Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix duringin vitroculture

Abstract

STUDY QUESTION How does in vitro culture alter the human ovarian cortical extracellular matrix (ECM) network structure? SUMMARY ANSWER The ECM composition and architecture vary in the different layers of the ovarian cortex and are remodelled during in vitro culture. WHAT IS KNOWN ALREADY The ovarian ECM is the scaffold within which follicles and stromal cells are organized. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Tissue preparation for culture of primordial follicles within the native ECM involves mechanical loosening; this induces undefined modifications in the ECM network and alters cell–cell contact, leading to spontaneous follicle activation. STUDY DESIGN, SIZE, DURATION Fresh ovarian cortical biopsies were obtained from six women aged 28–38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1 × 1 mm and cultured for 0, 2, 4, or 6 days (D). PARTICIPANTS/MATERIALS, SETTING, METHODS Primordial follicle activation, stromal cell density, and 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 content, collagen fibre characteristics, and follicle distribution within the tissue 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 Primordial follicle activation occurred concomitantly with a loosening of the ovarian cortex during culture, characterized by an early decrease in stromal cell density from 3.6 ± 0.2 × 106 at day 0 (D0) to 2.8 ± 0.1 × 106 cells/mm3 at D2 (P = 0.033) and a dynamic remodelling of the ECM. Notably, collagen content gradually fell from 55.5 ± 1.7% positive area at D0 to 42.3 ± 1.1% at D6 (P = 0.001), while elastin increased from 1.1 ± 0.2% at D0 to 1.9 ± 0.1% at D6 (P = 0.001). Fibronectin and laminin content remained stable. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Analysis at the sub-region level showed that collagen deposition was maximal in the outer cortex and the lowest in the mid-cortex (69.4 ± 1.2% versus 53.8 ± 0.8% positive area, respectively, P < 0.0001), and cortical collagen staining overall decreased from D0 to D2 (65.2 ± 2.4% versus 60.6 ± 1.8%, P = 0.033) then stabilized. Elastin showed the converse distribution, being most concentrated at the cortex–medulla junction (3.7 ± 0.6% versus 0.9 ± 0.2% in the outer cortex, P < 0.0001), and cortical elastin peaked at D6 compared to D0 (3.1 ± 0.5% versus 1.3 ± 0.2%, P < 0.0001). This was corroborated by a specific signature of the collagen fibre type across the cortex, indicating a distinct phenotype of the ovarian cortical ECM depending on region and culture period that might be responsible for the spatio-temporal and developmental pattern of follicular distribution observed within the cortex. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Ovarian cortical biopsies were obtained from women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution and structure of non-pregnant women. WIDER IMPLICATIONS OF THE FINDINGS Clarifying the composition and architecture signature of the human ovarian cortical ECM provides a foundation for further exploration of ovarian microenvironments. It is also critical for understanding the ECM–follicle interactions regulating follicle quiescence and awakening, leading to improvements in both in vitro activation and in vitro growth techniques. STUDY FUNDING/COMPETING INTEREST(S) Medical Research Council grant MR/R003246/1 and Wellcome Trust Collaborative Award in Science: 215625/Z/19/Z. The authors have no conflicts to declare. TRIAL REGISTRATION NUMBER N/A.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips

“…However, possible differences in the viscoelastic behaviour of the ovarian tissue and the mock wax may indeed bias the strain estimates that have been obtained. Another source of bias is the wide variations in tissue structure ( van Wezel and Rodgers, 1996 ; Grosbois et al, 2023 ) and mechanical behaviour from tissue donor to donor and from different harvesting zones of the same ovary. For the sake of an example, the Young’s modulus of the ovarian cortex has been reported to vary by as much as two orders of magnitude in different zones of the same ovary (Stewart et al, 2023).…”

Relationship between Amino Acid Metabolism and Bovine In Vitro Follicle Activation and Growth

“…These metabolites enter gluconeogenesis via the tricarboxylic acid cycle (TCA) cycle, while Hypro is an abundant constituent of collagen and elastin [ 28 ], which are the major components of the extracellular matrix (ECM) [ 29 ]. ECM, such as collagen, has important roles in folliculogenesis [ 30 ] and is altered in human ovarian cortical strips during culture [ 31 ]. Collagen (type IV) is known to be expressed in primordial to antral follicles in mice [ 32 , 33 ], humans [ 34 , 35 , 36 ], and cattle [ 37 ].…”

Bioengineered 3D Ovarian Models as Paramount Technology for Female Health Management and Reproduction

“…Del Valle and colleagues reported a significant increase in collagen IV in the stromal compartment of human ovarian cortex tissue cultured for 8 days, indicating that the ovarian cortex became more fibrotic during culture [51]. By contrast, Groibos and colleagues reported that human ovarian cortex tissue cultured for 6 days showed decreased collagen and increased elastin deposition [65]. This discrepancy may have resulted from differences in either the culture protocols or the donor material used.…”