Activation Or Apoptosis: Combined Suppression of Intrinsic and Extrinsic Follicle Activation Pathways Completely Prevents Cyclophosphamide-induced Follicle Loss, while DNA Damage doesn’t end in Oocyte Apoptosis

Oren Kashi 1 Hadassa Roness 1 Itay Spector 1 Sanaz Dereh Haim 1 Elina Tsirulnikov 1 Dror Meirow 1,2
1Kahn Center for Fertility Preservation, "Sheba" Medical Center, Israel
2Sackler Faculty of Medicine, Tel Aviv University, Israel

Introduction:
Follicle activation is suggested as major mechanism of chemotherapy-induced ovarian reserve depletion. Temsirolimus (Tem), an mTOR inhibitor, and rAMH were separately shown to partially reduce cyclophosphamide (Cy) primordial follicle (PMF) loss. Direct oocyte death following Cy is another mechanism proposed to destroy ovarian reserve.

Aim:
To determine chemotherapy-induced follicle loss mechanism in mice.

Methods:
Mice were treated with Cy vs. control, with/without TEM, rAMH or combined. Ovaries removed 1d following Cy treatment were analyzed for PI3K pathway proteins or immunostained for DNA damage (γH2AX, 12h after exposure), apoptosis (PARP, 24h), and proliferation (Ki-67, 24h). Total follicles were counted in ovaries removed 21d after exposure.

Results:
Cy caused significant loss of PMF (decrease of 78%), while combined Tem+rAMH co-treatment completely protected the PMF reserve, to control levels. Granulosa cell proliferation in primary follicles increased markedly following exposure to Cy, and significantly reduced in Cy+Tem+rAMH treated ovaries. Protein analysis demonstrated increased phosphorylation of rpS6 and mTOR following Cy, which was reduced significantly in Cy+Tem+rAMH group. High proportion of PMF oocytes (94%) stained positively to γH2AX 12h post Cy exposure. However, only 22% of PMF stained positive for cPARP indicating apoptosis. Both DNA damage and apoptosis were significantly reduced in Cy+Tem+rAMH treated ovaries.

Conclusions:
This study demonstrates that combined intrinsic-extrinsic inhibition of follicle activation completely protected against chemotherapy-induced follicle depletion.

Our data also indicates high baseline level of non-lethal DNA damage in PMF oocytes as shown by γH2AX, while only a small proportion of these oocytes also stained for apoptosis marker cPARP.