Novel extra cellular-like matrices to improve human ovarian grafting

Abstract

Purpose To investigate if human ovarian grafting with pure virgin human recombinant collagen type-1 from bioengineered plant lines (CollPlant™) or small intestine submucosa (SIS) yields better implantation results for human ovarian tissue and which method benefits more when combined with the host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. Methods Human ovarian tissue wrapped in CollPlant or SIS was transplanted into immunodeficient mice with/without host/graft treatment. The tissue was assessed by follicle counts (including atretic), for apoptosis evaluation by terminal deoxynucleotidyl transferase assay and for immunohistochemical evaluation of neovascularization by platelet endothelial cell adhesion molecule (PECAM) expression, and for identification of proliferating granulosa cells by Ki67 expression. Results Human ovarian tissue transplanted with CollPlant or SIS fused with the surrounding tissue and promoted neovascularization. In general, implantation with CollPlant even without additives promoted better results than with SIS: significantly higher number of recovered follicles, significantly fewer atretic follicles, and significantly more granulosa cell proliferation. Moreover, results with CollPlant alone seemed to be at least as good as those after host and graft treatments. Conclusions CollPlant is a biomaterial without any potential risks, and grafting ovarian tissue with CollPlant is easy and the procedure may be easily modified, with limited or no foreseeable risks, for auto-transplantation in cancer survivors. Further studies are needed using other novel methods capable of enhancing neovascularization and reducing apoptosis and follicle atresia.

Use of Simvastatin, Fibrin Clots, and Their Combination to Improve Human Ovarian Tissue Grafting for Fertility Restoration After Anti-Cancer Therapy

“…It is possible that the combined protocol of host treatment with simvastatin and graft embedding in fibrin clots may also be tested putatively to improve implantation in clinical ovarian transplantations to former cancer patients. Moreover, novel methods to improve ovarian tissue grafting with various pharmacological agents are being investigated (2,20) with the objective of increasing post-implantation follicle viability and reducing follicle loss. In future studies, fibrin clots can be supplemented during preparation with pharmacological agents supporting ovarian implantation, thus serving as an efficient vehicle for delivery of these substances to the graft to further improve human ovarian implantation outcomes (12).…”

“…In all specimens, preantral follicles (atretic and non-atretic) were counted throughout the field (magnification X100) in two levels (with at least 50 µm between levels to avoid counting the same follicle twice) using a computerized image analyzer (analySIS, Soft Imaging System, Digital Solutions for Imaging and Microscopy, System GmbH, Munster, Germany). As we conducted various evaluation methods we were unable to calculate follicle density, as in our previous studies (17,18,20).…”

“…None of the patients had ovarian diseases. We used similar sized groups in our previous studies (17)(18)(19)(20). Informed consent to donate tissue for this study was obtained from the patients or, for minors, from their parents.…”

“…As the study was conducted with human ovarian tissue, and the donated tissue per patient was very limited, we did not have sufficient tissue amounts from the same patient to allocate to all groups in parallel. In this study as well as in our previous similar ones (17,18,20) we attempted to utilize ovarian tissue from young patients all before chemotherapy, mostly similarly aged (Table I). In this manner we utilized samples with relatively high follicle numbers.…”

“…The ovarian samples were sliced thinly and placed in a 1.5M dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA) solution (17,18,20) for slow gradual freezing. Samples were then kept on ice for 30 min to allow equilibration, frozen slowly and gradually in a programmable freezer (Kryo 360-1/7, Planer Biomed, Sunbury on Thames, UK) and immediately placed in liquid nitrogen.…”