CO-TRANSPLANTATION OF HUMAN OVARIAN TISSUE WITH AMH-PRODUCING

CELLS INHIBITS RECRUITMENT OF PRIMORDIAL FOLLICLES IN A SHORT AND

LONG TERM GRAFTS

Limor Man M.D, M.Med.Sc

Assistant Professor of Research in Obstetrics and Gynecology

Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine

Weill Cornell Medicine, NYC, NY, USA

Major Obstacle Undermining Viability and Endocrine Output of Ovarian

Tissue Post-Transplantation

• Loss of oocytes during transplantation occurs due to ischemia

before revascularization

• Massive global activation of follicles upon transplantation is an

obstacle to maintaining long-term endocrine output and

salvaging a robust pool

- Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C. Endocrinology. 1999 Jan;140(1):462-71.

- Dolmans MM, Martinez-Madrid B, Gadisseux E, Guiot Y, Yuan WY, Torre A, Camboni A, Van Langendonckt A, Donnez J. Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude

mice. Reproduction. 2007 Aug;134(2):253-62.

Loss of Oocytes After Transplantation due to Ischemia

An Exogenous Cell Impetus Improves the Survival of

Co-Transplanted Ovarian Tissue

000

Long-Term Viability and Function of Ovarian Tissue Grafts are Improved by ExECs

Massive Global Activation of Follicles upon Transplantation

%

0

10

20

30

40

50

60

70

Primordial Primary Secondary

2W

3W

14W

%

AMH Exert a Repressive Input on Activation and/or Growth of Follicles

Anti-Mullerian Hormone AMH

• Mullerian Inhibiting Substance/Factor-MIS/MIF

• A dimeric glycoprotein, member of the transforming growth

factor beta superfamily –TGFb

• In females , Not expressed during ovarian sex differentiation

• Produced exclusively by granulosa cells of growing follicles from

the late antenatal stage till menopause

• Pool of the primordial follicles decreased faster In AMH-

Knockout than WT mice

-Rajpert-De Meyts E, Jørgensen N, Graem N, Müller J, Cate RL, Skakkebaek NE. Expression of anti-Müllerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells. J Clin Endocrinol Metab. 1999 Oct;84(10):3836-44. -Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA, Themmen AP. Control of primordial follicle recruitment by anti-Müllerian hormone in the mouse ovary. Endocrinology. 1999 Dec;140(12):5789-96

AMH Dual Effect on Follicular Development

primordial primary preantral antral

AMH AMH

FSH FSH

Metabolic demands

-Durlinger AL, Visser JA, Themmen AP. Regulation of ovarian function: the role of anti-Müllerian hormone. Reproduction. 2002 Nov;124(5):601-9.

Vectors-AMH

Human Endothelial Cells

Efficient Transduction of Cultured ECs/MSC

-Man L, Park L, Bodine R, Ginsberg M, Zaninovic N, Man O.A, Schattman G, Rosenwaks Z, James D.(2017). Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue. Sci Rep. 2017

Experimental Design

Muscle

Fibrin Clot

Ovarian Graft

Confocal Microscopy

Endothelial Cells Lectin-Functional Vessels

AMH-Ab Colocalization

Most Significant Benefit to the Retention of the Quiescent Follicular Pool

AMH-ECs AMH-MSCs

Highest Percentage of Primordial Follicles AMH Producing ECs

A Significant Retention of Primordial Follicles was Noted Also at 14 Weeks

0

10

20

30

40

50

60

Primordial Primary Secondary

Blank-Ecs

AMH-Ecs

P=0.05

P=0.01

Conclusions

• Comparison of different treatments AMH-ExECs revealed the most

significant benefit to the retention of the quiescent follicular pool in

short and long term grafts

• These findings present a cell-based strategy that combines

accelerated perfusion with a direct paracrine delivery of a bioactive

payload to transplanted ovarian tissue

• Improved tissue viability and enforced retention of quiescent follicles

can be combined to increase productivity and longevity of ovarian

tissue grafts

Future Directions

• Utilizing engineered ECs expressing secreted factors to interrogate their impact on human ovarian physiology, using our unique In vivo model

• Using a non-cellular method of delivering secreted factors for future clinical use

• Testing AMH as a chemo protectant agent

Supported by

• ASRM grant

• Clinical and Translational Science Center (CTSC) – Weill Cornell

• Internal (CRMI funding)

Acknowledgements

Laura Park

Richard Bodine

Nikica Zaninovic

Glenn Schattman

Zev Rosenwaks

Daylon James

Omar Alexander Man