Fertility preservation: improved neovascularization and follicle viability in cryopreserved bovine ovarian cortex transplants by remaining medulla tissue

Abstract

To investigate the advantages of cryopreserved medulla-containing ovarian cortex grafts with those of commonly used sole cortex grafts for fertility preservation by analyzing tissue quality, neovascularization processes, and the number of vital follicles. Experimental setting of cryopreserved bovine ovarian cortex tissue grafts with or without medulla tissue. Laboratory animal research at Ulm University, Ulm, Germany. Bovine ovaries and fertilized chicken eggs. Experimental setting of bovine ovarian tissue samples grafted on the chicken chorioallantoic membrane (CAM) after cryopreservation and thawing to examine histologic tissue integrity, apoptosis and proliferation immunohistochemically, blood vessel counts and determine the presence of neutral red-stained vital follicles. We used hematoxylin and eosin staining to visualize tissue structures, immunohistochemistry with anti-caspase 3 to detect apoptosis, anti-Ki67 to examine proliferation, blood vessel count on the chicken CAM to investigate neovascularization processes, and neutral red staining to evaluate vital follicles. We demonstrated that in all analyzed tissue samples, after cryopreservation, thawing, and grafting on the chicken CAM, there was excellent tissue integrity and quality, as shown by extremely rare apoptosis processes analyzed using immunohistochemical caspase 3 staining (sole cortex, 0.54%; thin medulla-containing cortex, 0.43%; thick medulla-containing cortex, 0.13%; and sole medulla, 2.82%). Moreover, we detected increased neovascularization in the vicinity of medulla and medulla-containing grafts (small blood vessels: cortex 8.7, thin medulla-containing cortex 9.9, thick medulla-containing cortex 9.7, and medulla 9.8; very small blood vessels: cortex 7.0, thin medulla-containing cortex 13.0, thick medulla-containing cortex 12.0, and medulla 15.0), with higher Ki67-detected proliferation (cortex, 17.58%; thin medulla-containing cortex, 20.28%; thick medulla-containing cortex, 20.56%; and medulla, 29.9%). Additionally, we identified an increased number of vital follicles in medulla-containing cortex grafts compared with the number of vital follicles in sole cortex tissue (cortex, 256.1; thin medulla-containing cortex, 338.2; thick medulla-containing cortex, 346.6; and medulla, 8.1). In this experimental setting, bovine medulla-containing cortex tissue had excellent tissue structure and quality after cryopreservation and thawing and increased neovascularization and an augmented vital follicle count after grafting than the commonly used sole cortex tissue. Therefore, we suggest reconsidering the current cryopreservation and grafting processes in humans for fertility preservation by favoring retain medulla tissue at the ovarian cortex.