Is oxygen availability a limiting factor for in vitro folliculogenesis?

Abstract

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles’ health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

Do Bioreactor Designs with More Efficient Oxygen Supply to Ovarian Cortical Tissue Fragments Enhance Follicle Viability and Growth In Vitro?

“…Figure 1. Schemes of the bioreactor configurations used for the in vitro culture of fragments of ovarian cortical tissue in the literature reports analyzed in this study: (a) conventional dish (CD), (b) CD with medium mixed by holding the CD on an orbital shaker during culturing [17], (c) CD with medium mixed by recirculating medium above the tissue fragments with an external pump [18], (d) submerged fragment culture on a permeable membrane at the bottom of an insert suspended in a well of a 24-well plate [15,16,33], (e) fragment culture on a permeable membrane at the bottom of an insert suspended in a well of a 24-well plate under a drop of medium [16], and (f) dish with a gas-permeable bottom (PD) [19]. Gas supply portrayed as green arrows.…”

“…Alternatively, it has been that the O 2 be supplied from both the upper and the lower surface of the tissue fragments. This has been pursued either by culturing the fragments on permeable inserts suspended in a well of a multi-well plate submerged in static medium (Figure 1d-e) [15,16], or in dishes with a gas-permeable bottom (PD) (Figure 1f) [19]. In both cases, minimization of the resistance to O 2 transport external to the fragments has been pursued using a culture under medium layers of decreasing thicknesses.…”

“…Various bioreactor designs have been explored to overcome the limitations associated with the use of CDs [14], which were intended to enhance the transport of dissolved gases (i.e., O 2 and CO 2 ) from the gaseous atmosphere in the incubator to the follicles in the tissue. The inconsistent results reported [15][16][17][18][19] cast doubt on the fact that the bioreactor designs that enhance gas transport may significantly influence follicle viability and growth in the in vitro culture of ovarian cortical tissue.…”

“…In the in vitro culture of fragments of human ovarian cortical tissue, it has been reported that gaseous O 2 concentrations higher than in air ensure adequate oxygenation and promote follicle viability long-term, as well as follicle activation and progression [32]. Recently, it has been shown that too low or too high dissolved O 2 concentrations in tissue are detrimental to both follicle viability and development [19]. In this analysis, the effect of gaseous CO 2 transport was not considered because in Talevi et al [19], it was shown to be less relevant than O 2 , at least for the bioreactors and the conditions investigated therein.…”

“…Recently, it has been shown that too low or too high dissolved O 2 concentrations in tissue are detrimental to both follicle viability and development [19]. In this analysis, the effect of gaseous CO 2 transport was not considered because in Talevi et al [19], it was shown to be less relevant than O 2 , at least for the bioreactors and the conditions investigated therein.…”