Scientific challenge 1

Thanks to the authors of this scientific challenge to share their protocols. I would like to raise two questions for discussion:

1. Do we currently have enough published or ongoing comparative studies to support the choice of one cryoprotectant over another in ovarian tissue cryopreservation?
2. While preserving follicle integrity is clearly a primary goal, how important is stromal preservation for the success of transplantation in terms of graft revascularization and longevity? Is there any clinical or experimental evidence supporting its role?

1. Do we currently have enough published or ongoing comparative studies to support the choice of one cryoprotectant over another in ovarian tissue cryopreservation?

The key problem is selecting an appropriate endpoint for the investigation. Ideally, pregnancy after transplantation would be the primary outcome, but a study powered for this endpoint would require many years to complete, so surrogate endpoints must be used. One possibility is quantitative assessment of follicle survival after thawing (e.g. Neutral Red staining). However, thaw survival per se is high with current protocols (we observe only ~7% loss of viable follicles following freezing/thawing with ethylene glycol, sucrose and hSA as CPA's), so differences between different CPA's are likely to be too small to guide a definitive choice. Small, non-growing human follicles appear relatively resistant to cooling and freezing; the principal challenge is therefore securing follicle survival during the immediate post-transplantation period, when a substantial proportion of follicles are lost.

1. While preserving follicle integrity is clearly a primary goal, how important is stromal preservation for the success of transplantation in terms of graft revascularization and longevity? Is there any clinical or experimental evidence supporting its role?

That is an important question and several arguments can be made. The majority of follicles that survive freezing/thawing and grafting are non-growing primordial follicles that lack a theca cell layer. When these follicles activate and enter growth, the theca lineage is recruited from local stromal precursors; this recruitment will only occur once the graft has re-established itself within the host. One could therefore argue that, provided stromal recovery is adequate after transplantation, the theca compartment will be available when needed. Conversely, impaired stromal viability caused by the transplantation injury could lead to locally persistent defects in the graft and compromise follicle maturation in some regions. This returns us to the central methodological difficulty: choosing appropriate, feasible study end-points that reflect the clinically relevant biology.