Firas K Albuz, Francois J Richard, Robert B Gilchrist. (2008.)

Evidences for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle.

Proceedings of the thirty-ninth annual conference of The Society for Reproductive Biology,

Melbourne, Australia. (Abstract 254)


Thompson and Robert B Gilchrist. (2009). Induced oocyte in vitro maturation (IVM)

substantially improves embryo yield and pregnancy outcomes. Proceedings of the fortieth

annual conference of The Society for Reproductive Biology, Adelaide, Australia. (Abstract

131)

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3. Local

1. Firas K Albuz. (2008). Improving the efficiency of oocyte maturation in vitro. The 2008

AusBiotech-GSK Student Excellence Awards National Lauch: Research to Revenue,

Adelaide, Australia.


Thompson and Robert B Gilchrist. (2009). Regulation of cAMP during oocyte maturation

substantially improves developmental outcomes. Proceedings of the Australian Society for

Medical Research SA conference, Adelaide, Australia. (Abstract RW1.)

3. Firas K Albuz (2009). IVF without hormones. The 2009 Women and Children`s Hospital

Young Investigator Award (YIA) Semi-finals-Scientific Presentation, Adelaide, Australia.

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Provisional Patent

Firas K Albuz, Robert B Gilchrist, Jeremy G Thompson. “Methods for the collection and

maturation of oocytes” US Provisional Patent Application No. IP10922/473 (14/05/2009).

Visits to Overseas Laboratories

2009 Prof. Johan Smitz, Follicle Biology lab, Vrije Universitiet Brussel (VUB),

Brussels, Belgium.

Invited Guest Seminar - International

2009 Induced oocyte in vitro maturation (IVM) substantially improves

developmental outcomes. Vrije Universitiet Brussel (VUB), Brussels, Belgium (24/06/2009).

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Awards, Scholarship & Prizes

2006-2009 Australian Postgraduate Award Industry (APAI) under the Australian

Research Council (ARC) Linkage Project (PhD scholarship)

2008 AusBiotech-GSK Student Excellence Award Finalist

2008 The Society for Reproductive Biology (SRB) Meat and Livestock

Australia New Investigator Award Finalist

2008 Society for Reproductive Biology Travel Scholarship

2009 Ross Wishart Memorial Award Finalist

2009 AUGU/RC Heddle Award Finalist

2009 The Society for Reproductive Biology (SRB) Meat and Livestock

Australia New Investigator Award Finalist

2009 Research Centre for Reproductive Health Research Scholarship

The University of Adelaide

2009 Faculty of Health Science Travelling Fellowship

The University of Adelaide

2009 Network in Genes and Environment in Development

Conference Participation Award

2009 The Women & Children`s Hospital Young Investigator Award Semi-

Finalist

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