P-412 Long-term in vitro dynamic culture of bovine ovarian cortical tissue (BOCT) increases follicle growth, viability and AMH secretion
Authors: Barbato V , Genovese V , De Gregorio V , Travaglione A , Candela A , Catapano G , Amoresano A , Serpico S , Pinto G , Mondrone G , D'Hooghe T , Longobardi S , Zheng W , Gualtieri R , Talevi R 
Published: June 2023

Abstract

Study question
Does improved oxygen availability and biomechanical stimulation during long-term dynamic in vitro culture of BOCT enhance secondary follicles growth and health?

Summary answer
Long-term dynamic versus static in vitro culture of BOCT increases the proportion, viability and AMH secretion of growing follicles.

What is known already
A limiting factor in multistep in vitro folliculogenesis is the low yeld of healthy secondary follicles after ovarian cortical tissue culture. We previously reported that dynamic in vitro culture of ovarian cortical tissue for 7 days in a newly designed perifusion bioreactor (patent 102020000027290) enhances follicle growth and health compared to conventional static culture. The bovine represents a valuable animal model to study early folliculogenesis in vitro.

Study design, size, duration
Bovine ovaries (age 8-24 months) were collected from slaughterhouse. BOCT strips from the same ovary were cultured for 14 days in perifusion bioreactors (PB, dynamic culture) and conventional dishes (CD, static culture). BOCT cultured in both PB and CD was monitored by collecting spent media every 2 days and through histology, live-dead confocal analysis at the end of culture.

Participants/materials, setting, methods
BOCT slices (0.5mm thick) were obtained by a custom tissue slicer, chopped into 1x1mm strips and cultured in group of 10 under in PB and CD. Follicle stages and quality in fresh (D0) and cultured tissues were evaluated by histology (hematoxylin-eosin staining), follicle viability was estimated by labelling with live-dead far-red and propidium iodide at the confocal microscope. Whole tissue viability (spectrophotometric LDH assays) and AMH secretion (mass spectrometry) were evaluated on spent media.

Main results and the role of chance
Overall, 939 follicles were analyzed (histology, 338; viability, 601). Day 0-Follicle stages: primordial, 82.4%; primary, 13.2%; secondary, 4.4%; follicle quality: grade 1-2, 37,6%; grade 3, 62,4%; live follicles: 94.3%. Day 14: PB vs CD - Follicle stages: primordial, 3.8 vs 5.7%,NS; primary, 75 vs 86.1%, NS; secondary, 21.2 vs 8.2%, P &lt; 0.05; follicle quality: grade I/II, 70 vs 18.9%, P &lt; 0.01; grade III, 30 vs 81.1%, P &lt; 0.01; live follicles 62.6 vs 35.3%, P &lt; 0.01. LDH activity in PB was markedly lower than in CD at all culture times (PB14d 150.5 nmol vs CD14d 233.1 nmol). During 14 days culture, AMH secretion continuously increases in PB (1700 picomol/Vtot) whereas in CD it markedly decreases during culture, falling down at very low values at the end of culture (67 picomol/Vtot). Findings indicate that the increased transport of solutes and dissolved oxygen, and the biomechanical stimulation in our novel dynamic bioreactor are key factors during ovarian tissue culture. Analysis of LDH and AMH levels in spent media confirm that dynamic culture better mantains general tissue health and promote a better functionality of growing follicles.

Limitations, reasons for caution
Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue.

Wider implications of the findings
Poor yield of secondary follicles during organ culture is a limiting step in the in vitro production of mature oocytes from primordial follicles. The use of a newly developed dynamic bioreactor for long-term culture could represent a valuable tool for in vitro multistep folliculogenesis.

Trial registration number
not applicable