Optimizing ovarian tissue quality before cryopreservation: comparing outcomes of three decortication methods on stromal and follicular viability.

OBJECTIVE: To evaluate whether specific ovarian decortication techniques vary in promoting ovarian cortex cryopreservation and transplant outcomes. DESIGN: Experimental design. SETTING: University hospital. ANIMAL(S): Nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice. INTERVENTION(S): Human ovarian biopsy samples allocated to one of the following decortication procedures: scratching with scalpel blade (B), cutting with microsurgical scissors (M), separation with slicer (S), or no-separation (control, C). Parallel, in vivo experiment: decortication techniques combined with slow freezing (SF) and vitrification (VT) before xenograft into immunodeficient mice. MAIN OUTCOME MEASURE(S): Follicular counts, apoptosis, shear stress, Hippo pathway and inflammation. In vivo: recovered grafts analyzed for follicular counts, angiogenesis, proliferation, and fibrosis. RESULT(S): There were no differences in follicular density or number of damaged follicles between the decortication techniques in the in vitro study. Nevertheless, the M samples showed statistically significantly increased stromal damage compared with the controls and S samples, and up-regulation of Hsp60 shear stress gene expression. Decortication by both M and S inhibited the Hippo pathway, promoting gene expression changes. In the 21-day xenograft, total follicular density statistically significantly decreased compared with the nongrafted controls in all groups. Nevertheless, no differences were observed between the decortication techniques. Ovarian stroma vascularization was increased in the vitrified samples, but among the slow-freezing samples, the B samples had the lowest microvessel density. The M decorticated xenografts had increased fibrosis. CONCLUSION(S): Decortication with a slicer causes less damage to ovarian tissue than other commonly used methods although microsurgical scissors seem to preserve slightly increased follicular numbers. Nevertheless, blade decortication seems to be a reliable technique for maintaining acceptable follicular conditions without inducing serious stromal impairment.