Impact of human ovarian tissue manipulation on follicles: evidence of a potential first wave of follicle activation during fertility preservation procedures

Abstract

Purpose: The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics.

Methods: Fresh ovarian tissue was retrieved from 9 women undergoing laparoscopic surgery for benign conditions. Biopsies from each patient were divided into 3 fragments, the first of which was immediately fixed in the operating room (T0) and the second and third just after processing at 25 (T25) and (T90) 90 min. To evaluate follicle activation, markers of the PI3K and Hippo signaling pathways were immunolabeled at each time point, targeting phospho-Akt (p-Akt) by immunohistochemistry and yes-associated protein (YAP) cellular localization in the granulosa cell layer by immunofluorescence.

Results: Four hundred forty primordial follicles were evaluated for p-Akt and 420 for YAP. Significantly stronger p-Akt expression was observed at T25 (23.01 ± 13.45%; p=0.04) and T90 (38.99 ± 25.21%; p<0.001) than at T0 (2.72 ± 3.35%). A significant nucleus-to-cytoplasm shift in YAP was detected at T25 (1.21 ± 0.25; p=0.015 compared to T0 (0.95 ± 0.09), while T90 (1.10 ± 0.16) values were similar to T25.

Conclusion: Our data prove that ovarian tissue manipulation significantly impacts follicle dynamics by stimulating the PI3K and Hippo signaling pathways involved in primordial follicle activation. Further experimental evidence must nevertheless be gathered to understand and gain control of follicle activation mechanisms in non-physiological conditions (like ovarian tissue manipulation), in order to optimize fertility preservation and restoration strategies.

Keywords: Fertility preservation; Follicle activation; Hippo pathway; Ovarian tissue processing; PI3K/Akt pathway.

Fig. 1

Experimental design. Fresh ovarian tissue was collected from 9 women undergoing laparoscopic surgery and divided into 3 fragments apiece. The first was immediately fixed in the operating room (T0). The other two were processed by removing the medulla and fragmenting the cortex before fixation 25 (T25) and 90 (T90) min following biopsy retrieval. All cortical fragments were fixed in 4% formaldehyde and embedded in paraffin for histological analyses and immunolabeling

Fig. 2

PI3K pathway activation. A P-Akt immunostaining on primordial follicles at different time points. (1) T0 (no staining), (2) T25 (weak staining of the GC layer and oocyte cytoplasm), (3) and T90 (intense staining of the GC layer and oocyte nucleus and cytoplasm). Scale bar = 20 μm. B P-Akt immunostaining in human lung cancer served as a positive control. Scale bar = 100 μm. C P-Akt staining analysis on primordial follicles (as a percentage, mean ± SD) analyzed using one-way ANOVA and Tukey’s post hoc test. Significant differences between time points are indicated by *p <0.05 and ***p<0.001. T0, time zero; T25, time 25 min; T90, time 90 min

Fig. 3

Hippo pathway disruption. A Immunofluorescence targeting YAP (green) and subcellular localization analysis in GC nucleus (DAPI, blue) and cytoplasm, delimited by plasmatic membrane counterstaining (red). (1) T0: predominantly cytoplasmic YAP localization; (2) T25: predominantly nuclear YAP localization; (3) T90: Both cytoplasmic and nuclear YAP localization, with nuclear preponderance. Scale bar = 20 μm. B Immunofluorescence targeting YAP (green), nucleus (DAPI, blue), and cytoplasm, delimited by plasmatic membrane counterstaining (red) in ovarian cancer tissue served as positive control. Scale bar = 20 μm. C YAP distribution trend at different time points (nuclear mean intensity staining/total mean intensity staining ± SD) in primordial follicles evaluated by one-way ANOVA and Tukey’s post hoc test. A significant difference between time points is indicated by * (p< 0.05). D Total YAP concentration levels (nucleus and cytoplasm of the GC layer). No significant differences were found between time points. T0, time zero; T25, time 25 min; T90, time 90 min