P-380 Improvement of follicle growth and health during dynamic culture of bovine ovarian cortical tissue (BOCT) is associated to a higher remodeling of the extracellular matrix
Authors: Genovese V , De Gregorio V , Travaglione A , Candela A , Gualtieri R , Talevi R 
Published: July 2024

Abstract

Study question
Is improvement of follicle growth and health during dynamic culture in Perifusion Bioreactor (PB) associated to a healthier condition and remodeling of extracellular matrix?

Summary answer
The dynamic culture in PB allows the extracellular matrix (ECM) network rearrangement improving follicle growth and viability compared to static culture

What is known already
Over the past two decades, in vitro folliculogenesis has achieved moderate success in large mammals, emerging as a distinctive and reliable method for obtaining secondary follicles. Recently, although dynamic culture in bioreactors has been demonstrated to enhance follicle progression, quality, and viability compared to static culture, less emphasis has been devoted to ECM remodeling. Ovarian tissue ECM plays a crucial role in cell–cell, and cell-ECM interactions involved in follicle activation and growth. The comprehension of ECM function deepens our understanding of follicular development and holds significant promise for future reproductive technologies

Study design, size, duration
Bovine ovaries (age 8-24 months) were collected at slaughterhouse. BOCT strips (1x1x0.5mm) from the same ovary were cultured in groups of 10 for 14 days in PB (dynamic culture) and conventional dishes (CD, static culture). At the end of culture, collagen thickness and packing density was assessed through PicroSirius red (PSR) staining, follicle stages and quality through histology, and follicle viability through live-dead far-red and propidium iodide labeling under confocal microscopy

Participants/materials, setting, methods
Neosynthesis and remodeling of collagen fibers was analyzed on PSR-stained samples under polarized light measuring the color of each pixel. A color threshold was set to isolate the four colors seen in PSR-stained samples under polarized light: green, thin fibers (neosynthesized); yellow, mid-sized fibers (low assembly); orange, mid-sized fibers (medium assembly); red, mature thick fibers (high assembly). The color threshold was set as follows: red 2–9, yellow 39-51, orange 10–38, green 52–128

Main results and the role of chance
In fresh tissue, analysis of PSR-stained samples showed the following values: Red 1.35; Orange 4.93; Yellow 0.18; Green 0.15. At Day 14, compared to the static culture, culture in PB, exhibits a significantly higher production of green (PB 0.22 vs CD 0.02 P &lt; 0.01) and yellow (PB 0.56 vs CD 0.26 P &lt; 0.05) fibers indicating an improved neosynthesis and remodeling of collagen and ECM health condition. Such higher ECM remodeling after dynamic culture is associated to a significantly higher percentage of secondary follicles (PB 15.4– CD 4.1%, P &lt; 0.01 ), superior follicle quality (grade I/II, PB 70,5 - CD 28.4%, P &lt; 0.01) and viability (PB 70 – CD 41.1%, P &lt; 0.01). PSR analysis suggests that the continuous nutrients and oxygen supply, catabolites removal, and biomechanical stimulation during dynamic PB culture, stimulates the deposition of newly synthesized collagen which in turn, promotes follicle growth

Limitations, reasons for caution
Although the bovine is a suitable reproductive model for human, such findings should be replicated on human ovarian tissue

Wider implications of the findings
The aim of ovarian tissue culture is to promote the growth of healthy secondary follicles that can be isolated for further culture to generate MII oocytes. The present findings suggest that adoption of dynamic ovarian tissue culture could enhance the outcome of folliculogenesis in vitro

Trial registration number
not applicable