Mitochondrial supplementation to enhance fertilisation and embryo development. Justin St. John Centre for Genetic Diseases
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2 Mitochondrial supplementation to enhance fertilisation and embryo development Justin St. John Centre for Genetic Diseases
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4 Declaration of competing interest This work was primarily funded by OvaScience Inc., Waltham, MA, USA Research grant awarded to Jus St. John, Hudson Institute of Medical Research
5 The mitochondrial genome High degree of homology between human and pig genome Human = 16,569 bp Mouse = 16,295 bp Pig = 16,613 bp
6 Regulation of mtdna copy number during development from oocyte to embryo
7 Higher mtdna copy number in fertilised oocytes Mean mtdna copy number ** = p < (t-test)
8 Higher mtdna copy number in fertilised oocytes Mean mtdna copy number Mean mtdna copy number ** = p < (t-test) * = p < 0.02 (one-way analysis of variance)
9 Third-party mitochondrial supplementation results in heteroplasmic offspring Proposed to enhance embryonic developmental outcome Resulted in heteroplasmic offspring 3-parents Developmental disorders reported in human and mouse Cohen et al. Lancet 1997; 350: Brenner et al. Fertil Steril 2000; 74: Barritt et al. Reprod Biomed Online 2001; 3: 47-48; Acton et al. Biol Reprod 2007; 77:
10 Mitochondrial homoplasmy maintained by autologous mitochondrial supplementation El Shourbagy et al. Reproduction 2006; 131:
11 Mitochondrial homoplasmy maintained by autologous mitochondrial supplementation Mitochondria extracted from brilliant cresyl blue (BCB) + sister oocytes El Shourbagy et al. Reproduction 2006; 131:
12 BCB model identifies fertilisable and non-fertilisable oocytes BCB + oocyte (high quality) BCB oocyte (low quality) G6PDH activity Intensity of BCB staining Oocyte Growth El Shourbagy et al. Reproduction 2006; 131:
13 Higher mtdna copy numbers in developmentally competent oocytes BCB +
14 Higher mtdna copy numbers in developmentally competent oocytes BCB + BCB maturing (0 hr and 22 hr) immature (44 hr) mature (44 hr) oocyte
15 Higher mtdna copy numbers in developmentally competent oocytes BCB + BCB BCB + and BCB - maturing (0 hr and 22 hr) immature (44 hr) mature (44 hr) oocyte Comparison of MII oocytes (T-Test)
16 Depletion of mitochondria results in poor fertilization and embryo development 22 hr IVM ddc - 22 hr IVM Depleted oocytes do not fertilise or embryos arrest 44 hr IVM ddc - 44 hr IVM Red: MitoTracker Red; Blue: DAPI ddc: mtdna replication inhibitor, 2',3'-dideoxycytidine Spikings et al. Biol Reprod 2007; 76:
17 Minimum threshold of mtdna copy number required for fertilisation >200,000 ~150,000 mtdna copy number/cell BCB + BCB - Threshold Mitochondrial supplementation Fertilisation Blastocyst Trophectoderm mtdna Set Point Heart Muscle Neurons Blood 200 Primordial Germ Cells Primordial germ cells Primordial follicle Preimplantation Development Postimplantation Development Birth
18 Supplementation with genetically identical mitochondria (micsi) to overcome threshold ~ 800 copies of mtdna Mitochondria extracted from brilliant cresyl blue (BCB) + oocytes BCB - oocyte
19 Supplementation MitoTracker Green TMRM MitoTracker Deep Red Merged Brightfield Zoom 1 hr 40um 5um 24 hr 40um 5um
20 Improvement in fertilisation and development of BCB - embryos following micsi Insemination Total oocyte*/ MII number % Fertilisation (total) % Blastocyst/ Fert (total) % Blastocyst/ Fert (±S.D) IVF 764 * ± 13.9 BCB + ICSI ± 15.3 micsi ± 13.8 IVF 507 * ± 5.8 a BCB - ICSI ± 11.0 a.b micsi ± 15.6 b Cagnone et al. 2016
21 Improvement in fertilisation and development of BCB - embryos following micsi Insemination Total oocyte*/ MII number % Fertilisation (total) % Blastocyst/ Fert (total) % Blastocyst/ Fert (±S.D) IVF 764 * ± 13.9 BCB + ICSI ± 15.3 micsi ± 13.8 IVF 507 * ± 5.8 a BCB - ICSI ± 11.0 a.b micsi ± 15.6 b Cagnone et al. 2016
22 Improvement in fertilisation and development of BCB - embryos following micsi Insemination Total oocyte*/ MII number % Fertilisation (total) % Blastocyst/ Fert (total) % Blastocyst/ Fert (±S.D) IVF 764 * ± 13.9 BCB + ICSI ± 15.3 micsi ± 13.8 IVF 507 * ± 5.8 a BCB - ICSI ± 11.0 a.b micsi ± 15.6 b Cagnone et al. 2016
23 Improvement in fertilisation and development of BCB - embryos following micsi Insemination Total oocyte*/ MII number % Fertilisation (total) % Blastocyst/ Fert (total) % Blastocyst/ Fert (±S.D) IVF 764 * ± 13.9 BCB + ICSI ± 15.3 micsi ± 13.8 IVF 507 * ± 5.8 a BCB - ICSI ± 11.0 a.b micsi ± 15.6 b Cagnone et al. 2016
24 Improvement in development of BCB - embryos following micsi as evidenced by increased cell number Cagnone et al. 2016
25 Cagnone et al Regulation of mtdna copy number during embryo development
26 Cagnone et al Regulation of mtdna copy number during embryo development
27 Cagnone et al Regulation of mtdna copy number during embryo development
28 Cagnone et al Regulation of mtdna copy number during embryo development
29 Cagnone et al Early rescue supports blastocyst development in BCB - embryos
30 Genes differentially expressed between micsi BCB - and ICSI BCB - blastocysts Cagnone et al. 2016
31 Conclusion Blastocyst Baby mtdna copy number Resetting of embryonic genome Blastocyst Baby
32 Acknowledgements Centre for Genetic Diseases Gael Cagnone Te-Sha Tsai Yogeshwar Makanji Pam Matthews Mat McKenzie Shahy El Shourbagy Emma Spikings Harvard, USA David Sinclair Michael Bonkowski UNSW Ashley Wong Lindsay Wu Monash University Kirstin Elgass MHTP Jodee Gould
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