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

OBJECTIVE: To assess whether co-culture with vitrified-warmed cumulus cells (CCs) in media drops improves rescue in vitro maturation (IVM) of previously vitrified immature oocytes. Previous studies have shown improved rescue IVM of fresh immature oocytes when cocultured with CCs in a three-dimensional matrix. However, the scheduling and workload of embryologists would benefit from a simpler IVM approach, particularly in the setting of time-sensitive oncofertility oocyte cryopreservation (OC) cases. Although the yield of developmentally competent mature metaphase II (MII) oocytes is increased when rescue IVM is performed before cryopreservation, it is unknown whether maturation of previously vitrified immature oocytes is improved after coculture with CCs in a simple system not involving a three-dimensional matrix. DESIGN: Randomized controlled trial. SETTING: Academic hospital. PATIENTS: A total of 320 (160 germinal vesicles [GVs] and 160 metaphase I [MI]) immature oocytes and autologous CC clumps were vitrified from patients who were undergoing planned OC or intracytoplasmic sperm injection from July 2020 until September 2021. INTERVENTIONS: On warming, the oocytes were randomized to culture in IVM media with CCs (+CC) or without CCs (-CC). Germinal vesicles and MI oocytes were cultured in 25 µL (SAGE IVM medium) for 32 hours and 20-22 hours, respectively. MAIN OUTCOME MEASURES: Oocytes with a polar body (MII) were randomized to confocal microscopy for analysis of spindle integrity and chromosomal alignment to assess nuclear maturity or to parthenogenetic activation to assess cytoplasmic maturity. Wilcoxon rank sum tests for continuous variables and the chi square or Fisher's exact test for categorical variables assessed statistical significance. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated. RESULTS: Patient demographic characteristics were similar for both the GV and MI groups after randomization to +CC vs. -CC. No statistically significant differences were observed between +CC vs. -CC groups regarding the percentage of 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. An increased percentage of GV-matured MIIs underwent parthenogenetic activation in the +CC group (92.3% [12/13] vs. 70.8% [17/24]), but the difference was not statistically significant (RR 1.30; 95% CI: 0.97-1.75), whereas the activation rate was identical for MI-matured oocytes (74.3% [26/35] vs. 75.0% [18/24], CC+ vs. CC-; RR 0.99; 95% CI: 0.74-1.32). No significant differences were observed between +CC vs. -CC groups for cleavage of parthenotes from GV-matured oocytes (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]). CONCLUSIONS: Cumulus cell co-culture in this simple two-dimensional system does not improve rescue IVM of vitrified, warmed immature oocytes, 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, in vitro fertilization clinic.

The fate of bisphenol A, bisphenol S, and their respective glucuronide metabolites in ovarian cells.

Ovarian cells are critical for reproduction and steroidogenesis, which are functions that can be impacted by exposure to xenobiotics. As in other extra-hepatic tissues, biotransformation events may occur at the ovarian level. Such metabolic events deserve interest, notably as they may modulate the overall exposure and toxicity of xenobiotics. In this study, the comparative metabolic fate of two bisphenols was investigated in ovarian cells. Bisphenol A (BPA), a model endocrine disruptor, and its major substitute bisphenol S (BPS) were selected. Bovine granulosa cells (primary cultures) and theca explants (ex vivo tissue) were exposed for 24 hr to tritium-labeled BPA, BPS and their respective glucuronides (i.e. their major circulating forms), at concentrations consistent with low-dose exposure scenarios. Mass balance studies were performed, followed by radio-HPLC profiling. The capability of both cell compartments to biotransform BPA and BPS into their respective sulfo-conjugates was demonstrated, with sulfation being the predominant metabolic route. In theca, there was a significantly higher persistence of BPA (compared to BPS) residues over 24 hr. Moreover, only theca explants were able to deconjugate inactive BPA-glucuronide and BPS-glucuronide back into their biologically active aglycone forms. Deconjugation rates were demonstrated to be higher for BPS-G than for BPA-G. These findings raise concerns about the in situ direct release of bisphenols at the level of the ovary and demonstrate the relevance of exploring the biotransformation of bisphenols and their circulating metabolites in different ovarian cells with specific metabolic capabilities. This work also provides essential knowledge for the improved risk assessment of bisphenols.

Susceptibility of human cumulus cells to bisphenol a In vitro.

Bisphenol A (BPA) is detectable in follicular fluid. However, the effect of BPA exposure on human cumulus cells (CC) that surround the oocyte and are crucial for oocyte competence has been largely unexplored. We exposed primary cultures of CC to increasing concentrations of BPA [0,0.002, 0.02 and 20µg/mL] and tested the effects of BPA on the expression of genes associated with apoptosis using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); we also assessed the effect of BPA on apoptosis by staining with anti-caspase 3. Exposure to 20µg/mL BPA led to significantly decreased expression of CDC20, BUB1B and HAS2 (p<0.03), increased expression of TRIB3 and LUM (p≤0.005), and increased frequency of cells positive for anti-CASP3 (p=0.03), compared to control. Our results imply that BPA may lead to ovarian toxicity by increasing CC apoptosis and provide an important molecular mechanism for the effect of BPA on human CC in vitro.