Abstract
BackgroundOvarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy. However, clinicians strongly advise against reimplantation of one’s own ovarian tissue when there is a high risk of recurrence after grafting. For these patients, development of an alternative strategy, namely a transplantable artificial ovary, offers future hope of conceiving. The first essential requirement for an artificial ovary is the set-up of a safe and effective follicle isolation procedure. Despite encouraging results with different variants of this technique, none of them take into the account the physiology and great variability in follicular density inside individual tissue fragments and between different patients. The goal of this study was to improve our previously applied follicle isolation procedure in order to develop a tailored isolation procedure for human follicles according to individual tissue properties. To this end, enzymatic digestion was divided into three time intervals in order to initially recover the first follicles to be isolated, and then further dissociate undigested fragments of tissue containing entrapped follicles.ResultsAfter thawing frozen human ovarian tissue using a modified and tailored follicle isolation method, already 35% of follicles were fully isolated and recovered after 30 min of enzymatic digestion. Indeed, this protocol resulted in a higher follicle yield (p < 0.01) and greater numbers of primordial and primary follicles (p < 0.05) than the previous approach. However, no significant difference was found in caspase-3-positive and Ki67-positive staining between the two isolation protocols. In addition, greater follicle quality was demonstrated. When human follicles isolated using the modified protocol were encapsulated in a fibrin matrix with high concentrations of fibrinogen and thrombin and xenografted to a SCID mouse, more follicles were found to be healthy after one week of transplantation than in a previous our study.ConclusionsWith the modified follicle isolation method, we were able to maximize the number and quality of isolated primordial and primary follicles, and develop a tailored follicle isolation procedure according to individual tissue properties. Moreover, improved follicle survival inside an artificial ovary prototype was detected after one week of xenografting.
Highlights
Ovarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy
Enzymatic digestion would be divided into several time intervals, in order to recover already isolated follicles, which would reduce the proportion of damaged follicles due to prolonged digestion
One can assume that follicles isolated during the first few minutes of enzymatic digestion could have their basement membrane progressively damaged over the course of 75 min of exposure to the enzyme, which would probably explain the presence of extruded oocytes in our previous studies [9, 10]
Summary
Ovarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy. Previous reports have already demonstrated that structurally intact human follicles can be isolated [8,9,10,11,12] and shown to be free of malignant cell contamination, an essential requirement for future clinical application of an artificial ovary [13, 14] Such protocols do not take into account the great heterogeneity of follicular density [15, 16] and extracellular matrix composition [17, 18] between patients, which inevitably influences isolation procedure success rates. Enzymatic digestion is usually performed over a single duration of time, without considering the possible negative impact of prolonged digestion on the first fully isolated follicles With this in mind, the aim of the present study was to improve the follicle isolation procedure in order to maximize the number and quality of isolated follicles, and develop a tailored follicle isolation approach according to each individual ovarian tissue sample
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