Autologous mitochondrial injection to increase

energy in oocytes

Robert Casper, M.D., F.R.C.S.C.

University of Toronto

TCART Fertility Partners

Global Demand for Fertility Treatments is Growing

• Approximately 1.5 million in vitro fertilization (IVF) cycles reported per year worldwide

• IVF has been in clinical practice for 35+ years

• Despite significant gains in the live birth rate in good prognosis patients, there are few treatment options for repeated IVF failures

2

Preclinical Support for Safety and Efficacy of Mitochondrial Transfer

3

Convincing evidence that adequate oocyte mitochondrial activity is necessary for embryo development to blastocyst and for successful implantation1

Goals:

• Improve oocyte and embryo quality

• Increase healthy live birth rate

4

New Option: AUGMENTSM (Autologous Mitochondrial Treatment)

1. Chappel, Obst and Gyn Intl 2013; Tilly et al, Cell Metab 2013

Discovery of Egg Precursor Cells

1. White, Y. A. R. et al. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.

Nat. Med. 18, 413–421 (2012).

2. Woods, D. C. & Tilly, J. L. Isolation, characterization and propagation of mitotically active germ cells from adult mouse

and human ovaries. Nat. Protoc. 8, 966–988 (2013).

3. Zou, K. et al. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat. Cell Biol. 11,

631–636 (2009).

4. Zhang, Y. et al. Production of transgenic mice by random recombination of targeted genes in female germline stem

cells. J. Mol. Cell Biol. 3, 132–141 (2011).

5. Zhou, L. et al. Production of fat-1 transgenic rats using a post-natal female germline stem cell line. Mol. Hum. Reprod.

20, 271–281 (2014).

6. Wolff, E. F. et al. Oogonial stem cells generate mature oocytes in an autologous Rhesus macaque transplantation

model. Reprod. Sci. 21, 119A (2014).

7. Anderson, R. A. et al. Evaluation of oogonial stem cells and neo-oogenesis in ovaries of girls and women with Turner

syndrome: a pilot study. Hum. Reprod. 2013; 28 (Suppl.): 52 (2013).

8. Dunlop, C. E., Bayne, R. A., McLaughlin M., Telfer, E. E. & Anderson, R. A. Isolation, purification, and culture of oogonial

stem cells from adult human and bovine ovarian cortex. Lancet 383, S45 (2014).

9. Zou, K., Hou, L., Sun, K., Xie, W. & Wu, J. Improved efficiency of female germline stem cell purification using Fragilis-

based magnetic bead sorting. Stem Cells Dev. 20, 2197–2204 (2011).

10. Park, E. S., Woods, D. C., White, Y. A. R. & Tilly, J. L. Oogonial stem cells isolated from ovaries of adult transgenic

female mice generate functional eggs and offspring following intraovarian transplantation. Reprod. Sci. 20, 75A–76A (2013).

11. Imudia, A. N. et al. Comparative gene expression profiling of adult mouse ovary-derived oogonial stem cells supports a

distinct cellular identity. Fertil. Steril. 100, 1451–1458 (2013).

12. Park, E. S., Woods, D. C. & Tilly. J. L. Bone morphogenetic protein 4 (BMP4) promotes mammalian oogonial stem cell

differentiation via SMAD1/5/8 signaling. Fertil. Steril. 100, 1468–1475 (2013).

13. Xie, W., Wang, H. & Wu, J. Similar morphological and molecular signatures shared by female and male germline stem

cells. Sci. Reports 4, 5580 (2014).

14. Khosravi-Farsani, S., Amidi, F., Roudkenar, M. H. & Sobhani, A. Isolation and enrichment of mouse female germ line

stem cells. Cell J. 16, 406–415 (2015).

15. Park, E. S. & Tilly, J. L. Use of DEAD-box polypeptide 4 (Ddx4) gene promoter-driven fluorescent reporter mice to

identify mitotically active germ cells in postnatal mouse ovaries. Mol. Hum. Reprod. 21, 58–65 (2015).

5

Egg Precursor Cell Mitochondria are Similar to

Oocyte Mitochondria

• Egg precursor cells are unipotent, germline cells in the outer cortex of the ovary

Human Brain Cell Human Oocyte

Human Egg Precursor Cell

Human brain: http://dx.doi.org/10.1093/brain/awu138, 2329-2345 2014

Human oocyte: Nottola et al., Hum Reprod 22:1123–1133, 2007

Human egg precursor cell: Harvard Medical Schoool

500 nm 500 nm 500 nm

6

Human Egg PC

Autologous Mitochondrial Transfer Possible

• Maintains mitochondrial homoplasmy and nuclear and mitochondrial DNA communications

Egg Precursor Cells

1

Nucleus

Mitochondria

(maternally inherited)

Ovarian Cortex

7

1. White et al., 2012

AUGMENTSM Process

Ovarian Tissue Biopsy

Controlled Ovarian Hyperstimulation

Oocyte Retrieval

Embryo Transfer

Mitochondria

ICSI

8

Oocyte precursor cells isolated

Oocyte precursor cell mitochondria

isolated

1. Performed by OvaScience

1 1

• Age

• Oocyte/embryo quality

• Antral follicle count

• Hormone levels: FSH (follicle stimulating hormone), AMH (anti-mullerian hormone)

• BMI (body mass index)

• Environmental factors

• Previous diagnoses: diminished ovarian reserve, endometriosis, etc.

• Previous failed IVF

Lack of agreement on how to rank significance of risk factors

9

Many Conditions are Prognostic for Live Birth

Baseline Clinical Pregnancy Rate

Metric Previous IVF History Data

Number of patients 34

Average current age 36.0 (Range: 26-44)

Background/Diagnoses Poor oocyte & embryo quality with one of

the following diagnoses:

Diminished Ovarian Reserve, Ovulatory

Dysfunction, Polycystic Ovarian Syndrome,

Tubal Factor, Endometriosis, Unexplained

Total previous IVF cycles initiated 71

Average cycles per patient 2 (Range: 1-5)

Total previous embryo transfers

(fresh & frozen)

79

Historical clinical pregnancy rate:

• per cycle initiated

• per embryo transfer

11% (8/71)

10% (8/79)

Historical live birth rate:

• per cycle initiated

• per embryo transfer

1.4% (1/71)

1.3% (1/79)

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Metric AUGMENT

Number of patients 34

Average current age 36.0 (Range: 26-44)

Background/Diagnoses Poor oocyte & embryo quality with one of

the following diagnoses:

Diminished Ovarian Reserve, Ovulatory

Dysfunction, Polycystic Ovarian Syndrome,

Tubal Factor, Endometriosis, Unexplained

Total mitochondrial injection cycles initiated 34

Average cycles per patient 1

Total embryo transfers (fresh & frozen) 26

Clinical pregnancy rate:

• per cycle initiated

• per embryo transfer

35% (12/34)

46% (12/26)

Ongoing clinical pregnancy and live birth rate:

• per cycle initiated

• per embryo transfer

26% (9/34) includes 1 live birth

35% (9/26) includes 1 live birth

Clinical Experience with AUGMENTSM

9 patients have 23 total frozen embryos remaining for transfer

11

Clinical Experience with AUGMENTSM

Patients with embryo transfers (3 had multiple transfers)

30

22

15

Total patients initiated treatment

Patients completed cycles with mitochondrial injection

Positive pregnancy tests (confirmed by quantitative hCG)

26 (42 total transferred embryos)

34

Number of embryo transfers

12

9

1 no sperm obtained, 1 no oocytes retrieved, 2 oocyte banking

3 no fertilization, 4 arrested embryo development, 1 pending frozen embryo transfer

3 chemical pregnancies

3 spontaneous abortions Ongoing clinical pregnancies

and live births*

Clinical pregnancies (confirmed by ultrasound)

12 * Includes 1 live birth

Clinical Experience Shows Improved Pregnancy

Rates Compared to Baseline

Previous IVF

History

AUGMENTSM

Total cycles initiated 71 34

Average cycles initiated per patient 2 1

Total embryo transfers (fresh & frozen) 79 26

Clinical pregnancies 8 12

Clinical pregnancy rate per cycle initiated 11% 35%

Clinical pregnancy rate per embryo

transfer

10% 46%

Ongoing clinical pregnancies and live

births (rate per cycle initiated)

No ongoing and

1 live birth (1%)

9 including 1 live

birth (26%)

13

Conclusion

• Clinical experience with AUGMENTSM during ICSI appears promising

– One live birth and 8 ongoing clinical pregnancies

– Ongoing clinical pregnancy rate of 26% per cycle initiated and 35% per embryo transfer

• Women included in this clinical experience were poor prognosis for live birth

– Failed previous IVF cycles, poor embryo development, and 1.4% live birth rate per previous cycle

• Nine patients have frozen embryos remaining for transfer

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• Dr Yaakov Bentov

• Dr Paul Chang

• Dr Dan Nayot

• Deborah Myers

• Caroline Lux

First Steps

• Dr Marjorie Dixon

• Dr Fay Weisberg

• Dr Navid Esfandiari

• Dr Julia Szeptycki

• Dennis De la Cruz

• Khalid Rao

TCART Clinical TCART Lab

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Acknowledgements