Di Nardo, Maddalena (2020) Innovative strategies for female fertility preservation: development of a more efficient system for ovarian tissue cryopreservation and in vitro culture. [Tesi di dottorato]

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Abstract

Over the last decades, ovarian tissue cryopreservation has been reported as a promising fertility preservation strategy and the only option available for young women with primarily cancer diseases who need immediate anticancer therapies. To date, a pregnancy rate of about 30% has been reported after auto-transplantation of frozen-thawed tissue, corresponding to more than 130 live births [1]. However, the possible risk of reintroducing malignant cells in ovarian cortical grafts remains the major problem associated with this procedure [2]. To overcome this limitation, the only possibility could be represented by a complete in vitro folliculogenesis, in a well-designed culture system. The possibility of obtaining in vitro mature oocytes in metaphase II starting from the huge reserve of primordial follicles present in the ovary, was born in 1996; when Eppig [3] obtained the birth of mice for the first time after the fertilization of mature oocytes derived from the development of follicles primordial in vitro, and improved in 2003 [4]. Although this promising technology has attracted numerous researchers over the past twenty years, attempts to reproduce these results in superior animals and humans have been largely unsuccessful. Ovarian strip in situ culture has tremendous advantages over isolated primordial follicle culture systems because follicles are maintained within their natural environment. Only recently, mature human oocytes have been developed from primordial follicles through a culture method that involves a multi-stage protocol [5]. Although these results are encouraging, the number and quality of the mature oocytes obtained is still very low. The causes for this can be traced back to several reasons listed below: 1. The inadequate cryopreservation of the tissue is one reason. It is a known fact that cryopreservation represents a crucial step of fertility preservation. The American Society for Reproductive Medicine still considers it experimental, despite several pioneers considering ovarian cryopreservation an established technique for preservation of gametes and embryos [6]. Nonetheless, to date we know that almost all live births in cancer patients have been derived from auto-transplantation of cryopreserved ovarian tissue by slow freezing/rapid thawing [7–12] and only two live births have been reported by Suzuki et al. in 2015 [13] following ovarian tissue vitrification in patients with primary ovarian insufficiency. Ovarian cryopreservation needs to be refined to reduce cryo-damage and the consequent depletion of follicles and stromal cells (SCs) that may impair the full recovery of ovarian function. Some studies suggest that slow freezing may cause extensive DNA fragmentation in primordial follicles and cause injury to stromal cells as well [14–17]. Starting with this knowledge, numerous researchers have tried to improve the efficiency of ovarian tissue cryopreservation. The research group, with which I worked for my doctorate had already shown that ultra-rapid vitrification of human ovarian tissue with slush nitrogen (SN) improves recovery of healthy follicles in 2016. The group had also demonstrated preservation of granulosa cells (GCs) and stromal cells ultrastructure, DNA integrity of SCs and viability of oocytes, GCs, and SCs over conventional vitrification with liquid nitrogen (LN) [18]. During my PhD I attempted to merge optimal protocols for cryopreservation and culture and assess the functional potential of SN vitrified strips during long term culture [19]. 2. The second reason is the insufficient number of secondary follicles that are obtained in the early stages of culture or after the first step of culture, that rarely exceeded 10% [5-20]. This could be due to inadequate culture conditions that limit the number and health of secondary follicles produced during the first step of culture. Despite several studies having investigated the nutritional and endocrine requirements of ovarian tissue to optimize media for cortical strip culture, only a few studies have investigated the role of oxygen supply for follicular growth. Therefore, during my second year of PhD, the aim of my project was to study the effects of oxygen on follicular morphology, progression and viability during the culture of the ovarian cortical strips. We made use of oxygen permeable petri dishes to modulate the local oxygen tension in the vicinity of the tissues. To study the effect of media volumes on the performance of the grown strips, we attempted with change in the conditions of ambient oxygen i.e. 5% and 20% oxygen concentration. This was followed by analysis of the influence of the optimal diffusion of oxygen in the in situ culture of the ovarian strip. 3. The third reason is the culture conditions of the current techniques which are still sub-optimal. We showed that the in vitro culture of human ovarian cortical strips in gas-Permeable Dishes (PD) enhances follicular health and secondary follicle growth over conventional dishes by improving oxygen availability in situ [4,5]. However the static culture systems adopted so far, lack physiological and biomechanical cues which generates stagnant media layers around the tissue with a consequent deprivation of nutrients and accumulation of toxic/waste metabolites. Environmental cues such as oxygen tension and mechanical stimuli may be further enhanced to match the physiological requirements. In my last year of PhD, we investigated dynamic culture systems in continuous perfusion bioreactors (PB) that enabled the disruption of solute concentration gradients and application of physiological fluid mechanic stresses on tissue which improved the ovarian cortical tissue culture.

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