A modified and tailored human follicle isolation procedure improves follicle recovery and survival

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.Electronic supplementary materialThe online version of this article (10.1186/s13048-017-0366-8) contains supplementary material, which is available to authorized users.

FERTILITY PRESERVATION: Construction and use of artificial ovaries

“…Dolmans et al (2008) showed that 5 months after xenografting, human follicles were able to reach the antral stage. Follicle recovery rates in fibrin and decellularized human ovarian matrices after 1 week of xenografting were found to be comparable to those reported for human ovarian tissue in similar grafting experiments (Nisolle et al 2000, Paulini et al 2016, Chiti et al 2017a, Pors et al 2018. Formation of an ovary-like structure, which is necessary for further follicle development, was also demonstrated in several studies (Dolmans et al 2007, Paulini et al 2016, Chiti et al 2017a.…”

“…We found that by subjecting the remaining fragments to fresh enzymatic solution every 30 min, we were able to successfully digest any type of ovarian tissue, from the softest to the hardest. This ensured that all the tissue was processed without affecting the quality of the isolated follicles, as demonstrated by our findings after encapsulation in fibrin and xenografting (Chiti et al 2017a). This new protocol allows personalization of ovarian follicle isolation according to individual tissue properties and should be implemented on a case-by-case basis to make best use of precious ovarian follicles and maximize the chances of pregnancy after artificial ovary transplantation.…”

“…With this in mind, we optimized our isolation protocol (Vanacker et al 2011) by inactivating the enzymatic digestion process every 30 min and filtering the suspension, so that fully isolated follicles would not be continuously exposed to enzymes, which could damage their basement membrane and result in their death. The remaining fragments of ovarian tissue left behind in the filter would then be digested in fresh enzymatic solution in order to increase the number of isolated follicles (Chiti et al 2017a). This filtrationdigestion step was repeated until all the ovarian fragments were completely digested.…”

“…It is important to stress that in order to allow its application in patients, reagents used in isolation protocols must fully comply with good manufacturing practice (GMP) guidelines. We therefore developed protocols associating two GMP-produced enzymes: Liberase DH and DNase (Vanacker et al 2011, Chiti et al 2017a).…”

“…A few studies have demonstrated the ability of this polymer to support survival and growth of human follicles. However, fibrin formulations need to be much stiffer than those tested for mouse follicles (Paulini et al 2016, Chiti et al 2017a. Indeed, fibrin matrices containing 50 mg/mL fibrinogen most closely resemble human ovarian cortex in terms of ultrastructure ( Fig.…”