Comparison of Two Commercial In-Vitro Maturation Culture Systems with Standard IVM Media: What Works Best for Mouse Oocytes

Abstract

Background: Despite many variations made to the standard IVM protocol and to the culture conditions the rate of implantation ranges between 5% to 22% and the pregnancy rate ranges from around 8% to 40% [1]. The culture media is important as it provides the oocyte with vital metabolites such as ATP and pyruvate [2]. In order to improve the success rates with IVM, many changes have been made to the standard IVM media and studies have emerged that compare the various components of the IVM media based on maturation, developmental and fertilization rates of immature oocytes. Aim: To determine and device an optimal in-vitro maturation media which is best suited to enhance IVM outcome (maturation rates of MII oocytes and subsequent embryonic development post-IVF) by culturing immature F1 mouse oocytes at the GV stage using different commercially available and homemade IVM media recipe. This study was conducted based on the hypothesis that diversity in the various commercially available and in-house IVM media can have a significant potential effect of the maturation rate of oocytes. Methodology: A sum total of (273 ) GV oocytes were collected from F1 mice, out of which 261 GV oocytes were available for in-vitro maturation which were randomly allocated to and cultured in IVM media (LAG/NON-LAG) (n=93/85 ) and a control group (n=95) cultured in a standard or in-house IVM recipe (Appendix A) supplemented with FCS or fetal Calf Serum (0.5ml), PMSG (3µL), HCG (10µL) penicillin and streptomycin (1ml/100ml IVM media stock), EGF (2µL), Napyruvate (2µL), PMSG (10µg/ml), HCG (5µg/ml). We then proceeded to assess the maturation rates of the immature oocytes up to the M-II stage at 17 hours post culture in maturation media. Furthermore, the developmental competence of the in-vitro matured M-II stage oocytes were also assessed by performing IVF of these oocytes and assessing outcome measures such as fertilization potential and subsequent culture until day 5 with daily observation of cleavage rate and development up to blastocyst stage. All procedures were performed according to commercially available protocols as per the manufacturer’s instructions. Results: The following results were obtained in terms of maturation rate (34/95:35.79% & 31/93: 33.33% and 24/85: 28.2% respectively) and the fertilization rate (12/95: 35.29% & 9/93: 29.03% & 4/85: 16.7%) each of which met the expected values Chi-squared=1.99; with a degree of freedom=2. This result was found to be statistically not significant (p<0.6) in each of the groups respectively. Discussion/Interpretation: Variation in the IVM culture systems does not seem to affect the maturation rate and subsequent fertilization outcomes. Limitations: A small sample size and a limited number of replicates performed for each of the available IVM culture systems. Conclusion: Conclude that the modified Medicult IVM system may not be the best choice of media for maturing F1 mouse oocytes along with cumulus cells in-vitro.