Role of the PI3K and Hippo pathways in follicle activation after grafting of human ovarian tissue.

PURPOSE: Our aim was to elucidate the mechanisms involved in follicle activation of the ovarian reserve after human ovarian tissue transplantation, with specific focus on the role of the effectors of the PI3K (mTOR and FOXO1) and Hippo (YAP) signaling pathways and whether they are somehow altered. METHODS: Frozen-thawed ovarian tissue was collected from six women (age 25-35 years) undergoing surgery for non-ovarian pathologies and divided into 4 fragments in each case: one for non-grafted controls and three for grafting to immunodeficient mice for 3, 7 and 21 days. The tissue was processed for hematoxylin and eosin staining, immunohistochemistry and immunofluorescence at different timepoints before and after grafting. Activation of the PI3K and Hippo signaling pathways was investigated by analysis of mTOR phosphorylation, FOXO1 cytoplasmic localization and YAP nuclear localization. RESULTS: No change in mTOR levels was observed in primordial follicles post-transplantation, but a significant upturn was recorded in growing follicles compared with primordial follicles, irrespective of grafting time. A higher percentage of primordial follicles was also found with FOXO1 in the cytoplasm after 3 days of transplantation than in non-grafted controls. Finally, a greater proportion of primordial follicles was detected with YAP in the nucleus at all timepoints after grafting. CONCLUSIONS: This study supports the hypothesis that follicle activation may occur as an early event after transplantation, with follicle growth and death both contributing to the burnout phenomenon. This is the first time that the effectors of the PI3K and Hippo pathways have been investigated in grafted human ovarian tissue and their role in burnout documented.

In vivo characterization of metabolic activity and oxidative stress in grafted human ovarian tissue using microdialysis.

OBJECTIVE: To characterize oxidative stress and metabolic activity in xenografted human ovarian tissue using microdialysis. DESIGN: Prospective experimental study. SETTING: Gynecology research unit at a university hospital. PATIENT(S): Cryopreserved ovarian cortex from five women 27-35 years of age. INTERVENTION(S): Frozen-thawed human ovarian tissue fragments were xenografted to the back muscle of ten nude mice. Before grafting, a microdialysis probe was placed inside each fragment. MAIN OUTCOME MEASURE(S): Daily reactive oxygen species (ROS), lactate, and glucose levels were collected by means of microdialysis. Follicle loss (hematoxylin and eosin), murine and human vascularization, and vessel stability (CD31, von Willebrand factor, and α-smooth muscle actin triple immunofluorescence) were analyzed on post-grafting days 10 and 21. RESULT(S): Lactate levels were significantly higher than glucose levels until day 10, after which time the lactate-glucose ratio stabilized at ∼1:1. Regarding ROS generation, there were two peaks on post-grafting days 10 and 17. Total vascularization increased significantly up to day 10 and remained similar up to day 21. However, murine vessel area and stabilization significantly increased up to day 21. Major follicle loss occurred in the first 10 days after transplantation. CONCLUSION(S): Our data validated microdialysis as a tool to characterize metabolic behavior and oxidative stress in grafted ovarian tissue. Three different post-grafting periods were identified according to the metabolic activity of grafted tissue, showing a long progression from anaerobic to aerobic metabolism and a protracted period of ROS generation. Oxidative stress was observed relatively late, after the most critical period of follicle loss, and lasted until the tissue vasculature stabilized.