Production of ovine fertilizable oocytes using innovativein vitro (iv) technologies applied to preantral follicles growth

Study question: Considering the early pool of gametes potentially able to generate offspring, what actions should be taken to optimize ivF thus increasing fertilizable oocytes availability? Summary answer: The standardization of long-term gonadotropin stimulation is a key step to implement ivF in order to promote both reproductive targets, oogenesis and follicle steroidogenesis activation. What is known already: Increasing in childhood cancer survivors points up an urgent need in fertility preservation (FP) strategies to overcome the negative impact of cancer treatments on reproductive cell cycle. Because of a concern exists on the possible re-introduction of malignant cells following ovarian transplantation, in vitro (iv) culture of early stage follicles still remains a potential alternative to obtain fertilizable oocytes that contribute to biological offspring. Since technologies advances are required to transfer iv Follicologenesis (ivF) from bench to bedside, monovulatory large mammal becomes ideal translational models to validate cultural conditions enhabling synergic follicles-oocyte iv development. Study design, size, duration: The present research aimed to study the influence of 2 different gonadotropins (oFSH vs Pregnant Mare’s Serum Gonadotropin/PMSG) on ivF cultures of single ovine preantral follicle (PA). The ivF outcomes obtained by using the specie-specific (oFSH) or the trans-species chorionic gonadotropin (PMSG) were analyzed after 14 days of culture by comparing follicle/oocyte growth, timing and percentage (%) of antrum differentiation as well as % of Methaphase II (MII) oocytes after iv Maturation (IVM). Participants/materials, setting, methods: Preantral follicles, mechanically isolated from slaugterhoused prepubertal ovaries, were incubated as 3D single follicle culture and exposed to 25 ug oFSH or to different PMSG doses (from 0,4 to 40 IU/ml). In order to compare the degree of meiotic competence, MII oocytes obtained adopting an advanced IVM, consisting of cumulus oocytes complexes co-cultured with walls antral follicle-derived somatic cells, were analyzed. Main results and the role of chance: The PMSG influence on ivF performances was strictly dose-dependent. More in detail, 4 IU was the concentration of PMSG able to enhance follicular diameter increment (Δ%: 63.7±28.9 vs 43.4±19.3 and 52.7±33.4, respectively for 4, 0.40, 40 IU; p<0.05). Conversely, any significant increase in antrum differentiation upon PMSG treatment (4, 0.4, 40 IU) was observed. The chorionic gonadotropin was more effective than oFSH in promoting ivF outcomes. Indeed, preantral follicles stimulated with 4 IU PMSG displayed a significantly greater growth than those exposed to oFSH (Δ%: 63.7±28.9 vs 52±31; p=0,038). Moreover, PMSG promoted a complete meiotic competence in the majority of collected oocytes (58.3% MII, 2.8% GVBD, 38.8% GV). Of note, the MII oocytes were all isolated by early antral follicles with Δ%>40 (p<0.01). On the contrary, only a very small fraction of the oocytes isolated from oFSH-treated follicles was able to resume meiosis and no one reached the MII stage (83,30% GV, 16,70% GVBD; p<0.001). Limitations, reasons for caution: - Wider implications of the findings: These proof-of-concept PMSG experiments establish a way for new protocols optimizing ivF and allow to identify a trans-species hormone to functionalize biomaterials. Both strategies might support follicle development in large animal models and humans with the hope of translating this technology for fertility preservation purposes.