Anti-Mullerian Hormone (AMH) for prevention of ovarian

follicle depletion after pre-pubertal ovarian cortex

xenotransplantation

Laura Detti, Irene Peregrin-Alvarez, Robert Roman

University of Tennessee Health Science Center, Memphis, USA

Disclosures

Nothing to disclose

Introduction

Ovarian tissue preservation techniques have been primarily applied to post-pubertal females undergoing chemotherapy or radiotherapy Fertility is only one of the benefits achieved with autotransplantation of ovarian cortex Return to ovarian endocrine function is of great importance in pre-pubertal girls, who could regain their ability to undergo spontaneous puberty, in addition to preserving their bone, cardiovascular, and overall health

Jadoul P, et al. Hum Reprod Update 2010;16:617–30.

Introduction

In ovarian cortex transplant studies, there is massive depletion of primordial follicles during the first week after transplant. - Slow vascularization of the transplanted tissue? - Hypoxia-driven follicular damage? - Massive cellular activation with consequent apoptosis?

- Cryopreservation process itself?

Liu J, et al. Hum Reprod 2002;17:605–11.

Gook DA, , et al. Hum Reprod 1999;14:2938– 40.

Introduction

FSH and LH

GnRH-agonists - triptorelin pamoate, goserelin

Sphyngosine-1-phosphate

VEGF

mTOR inhibitors - rapamycin Metformin - ?

Gook DA, et al. Hum Reprod 2001;16:417–22. Flaws JA, et al. Biol Reprod 1997;57:1233–7.

Maltaris T, et al. Reproduction 2007;133:503–9.

Oktem O, et al. Reprod Scien 2007;14:358–66. Soleimani R, et al. PLoS One 2011;6:e19475.

Abir R, et al. Fertil Steril 2011;95:1205–10.

Yu J, et al PLoS One 2011 Zhang J, et al J Cell Physiol 2017

‘Follicular protectants’ at the time of ovarian cortex transplantation:

Introduction

In vitro studies

Exposing ovarian cortex tissue to increasing in vitro

concentrations of AMH caused downregulation of AMH

production and decreased sensitivity to FSH and LH by

downregulation of their receptors.

Inhibitory role on follicular development

Detti L et al. in Press, Reprod Sci 2017.

Introduction

In vitro studies

Exposing ovarian cortex tissue to increasing in vitro

concentrations of rAMH caused downregulation of GDF9

and of stemness markers such as Oct-4, Sox2, and

NANOG, while upregulating VEGF.

Inhibitory role on follicular development

Detti L et al. Submitted, in revision 2017.

Introduction

In vivo studies AMH stalled tissue activation by downregulating GDF9 and stemness markers, Oct-4, Sox2, NANOG, thus regulating ovarian tissue regenerative potential. AMH-treated mice preserved their reservoir of primordial follicles during administration of chemotherapeutic agents such as carboplatin, doxorubicin, or cyclophosphamide

Detti L et al. in Submission 2017.

Kano M et al. Proc Natl Acad Sci USA. 2017.

Can AMH have a role in preventing post-transplant follicular depletion in pre-pubertal ovarian cortex?

Hypothesis

AMH inhibits post-transplant follicular activation thus protecting the primordial follicle reservoir

Materials and Methods

12 NU/J nude mice

Control

(6 mice)

AMH Treated

(6 mice)

Materials and Methods Time 0: Blood draw, ovariectomy and pump placement

Alzet pumps delivered 1.23 mcg rAMH/day to reach a serum concentration of

17.5 ng/mL (x5 physiologic AMH level in humans), or placebo

Materials and Methods

• Time 7 days: Previously vitrified/warmed 2x2 mm ovarian cortex fragments from one pre-pubertal girl were transplanted

Materials and Methods

Time 14 days: Euthanasia, Blood draw, Ovarian cortex explant and Alzet pump retrieval.

Materials and Methods

• Fragments were fixed, cut in serial 5 µm sections, and stained with H&E, Ki67 and TUNEL.

• Ovarian follicles were counted in at least four serial sections, 25 µm apart, in each fragment.

• Computerized Spectrum by Aperio image analysis

Fresh Post-warming Post-warming Placebo

Post-warming Placebo

PDF PDF

PRF

SEF

Results

Results

Summary and Conclusions

• The vitrification-warming process caused a non-significant decrease in PDF in pre-pubertal ovarian cortex.

• rAMH in the peri-transplant period did not preserve the primordial follicle number.

• rAMH in the peri-transplant period prevented follicular activation to PRF and SEF, and post-transplant premature depletion.

• rAMH in the peri-transplant period decreased apoptosis and cellular activation.

AMH could be used as a ‘protectant’ of the ovarian cortex during the peri-transplant period.

Thank you!

Results Serum AMH, estradiol and FSH at time of ovariectomy/pump placement, xenotransplant, and euthanasia

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