P-350 Recombinant human anti-Müllerian hormone reduces doxorubicin-induced reproductive aging by regulating Id3 expression and promoting DNA repair
Authors: Chang E  M , Godin P , May P , Chauvin M , Kashiwagi A , Nagykery N , Pepin D 
Published: July 2024

Abstract

Study question
What is the mechanism of ovarian damage after doxorubicin (Dox) treatment and how recombinant human anti-Müllerian hormone (rhAMH) suppresses ovarian aging and prevents chemotherapy-induced damage?

Summary answer
Administration of rhAMH attenuates Dox-induced follicle depletion and protects the ovarian reserve in mice, primarily by reducing DNA damage via Id3 upregulation.

What is known already
Treatment with rhAMH results in reversible ovarian quiescence that can reduce primordial follicle depletion induced by Dox when it is used as an adjuvant. However, the exact mechanism of Dox-induced ovarian toxicity and the mechanisms by which rhAMH rescue this damage were unknown.

Study design, size, duration
For the in vivo experiments: 25-day-old mice were randomized into four treatment groups (Control, Dox, Dox+rhAMH, and rhAMH) for three different timepoints (n = 5 per group, total n = 60). Ovaries were removed for analysis at 4, 24 hours and and 1 week after treatment. For the in vitro studies: 21-day-old mice (n = 30) were treated with subcutaneous injection of diethylstilbestrol (DES) to enhance preantral follicle growth, and primary granulosa cells were collected by mechanical dissociation.

Participants/materials, setting, methods
Immunohistochemistry with DDX4 and counter staining with hematoxylin was used for differential follicle counts. DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and by TUNEL assay. MTS assay, clonogenic assay and siRNA Id3 RNA interference knockdown experiment were carried out to reveal the effects of doxorubicin and rhAMH on primary granulosa cell survival.

Main results and the role of chance
Treatment with Dox alone caused significant loss of PMF reserve (59% reduction, 5.25 versus 12.76 in control, P&lt;0.0001) and this was significantly attenuated by coadministration with rhAMH (33% rescue, 9.47 P&lt;0.001). The DNA damage marker γH2AX was elevated in primordial follicle (PMF) and primary follicle (PF) oocytes from Dox-treated ovaries 4 h after treatment, compared with controls (P&lt;0.0001) and was significantly reduced (95% reduction) in rhAMH cotreated ovaries (P&lt;0.001). Oocytes of secondary follicle (SF) and antral follicle (AF) were negative for γH2AX while granulosa cells (GCs) showed increased positivity for γH2AX after Dox treatment with significant reduction after rhAMH cotreatment. (24% reduction, P&lt;0.001). Oocytes and somatic cells of PMF and PF rarely showed positive TUNEL staining at any of the timepoints evaluated.In vitro study showed reduced double stranded breaks (DSBs) in Dox-rhAMH cotreated GC compared to Dox alone indicated by number and signal intensity of γH2AX positive cells. Finally, while rhAMH treatment increased Id3 expression in early stage oocyte and granulosa cells, siRNA Id3 knock-down in granulosa cells significantly reduced the protective effect of rhAMH, as seen by the increased DSBs and pyknotic morphology of cells after Dox suggesting Id3 is downstream of AMH and enhancing Dox-induced DSB DNA repair.

Limitations, reasons for caution
This study failed to detect fate of PMF after DNA damage. PMF distinguished without apoptotic finding and this might be because experiment was conducted in limited time point or mechanism other than apoptotic cell death are responsible for PMF loss such as shedding, phagocytosis or necrosis.

Wider implications of the findings
Understanding of the mechanism behind rhAMH prevention of chemotherapy-induced reproductive aging is important for reproductive health and longevity. Possibility of rhAMH as non-invasive ferto-protective agent during chemotherapy may overcome limitation of current fertility preservation method which is limited by time requirement, cost and success rate.

Trial registration number
’not applicable’