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Current laboratory standards for assessing vitrified-warmed blastocyst viability fail to accurately predict their implantation potential.

petri dish

Authors: Whitney JB, R vanTol, A Miller, K Mayer, MC Schiewe

Study question: Do current standard assessments of vitrified-warmed embryo survival and cellular viability accurately predict blastocyst functionality post-transfer? Do all embryos deemed viable at 3hr sustain development?

Summary answer: Blastocysts appearing viable at +180min may not continue development 24hr later, confirming that standard laboratory viability assessments are visual not functional determinants of cellular health.

What is known already: Vitrification has revolutionized cryopreservation in the field of reproductive medicine by achieving >95% complete blastocyst survival rates. A variety of open and closed device vitrification techniques use mixed solutions of cryoprotectants and concentrated macromolecules to form a supercooled vitreous solid. These methods rely on faster warming rates than cooling rates to avoid damaging ice formation and optimize post-warming/dilution survival. Pre-transfer embryo survival evaluation typically occurs between 0 and 3hr post-warming. Although high live rate rates are associated with vitrified embryo transfer cycles, it is unknown if current survival assessments at 3hrs accurately identify viability.

Study design, size, duration: Research consented vitrified blastocysts (n=60), from four Ovation laboratories, were warmed using standardized, published protocols.  Survivability and blastocoele expansion measurements were assessed at +1min, +30min, +60min, +120min, +180min, and +24hr post-warming.  Initial survival was determined at +30min, while two viability assessments were made, the first at +3hr post-warming using standard transfer viability criteria.  At +24hr, a second viability determination was based on complete hatching or positive growth from +180min to +24hr.

Participants/materials, setting, methods: Blastocysts were warmed using standardized, published protocols based on the vitrification system used: Cryolock, Cryotech, or µicroSecure devices. Post-thawing and dilution, all blastocysts were incubated in low O2, humidified, tri-gas environments at 37°C. At designated time intervals, blastocysts were analyzed for continued development and documented by photography (200X).  Subsequently, measurements of blastocoele diameter were estimated along the longest axis of the trophectoderm. Differences in survival and viability were determined using a χ2 test (p<0.05).

Main results and the role of chance:  Initial survival was 97% (58/60) at +30min post-thaw.  The first viability assessment at +3hr post-warming determined no change in developmental status, with a non-viable rate of 3.33% (2/60) maintained.  After +24hr, the second viability assessment resulted in a higher (p<0.05) non-viable rate of 13.8% (8/58). The latter 8 blastocysts failed to exhibited blastocoele growth past the +3hr mark and were deemed non-viable. Growth rates assessing increases in blastocoele diameter between time intervals and laboratories were significant between vitrification methods.  Not surprisingly, notable blastocoele recovery and trophectodermal expansion was evident between +30min to +180min, with non-DMSO exposed, microSecure-treated blastocysts tending (p<0.10) to be more expanded up to +120min. Overall, our preliminary data reveals that viability assessments between laboratories (non-DMSO, closed device, n=20; DMSO-EG, open devices, n=40) showed no difference at +24hr (50 of 60, 88.3%).

Limitations, reasons for caution: Survival and viability are two separate assessments that are commonly performed simultaneously to determine blastocyst implantation potential. Current embryo viability evaluations at 0-3 hours post-warming can be inaccurate predictors of implantation potential. In turn, alternative methods, perhaps integrating short-term time lapse imaging, are needed to improve our estimation of embryo viability.

Wider implications of the findings: The viability of embryos post-warming is difficult to assess as most laboratories artificially collapse or biopsy the trophectoderm of blastocysts prior to vitrification.  Robust expansion may take several hours. Time-lapse imagery may prove beneficial to identify key growth rates to more accurately identify viability.