P-147 Cumulus cell co-culture in media drops does not improve in vitro maturation of vitrified warmed immature oocytes

Abstract

Abstract Study question Does co-culture with vitrified warmed cumulus cells (CC) in media drops improve in vitro maturation (IVM) of previously vitrified immature oocytes? Summary answer CC co-culture in this simple two-dimensional system does not improve IVM of vitrified warmed immature oocytes. What is known already Previous studies have shown improved IVM of fresh immature oocytes when co-cultured with cumulus cells in a three-dimensional (3D) matrix. However, scheduling and workload of embryologists would benefit from a simpler IVM approach, particularly in the setting of time-sensitive oncofertility oocyte cryopreservation cases. Although yield of developmentally competent mature (metaphase II, MII) oocytes is increased if IVM is performed before cryopreservation, it is unknown whether maturation of previously vitrified immature oocytes is improved following co-culture with CCs in a simple system not involving a 3D matrix. Study design, size, duration A randomized controlled trial was performed where 320 immature oocytes (160 GV and 160 MI) and autologous CC clumps were vitrified from 7/2020 to 9/2021. Upon warming, the oocytes were randomized to culture in IVM media with cumulus cells (+CC) or without cumulus cells (-CC) and then assessed for nuclear maturation using confocal microscopy and for cytoplasmic maturation following parthenogenetic activation. Participants/materials, setting, methods GV and MI oocytes were cultured in 25µL (SAGE IVM medium) for 32 and 20-22 hours, respectively. Oocytes with a polar body (MII) were randomized to confocal microscopy for analysis of spindle integrity and chromosomal alignment or parthenogenetic activation to assess cytoplasmic maturity. Wilcoxon rank sum tests for continuous variables and chi square or Fisher’s Exact test for categorical variables assessed statistical significance. Relative risks (RR) and 95% confidence intervals (CI) were calculated. Main results and the role of chance Patient demographic characteristics were similar for both GV and MI groups after randomization to +CC vs -CC. No statistically significant differences were observed between +CC versus -CC groups regarding the % MII from either GV [42.5% (34/80) vs 52.5% (42/80); RR 0.81 95% CI: 0.57-1.15] or MI [76.3% (61/80;) vs 72.5% (58/80); RR 1.05 95% CI: 0.88-1.26] oocytes. There was more parthenogenetic activation of GV-matured MIIs in the +CC group [92.3% (12/13) vs 70.8% (17/24)], but the difference was not statistically significant (RR 0.77 95% CI: 0.57-1.03). There was no difference for MI-matured oocytes [74.3% (26/35) vs 75.0% (18/24), CC+ vs CC-; RR 1.01 95% CI: 0.76-1.35]. No significant differences were observed between +CC vs -CC groups for parthenotes from GV-matured oocytes for cleavage [91.7% (11/12) vs 82.4% (14/17)] or blastulation (0% for both); or for MI-matured oocytes [cleavage: 80.8% (21/26) vs 94.4% (17/18); blastulation: 0% (0/26) vs 16.7% (3/18)]. Further, no significant differences were observed between +CC vs -CC for GV-matured oocytes regarding incidence of bipolar spindles [38.9% (7/18) vs 33.3% (5/15)] or aligned chromosomes [22.2% (4/18) vs 0.0% (0/15)]; or for MI-matured oocytes [bipolar spindle: 38.9% (7/18) vs 42.9% (2/28); aligned chromosomes: 35.3% (6/17) vs 24.1% (7/29)]. Limitations, reasons for caution One person performed the vitrification, thaw, co-culture and parthenogenetic activation experiments so scheduling capacity required the culture duration to be shorter than the optimum of at least 36 hours. Small sample sizes for the parthenogenetic activation and confocal analyses limit reliability of definitive conclusions. Wider implications of the findings Warmed CCs and immature oocytes co-cultured in medium drops may not result in improvements in nuclear and cytoplasmic maturity, at least by the markers assessed here. Further work is required to assess the efficacy of this system given its potential to provide flexibility in a busy IVF clinic. Trial registration number not applicable