O-218 The effect of mTOR activation on human primordial follicle activation during in-situ culture
Authors: Ghezelayagh Z , Abtahi N , Valojerdi M. Rezazadeh , Ebrahimi B 
Published: August 2021

Abstract

Study question
What is the effect of mTOR pathway activation on human primordial follicles in-situ activation and subsequent development following tissue cryopreservation?

Summary answer
Temporary treatment of cryopreserved human ovarian tissue with mTOR activators cause the initiation of primordial follicle development and influence steroidogenesis.

What is known already
In-vitro activation of primordial follicles to produce mature oocytes provides an alternative technique for fertility preservation. The employment of different stimulators of PI3K pathway has been successfully used to activate resting follicles during culture or prior to grafting in patients with premature ovarian insufficiency. The addition of phosphatidic acid (PA) and propranolol (PP), as mTOR stimulators, in the culture medium has promoted primordial follicle activation morphologically in mouse and human ovaries. Molecular and functional evaluations of primordial follicle activation after treatment with the mentioned stimulators has not been conducted.

Study design, size, duration
Ovarian tissues which were donated from 6 transsexual patients (23-35 years old), were dissected and cryopreserved with slow-freezing technique. After thawing, they were cut into 1x1x1 mm fragments and incubated with different stimulators in three groups: 1) Control (without stimulators), 2) PA (200µM), and 3) PA+PP (200µM & 50µM respectively). In groups two and three, ovarian fragments were cultured for 24 hours in presence of stimulators and then cultured for additional 6 days without stimulators.

Participants/materials, setting, methods
The cultured ovarian fragments were directly processed for Hematoxylin and Eosin staining and Western blot analysis. The proportion of morphologically normal and degenerated primordial and growing follicles and 17β-estradiol (E2) level in the culture medium were compared after 1 and 7 days of culture to assess follicular development and function. Western blot analysis for phosphorylated and non-phosphorylated status of FOXO3a and RSP6 proteins expression were compared after 24 hours of incubation.

Main results and the role of chance
The proportion of primordial and growing follicles were not significantly different in the experimental groups after 24 hours of incubation with either of the stimulators. Western blot analyses indicated a significant reduction of FOXO3a in the PA+PP group compared to the control group. The phosphorylation level of RPS6 protein did not significantly change in either of the groups. The proportion of transitional follicles were significantly higher in the PA group compared to other groups after 7 days of culture. The E2 secretion level was significantly higher at the last day of culture compared to day 1 for all groups. At the end of the culture period, E2 levels was significantly higher in both PA and PA+PP groups compared to the control group.

Limitations, reasons for caution
Due to ovarian fragmentation before culture, the HIPPO pathway downstream molecules should have also been evaluated by western blot, which was not contributed in this study.

Wider implications of the findings
The results demonstrate the beneficial effect of mTOR signaling to accelerate early primordial follicle recruitment in cryopreserved-thawed human ovarian fragments.

Trial registration number
not applicable