REPROTEN for in vitro folliculogenesis: new perspectives for biotechnologies applied to animal reproduction.

Exploiting novel strategies aimed at boosting the reproductive performance of domestic animals or preserving biodiversity by counteracting the loss of mammalian endangered species remains a challenging goal of Assisted Reproductive Technology (ART)(1). Among ART techniques, in vitro recapitulation of early phases of folliculogenesis (ivF) represents an efficient way to recruit the reserve of immature and low competent female gametes towards ART. Importantly, the reproductive research area has been developed in the last decade to leading-edge advances based on the use biocompatible materials designed for engineering reproductive systems. The new field  of REPROductive Tissue ENgineering (REPROTEN) has been proposed to generate innovative solution for infertility (2). To this aim, the present research has been designed to assess the possibility to exploit a reproductive biomimetic material for innovating ivF protocols applied to prepubertal sheep.  The advancement of ivF protocol has been designed by comparing the culture of single ovine preantral follicles on a previously validated method (3D-oil method) (3) with a new one carried out on biomimetic electrospun poly(epsilon-caprolactone) (PCL) fibers made with different topology (random vs. patterned fibers)(4). Perfomances of both ivF systems (3D-oil vs PCL) were assessed by analyzing the morphological and functional outcomes of in vitro grown early antral (EA) follicles and oocytes. Gathered data showed how only PCL scaffold with patterned topology was able to support follicle growth under a constant eCG stimulation. In addition, PCL patterned system boosts ivF performances by increasing the rate of follicle growth (75% vs. 70% patterned PCL vs 3D-oil: p>0.05) and the incidence of antrum formation (88% vs. 63%: p<0.01), by doubling the expression of the aromatase enzyme (2-fold increase: p<0.05) which is responsible for the estradiol production, and, by improving the rate of oocyte maturation rate (80% vs. 68%: p<0.01). Overall, the present research has demonstrated for the first time that REPROTEN can be applied to ivF by generating concrete solutions for broadening the use of ART to the large source of immature gametes, a key step toward the improvement of reproduction performances in domestic animals and the preservation of endangered species.