P-410 First wave of primordial follicle activation during human ovarian tissue manipulation for fertility preservation
Authors: Barretta M , Cacciottola L , Hossay C , Donnez J , Dolmans M M 
Published: June 2023

Abstract

Study question
Does ovarian tissue biopsy, transportation and processing for fertility preservation and restoration trigger primordial follicle activation?

Summary answer
Early manipulation of ovarian tissue is sufficient to trigger follicle activation by stimulating PI3K/Akt and disrupting the Hippo pathway.

What is known already
Primordial follicle recruitment occurs continuously in physiological conditions by modulation of autocrine and paracrine factors, like the PI3K/Akt and Hippo pathways, to ensure follicle growth over time. During fertility preservation and restoration procedures, follicle activation may be disrupted and follow nonphysiological patterns. The ability to control activation dynamics by up- or downregulation of these pathways may enhance fertility restoration outcomes in a number of ways. Indeed, downregulation of follicle activation shortly after transplantation may protect the ovarian reserve from early depletion. Conversely, ovarian tissue in vitro culture may benefit from upregulation of primordial follicle activation to boost further growth.

Study design, size, duration
Fresh ovarian tissue was retrieved from nine women undergoing laparoscopic surgery for benign conditions. Three timepoints were investigated. One-third of collected tissue per patient was immediately fixed in the operating room, without any manipulation (time zero, T0). The remaining tissue was transferred to the laboratory and dissected to remove any surplus medulla. It was then cut into small cortical fragments, half of which were fixed after 25 minutes(T25) and the other half after 90 minutes(T90).

Participants/materials, setting, methods
All cortical fragments were fixed in 4%formaldehyde and embedded in paraffin for histology. In order to explore follicle activation,markers of the PI3K/Akt and Hippo signaling pathways were immunolabeled at each timepoint, targeting: (i) phospho-Akt (p-Akt) in primordial follicles by immunohistochemistry as a marker of early PI3K/Akt pathway activation; and (ii) Yes-associated protein (YAP) cellular localizationin the granulosa cell layer of primordial follicles by immunofluorescence as a marker of Hippo disruption.

Main results and the role of chance
An upturn in p-Akt expression was observed at T25 (22,34 ± 0.13%; p = 0.0233) and T90 (39,01 ± 0.22%, p = &lt; 0.0001) compared to T0 (2,87 ± 0,03%). In terms of YAP cellular localization, a significant nucleus-to-cytoplasm shift was detected at T25 (1.11 ± 0.09; p = 0.0428) compared to T0 (0.97 ± 0.10), while T90 (1.07± 0.14) values were similar to T25. Our data prove that ovarian tissue manipulation triggers primordial follicle activation very early, involving both the PI3K/Akt and Hippo signaling pathways, which appear to cooperate in primordial-to-primary follicle transition. Our results indicate that the first stages of any fertility preservation or restoration procedure involving ovarian tissue manipulation contribute to dysregulation of the very mechanisms responsible for the ovarian reserve maintenance and follicle growth. Additional strategies are required to gain the control of follicle activation mechanisms in nonphysiological conditions (ex vivo ovarian tissue manipulation), in order to exploit ovarian reserve dynamics to serve the need of patients.

Limitations, reasons for caution
Analyses in the study were limited to histology and immunolabeling to acquire a descriptive picture of pathway activation kinetics over time. Further investigations using dynamic experimental models are essential to advance our understanding of signaling pathway synergy in vivo.

Wider implications of the findings
Since dysregulation of follicle activation in nonphysiological conditions appears to be associated with poor oocyte quality, enhancing our ability to control the relevant signaling pathways is crucial to optimizing fertility preservation procedures.

Trial registration number
not applicable