In this pilot study, we demonstrated that warming blastocysts with an ultrafast 1-step warming protocol results in comparable embryology and clinical outcomes compared with conventional 3-step warming protocol. These encouraging indicators were maintained throughout the 3-month follow-up period at the same level than usually observed during previous period with conventional protocol. We also showed that the expression of main cell fate markers at the blastocyst stage remain unchanged with this new simplified warming protocol.

Vitrification was initially developed in the 90’s in order to overcome intracellular and extracellular ice crystal formation and subsequent poor survival rates in oocytes and embryos due to incomplete dehydration observed with slow freezing. Since then, vitrification demonstrated its superiority over slow-freezing in terms of clinical outcomes and cryosurvival rates for oocytes, cleavage-stage embryos and blastocysts, as elegantly reviewed in [2]. Biologically, the success of vitrification relies on ultrafast colling temperature and high concentration of cryoprotectant agent in order to skip ice formation and reach a vitreous, glass-like cellular state [10, 11]. Whatsoever the brand and the cryoprotectant molecule considered (DMSO, ethyleneglycol), the high concentration of cryoprotectant agent is reached progressively to allow cells achieve deep dehydration while maintaing structural integrity. Interestingly, it has been demonstrated that the intracellular concentration of cryoprotectants after vitrification was lower than after slow freezing despite exposure to higher concentration of cryoprotectant solutions [12]. Thawing and warming of cells has often been considered as less critical than cooling, and has therefore been less investigated. Overall, the same (but inverse) approach than for vitrification was used during warming to progressively remove cryoprotectant agent and rehydrate embryonic cells or oocyte, exposing them to successive media with decreasing sucrose concentration. This historical belief that multi-step cell recovery is preferable might originate from very early work on freezing/thawing which did not use sucrose but only decreasing concentrations of cryoprotectant, hence making stepwise thawing mandatory [13].

Actually, if not closely controlled, the exposure of cells to osmotic stress during the thawing process can result in the alteration of microtubules, organites, membrane and and ultimately cell lysis [10]. Warming rate has been explored in several studies, pointing at the importance of reaching the highest warming rate possible. Some authors even suggested that warming rate is significantly more critical for cell survival than cooling rate [14, 15]. In this context and quite surprisingly, the duration of exposure to warming solution(s) with decreasing CPA concentrations has hardly ever been reported in the literature. Actually, various CPA concentration and exposure time have been proposed, but very little data are available and evidence seem to be still missing. However, it can be speculated that the vast majority of cryoprotectant removal occurs rapidly, potentially questionning the relevance of culturing the embryo in successive baths with decreasing CPA concentration during warming procedure. The lower intracellular concentration of cryoprotectants found after vitrification than after slow freezing already cited above supports this reflexion [12]. Furthermore, high sucrose level, and subsequent concern on osmotic stress, might not be mandatory for blastocyst warming, as reported in early work by Lane and Gardner [16], where they demonstrated that warming could be performed efficiently with low sucrose concentration (0.25 M), instead of 1 M concentration as reported in various studies [17]. Therefore, the combination of of less intracellular CPA for vitrification, together with the experience of warming with low sucrose level might support the use of single step warming protocols for blastocysts.

In this context, a few authors recently proposed the use of a simplified warming protocol and reported preliminary data in various congresses during the last months. Manns et al. [6] reported a prospective randomized study conducted in 71 donated blastocysts, warmed with either ultra-fast (single-step) or standard protocol. No significant difference in terms of survival and re-expansion was found between the 2 protocols. Naert and al [4] reached the same conclusion when comparing survival and re-expansion rates of biopsied and non biopsied blastocyst with either ultra-fast or standard warming protocol, highlighting that neither the blastocyst stage at vitrification nor the biospy status affected these results. Interestingly, Guns & Ahlstrom [7] compared 2 different sucrose concentrations for ultrafast warming protocol. Both low and high sucrose concentrations yielded high survival and expansion rates, comparable to the standard procedure. Altogether, these 3 reports are in agreement with our results and confirm that ultrafast warming protocol does not affect blastocyst survival and re-expansion rates, and suggest that ultrafast warming protocol can be used with various sucrose concentrations at various temperature (i.e. 37 °C or room temperature).

We believe that this pilot study has some strengths. First, and as far as we know, it is the first of its kind with a pseudo-randomised design. Second, the prospective follow-up on a large sample of cycles brought reassuring clinical information on the clinical relevance and safety of this new simplified warming protocol. Third, the combination of a clinical trial with some more fundamental experiments brings more insight into the expected safety of the procedure. We however acknowledge that this pilot study also has some limitations. The limited number of cycles included the pseudo-randomized study advocates for further confirmation in larger well-designed RCTs, with pregnancy and delivery follow-up, including neonatal aspects. The monocentric design and the use of one brand of vitrification / warming media also prevent from drawing general conclusion in other settings and/or with other vitrification / warming media.

In conclusion, we report here some encouraging preliminary data on a simplified warming protocol for vitrified blastocysts. Our results illustrate that this protocol appear to perform equally well as conventional warming procedure, but with much shorter technical time, hence being a great opportunity for increasing flexibility and time-efficiency in busy IVF labs with heavy workload.