P-343 Assessing the effect of adipose tissue-derived stem cell-conditioned medium on follicles and stromal cells in cultured ovarian tissue
Authors: Vitale F , Camboni A , Donnez J , Dolmans M  M 
Published: July 2024

Abstract

Study question
Does addition of adipose tissue-derived stem cell-conditioned medium (ASC-CM) enhance follicle survival and development and stromal cell viability in ovarian tissue culture?

Summary answer
Adding ASC-CM to ovarian tissue in vitro culture (IVC) promotes follicle survival, development, estradiol (E2) production, and stromal cell viability.

What is known already
Despite extensive efforts to optimize ovarian tissue IVC conditions, outcomes of follicle in vitro development remain suboptimal, mainly showing poor follicle survival and growth. IVC conditions that best resemble the in vivo environment are likely to generate less cellular distress and yield better outcomes. ASCs are known for promoting cell proliferation and survival through secretion of growth factors and cytokines. CM obtained from ASC culture contains these beneficial biomolecules and could potentially be a valuable supplement for ovarian tissue IVC.

Study design, size, duration
This prospective experimental study used 8 fragments of bovine ovarian cortex, which were cultured for 8 days in two different groups: (i) standard ovarian tissue culture (OT Ctrl D8); and (ii) ovarian tissue cultured with ASC-CM supplementation (OT+CM D8). One fragment per sample served as a non-cultured control (D0). Half of the culture medium was replaced every other day, and medium from days 2 and 8 was stored for further analyses.

Participants/materials, setting, methods
E2 production was evaluated on days 2 and 8 by ELISA. Follicle classification was established using hematoxylin and eosin (H&E) staining. Follicle and stromal cell apoptosis rates were assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) labeling served as a follicle quality marker. ASC-CM was analyzed to identify and measure soluble factors like vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-β1), associated with protective effects.

Main results and the role of chance
The OT+CM D8 group showed significantly lower primordial follicle rates (p = 0.03) and higher secondary follicle rates (p = 0.02) than the OT Ctrl D8 group. Concerning apoptosis, the OT+CM D8 group demonstrated significantly lower follicle (p = 0.014) and stromal cell (p = 0.001) rates than the OT Ctrl D8 group. Furthermore, follicles in the OT+CM D8 group exhibited a significant increase (p = 0.002) in GDF-9 expression compared to those in the OT Ctrl D8 group. E2 production was also significantly higher (p = 0.04) after 8 days of culture in the OT+CM D8 group. All studied paracrine factors (VEGF, IL-6 and TGF-b1) were found to be present in the ASC-CM.

Limitations, reasons for caution
This study focuses only on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue, and not on subsequent steps.

Wider implications of the findings
Our study demonstrates that adding ASC-CM to ovarian tissue IVC medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. Moreover, our data suggest that ASC-derived factors VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects.

Trial registration number
not applicable