Vitrification of human oocytes employing a closed carrier with enhanced thermal efficiency and short times of exposure to synthetic cryoprotectant solutions

Resumen

In the first part of this thesis, the effectiveness of a vitrification system, developed by the spin-off of the University of Seville, Safepreservation, is studied. Two prospective studies with donor eggs, carried out in a fertility clinic (Ginemed Sevilla) were conducted. In the first study, we test a vitrification solution of fully synthetic composition, free of proteins of human origin such as albumin, which are substituted by the polymer hydroxypropyl cellulose. This type of molecules are generally used as surfactants and also perform osmotic activity. The clinical results of a group of oocytes vitrified with these solutions are compared against another group of oocytes, coming from the same donor, not subjected to vitrification. The results show that the synthetic alternative developed by Risco's group provides satisfactory embriology outcomes, similar to those obtained with classic vitrification solutions that incorporate in their formulation proteins of human or animal origin. Yet, without the disadvantages associated to them, such as the risk of contamination and a reduced lifespan. In the second work, an experimental design similar to that of the first study is used to test the effectiveness of the closed vitrification carrier "SafeSpeed". The carrier consists of an ultra-thin capillary attached to a straw of ionomeric resin. The capillary where the oocytes are loaded for vitrification allows to maximize the rates of cooling and warming, since its thermal efficiency is superior to that of other vitrification carriers currently available. It should also be noted that it is a closed carrier, in which biological samples do not come into contact with the liquid nitrogen used for cooling and storage, minimizing the risk of contamination by pathogens. The outcomes of this study show that the ovocytary survival rates to the vitrification procedure with this carrier meet and even exceed current standards, and that the development of the embryos resulting from the vitrified eggs is similar to that of embryos from eggs that have not undergone vitrification. The results of this two studies provide initial evidence that the vitrification system developed at the University of Seville is a very effective tool that allows obtaining benchmark results in the clinical context. However, the vitrification protocols are still susceptible to improvement: although the cooling and warming of the samples is ultra fast, the whole procedure is time consuming, since each group of up to 3 oocytes takes from 10 to 15 minutes of exposure to hypertonic solutions with cryoprotectants to be prepared for vitrification. A reduction in the duration of this phase is desirable to improve workflow in the IVF laboratory and reduce the exposure time to potentially toxic cryoprotectants. In order to explore this possibility, we decided to study the dynamics of the permeation of cryoprocectors in the human oocyte. Firstly, with an in silico approach, through a program developed in MatLab to integrate the two differential equations that describe the permeability of the plasma membrane of the human Metaphase-II oocyte, according to a 2-P model. These simulations were complemented with in vivo observations of the osmotic behavior of the oocytes. The standard protocol for the preparation of oocytes for vitrification consists on a prolonged exposure (10-15 minutes) to a solution with an intermediate concentration of cryoprotectants (≈25% w/w), called a non- vitrifying, or equilibration, solution. This is followed by a second short exposure to a vitrifying solution with higher CPA concentration (≈45% w/w). We compared the osmotic activity that occurs in the standard protocol against that of a short protocol, based on dehydration, in which the duration of exposure of the oocytes to both the non-vitrifying and vitrifying solutions was limited to 60 seconds We verified that the dehydration of the oocyte after exposure to vitrification solutions occurs very fast; the minimum volume point of the contraction and expansion curve resulting from the osmotic gradient is reached within the first 60 seconds. At that point, the intracellular water content is minimal, the penetration of low molecular weight cryoprotectants is almost complete and, as a result, the total concentration of intracellular solute is high. Therefore, prolonging the first exposure phase up to 15 minutes, as recommended in the current protocols, would presumably not reduce the chance of lethal ice formation at certain rates of cooling and warming. The results of tests on human oocytes and embryos, unsuitable for clinical use and donated for research, show that the vitrification survival rate is not compromised by reducing the exposure times, confirming that the desired osmotic effect occurs in a reduced time. Innovations in the techniques used to vitrify reproductive cells are governed by the premise that a compromise must be maintained between safety and efficacy: the technique must be as aseptic and effective as possible. It could be argued that SafeSpeed, as a closed support for virification, meets this criterion of improving efficiency —thermal efficiency, at least— without compromising safety, and as such could be considered a step in the right direction. Likewise, the use of the synthetic polymer hydroxypropyl cellulose also represents a safer and more stable alternative to proteins derived from human or animal. Finally, the same could be said about our attempts to shorten the vitrification protocol: if oocytes and embryos prove to be equally competent, reducing the duration of exposure to potentially cytotoxic cryoprotectants and at suboptimal temperatures should be safer. Ultimately, for each relevant modification of the procedure, a well designed validation route must be performed before its use in the clinical context, from the preclinical stage in models of mammals and donated human material, to prospective controlled clinical studies.