P-456 Differential distribution of the extracellular matrix components within the human ovarian cortex and remodelling during in vitro culture
Authors: Grosbois J , Bailie E , Anderson R , Telfer E 
Published: June 2022

Abstract

Study question
How is the extracellular matrix (ECM) network distributed within the human ovarian cortex, and how does in vitro culture alter its structure?

Summary answer
The ECM composition varies in the different layers of the ovarian cortex and is further remodelled during in vitro culture.

What is known already
The ovarian ECM is the scaffold within which follicles and stromal cells are organised. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Culturing primordial follicles within ovarian strips enables to reproduce the exact composition and stiffness of the native ECM. Yet, tissue preparation, which involves mechanical loosening, induces modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. Characterising the native ovarian cortical ECM and its dynamic changes during culture will help decipher its role during folliculogenesis and improve both in vitro activation (IVA) and in vitro growth (IVG) systems.

Study design, size, duration
Fresh ovarian cortical biopsies were obtained from 6 women aged 28–38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1×0.5 mm and cultured for 0, 2, 4 or 6 days.

Participants/materials, setting, methods
Stromal cell density as well as the percentage of ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin contents were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions.

Main results and the role of chance
Collagen content of the cortical ECM decreased from 55.5% ± 1.7% positive area at day 0 (D0) to 42.3% ± 1.1% at D6 (p = 0.001), and elastin increased from 1.1% ± 0.2% at D0 to 1.9% ± 0.1% at D6 (p = 0.001). Fibronectin and laminin remained stable. This suggests tissue loosening during culture, in accordance with the decreased stromal cell density from 3.6x106 ± 0.6 to 2.8x106 ± 0.3 cells/mm3 at D2 (p = 0.033) with no subsequent change. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Collagen deposition was maximal at the outer cortex and the lowest at the mid-cortex (69.4% ± 1.6% vs 53.8% ± 1.5% positive area, p &lt; 0.001), and decreased from D0 to D2 (65.2% ± 2.0% vs 60.6% ± 2.2%, p = 0.033) then stabilised. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7% ± 0.3% vs 0.9% ± 0.2% in the outer cortex, p &lt; 0.001), and peaked at D6 compared to D0 (3.1% ± 0.3% vs 1.3% ± 0.2%, p &lt; 0.001). These data indicate a distinct phenotype of the ovarian cortical ECM depending on both its region and the culture period. Further characterisation of the differences in ECM architecture is ongoing.

Limitations, reasons for caution
Ovarian cortical biopsies were obtained from pregnant women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution of non-pregnant women.

Wider implications of the findings
Clarifying the composition and architecture signature of the ovarian cortical ECM will not only provide a foundation for further exploration of ovarian microenvironments, but also be of importance for understanding of ECM-follicle interactions maintaining the primordial pool and early growth stages leading to improvements in IVA and IVG.

Trial registration number
not applicable