Systematic Development, Validation and Optimization of a Human Embryo Culture System

Abstract

Objective: To develop and validate a reliable in vitro culture system for human embryos. Design: Retrospective analyses of a series of four studies were conducted between 2006 and 2010 to assess the effect of incubator type (CO 2 box versus Tri-gas minibox), media type, oil type, and hyaluronate supplementation. Optimization of in vitro blastocyst development was verified by assessing our National CDC/ART Surveillance reports between 2010 and 2016. Material and Methods: All patients experienced controlled ovarian hyperstimulation, followed by egg retrieval 35 h post-hCG. Cumulus-oocyte complexes were temporarily cultured in P1 or LG Fert medium plus HSA. Eggs were moved to a more complex media (G-medium or Global ® -LG medium) containing a synthetic protein and embryo adhesion supplement (SPS and EAS, respectively; mLG) post-ICSI insemination. Zygotes were assigned to group culture in 25 µl droplets under oil (light mineral oil or paraffin oil; 37 • C) and embryo development was evaluated on Days 3, 5, and 6 and transferred on Day 3 to 5 depending on the number/quality of embryos available and the IVF history of the patient. Transfers were performed under ultrasound guidance, primarily using a Sureview-Wallace catheter, and enriched ET medium containing 500 µg/mL EAS. Results: Pilot study results (Expt. 1) showed that a mLG single-step medium could be effectively used in combination with Sanyo MCO-5 tri-gas (TG) incubators. Once adapted to SCIRS Lab in 2007 (Expt. 2), the latter culture system yielded improved blastocyst production and pregnancy outcomes compared to CO 2 in air sequential incubation in P1/Multi-blast medium. In Expt. 3, the mLG/TG system yielded high levels of ≥2BB quality blastocysts (51 to 66%) across all age groups, and greater (p < 0.05) pregnancy success/live birth rates using fewer embryos transferred on Day 5 versus Day 3. After validating its clinical effectiveness, mLG was then prospectively compared to a new generation G-media (1.5 & 2.5; Expt. 4) and determined that the crossover treatment using paraffin oil (Ovoil™) allowed the mLG system to be optimized. Subsequently, a compilation of our Annual CDC/ART reported data over six years verified the overall viability of in vitro cultured and vitrified blastocysts produced in the mLG/TG system. Conclusion: By systematically evaluating and implementing various components of an embryo culture system we were able to optimize blastocyst development over the last decade. Our mLG/TG culture system modified an exceptionally well designed KSOM AA LG medium using endotoxin-free EAS and SPS additives to support cellular membrane wellness under stressful in vitro conditions (e.g., culture, cell biopsy, vitrification). Our use of the mLG/TG culture system has proven to be effective, creating reliably high blastocyst production, implantation, and healthy live births.