Authors: U.S. Braun1, L. Watson2, M. VerMilyea3. (1Ovation Fertility, Embryology, Bryan, U.S.A., 2Fujifim Irvine Scientific, Research and Development, Austin, TX, U.S.A., 3Ovation Fertility, Embryology, Austin, TX, U.S.A.)

Study question: Do the base ingredients and total composition of vitrification media have an effect on meiotic spindle reformation in warmed donor oocytes?

Summary answer: Meiotic spindle reformation occurs more readily in donor oocytes vitrified and warmed with a contemporary culture media based vitrification formulation than in traditional vitrification media.

What is known already: Embryo culture trends continue towards more freeze-all cycles and oocyte preservation is becoming more prevalent across all age groups, thus vitrification continues to serve a pivotal role in today’s laboratory; However, the formulation of vitrification media remains largely unchanged from the m199 or mHTF base composition of antiquated slow freeze media. Literature identifies temperature as a key determiner in spindle reformation. Our preliminary data utilizing a robust vitrification medium with a basal formulation specifically designed for human embryo growth demonstrates a (positively) pronounced positive effect on oocytes post-warm in regard to spindle recovery time.

Study design, size, duration: In a prospective study, 30 oocytes obtained from a diverse population of 10 oocyte donors donating to an egg bank were imaged prior to vitrification to identify the meiotic spindle using the Oosight Imaging System (Hamilton Thorne). Donor oocytes were then split between two vitrification media groups, Vitrification Kit NX (FujiFilm Irvine Scientific) and Vitrification Media (Kitazato USA). After warming, oocytes were again imaged at various time points and meiotic spindle (retardance) reformation was noted.

Participants/materials, setting, methods: Oocytes from approved egg bank donors were imaged, vitrified and warmed using protocols recommended by the manufacturers. The oocyte donors were all cycled and retrieved in the same private IVF Clinic and Laboratory between October 2019 and February 2020. Temperature of media, culture dishes, incubators and work stations were all monitored and maintained. Retrieved oocytes were imaged prior to vitrification. Oocytes were imaged immediately upon warm, and at 1 and 3 hours post warm.

Main results and role of chance: Oocyte survival was similar across the two media groups, with Vitrification Kit NX (showing) at 86.7% and Vitrification Media at 80.0% survival. 100% of the degenerated oocytes in Vitrification Kit NX did not display an initial meiotic spindle when imaged prior to vitrification as opposed to 67% of the degenerate oocytes in Vitrification Media. Upon warming, immediate imaging showed oocytes from Vitrification Kit NX displayed a spindle at a rate of 41.7%, while Vitrification Media allowed this in 0% of the oocytes. At 1-hour post warm, Vitrification Kit NX and Vitrification Media displayed meiotic spindles in 83.3% and 22.2% of oocytes, respectively. The final time point was imaged at 3-hours post warm and showed meiotic spindle reformation in 91.7% of Vitrification Kit NX oocytes and 66.7% of Vitrification Media. While none of the time points imaged show significance, there is a defined trend towards a faster rate of meiotic spindle recovery in vitrification media formulated with a more modern culture media base.

Limitations/reasons for caution: The preliminary findings of this study offer that the composition and formulation of the vitrification medium perhaps do have an effect on the reformation of the meiotic spindle post-warm; (H)however, more information is needed and a larger population size must be examined prior to discerning any significance.

Wider implications of the findings: If, through further study and imaging, it is (determined) ascertained that meiotic spindle reformation is determined in part by the vitrification media composition, it could lead to potentially healthier warmed oocytes for the patient and less workload and scheduling stress on the embryologists due to shorter wait times (for) prior to ICSI.