When Electrospun Fiber Support Matters: In Vitro Ovine Long-Term Folliculogenesis on Poly (Epsilon Caprolactone) (PCL)-Patterned Fibers

Abstract

Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes.

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture

“…To fulfill this aim, the present research has taken advantage of the use of electrospun fibers composed of biodegradable biomaterials developed for reproductive tissue engineering (REPROTEN) applied to the ovarian tissue [ 78 , 85 , 86 ] or, alternatively, for performing 3D iv F (in vitro folliculogenesis) protocols aimed to grown PA follicles in culture up to the EA stage [ 78 ].…”

“…These innovative bioengineering approaches potentially enable the increasingly physiological in vitro modeling of reproductive key events availing of the use of a biocompatible scaffold to encapsulate the isolated follicles and the ovarian-derived cells, whose co-presence may be relevant for the survival and the maintenance of synchronous crosstalk between germinal and somatic follicular compartments. By exploiting the use of the PCL electrospun fibers applied for the fabrication of engineered reproductive materials [ 77 , 78 , 85 , 86 ], follicle culture systems can be developed by preserving the unaltered 3D structure, avoiding oocyte manipulation. This operative step can easily mechanically compromise oocyte integrity [ 119 ].…”

“…Patterned oriented scaffolds, previously validated for ovarian engineering protocols [ 77 , 78 ], were produced by using a polymeric solution for the electrospinning, prepared by dissolving PCL (average Mn 80,000) in glacial acetic acid (VWR, Darmstadt, Germany), with a concentration of 20% w / v , stirred overnight and electrospun directly after immersion for 1 h in an ultrasound bath. The process parameters used for the PCL-Patterned scaffold are standardized [ 77 ] and briefly reported here: the applied voltage was set at 15 kV, the distance between the tip of the needle and the collector was 11 cm, the flow rate of the solution was 0.4 mL/h, and the needle diameter was 23 G. The used electrospinning device was equipped with a climate chamber, which allowed the setting and control of temperature and relative humidity during the process (EC-CLI, IME Medical Electrospinning, Waalre, The Netherlands).…”

“…More in detail, both COCs and whole follicles derived from EA structures were incubated on confluent monolayers of cells isolated from the ovary (ovarian surface epithelium-OSE and granulosa cells-GC) or non-ovarian district (tenocytes-TC). More in detail, OSE cells, previously validated by our group as cell monolayer coculture (positive Ctr) [ 78 , 80 ], were isolated using a surgical scalpel from the ovarian cortex of pre-pubertal ovaries previously incubated in 0.25% Trypsin/EDTA 200 mg/L at 38.5 °C for 5 min. Cell suspensions were collected into a petri dish (6 cm) filled with DPBS solution containing 30% Fetal bovine serum (FBS) (Gibco, Darmstadt, Germany) to inactivate Trypsin, centrifuged, and the supernatant discarded.…”

“…To this aim, IVM of a follicle-enclosed oocyte (FEO) derived from early antral follicles (EA) was performed on patterned poly(epsilon-caprolactone) (PCL) electrospun scaffold, a fibrous construct composed of fibers mimicking the natural morphology of the ovarian tissue, obtained by following a bioinspired approach [ 77 , 78 ]. The feasibility of a biomimetic 3D IVM system was tested by assessing the oocyte in vitro meiotic competence and parthenogenetic activation (MII and parthenogenetic activation rates) by comparing the reproductive FEO outcomes with those obtained under a validated IVM system specifically developed for lamb low competent oocytes [ 39 , 78 , 79 , 80 , 81 ]. The comparative IVM approach has been further exploited to test the role of gonadotropin stimulation (eCG + hCG) and/or cell-based cocultures to generate a controlled IVM system standardized for low-competent oocytes.…”