Stress relief: can continuous culture in a low lactate culture media reduce numerical chromosomal abnormalities and therefore improve euploidy rates?

Abstract

Research Study. MATERIALS AND METHODS: Human zygotes with 3 pronuclei (PN) were cryopreserved with consent, thawed, and placed into control (CON; n¼23) or reduced nutrient (RN; n¼25) sequential culture medium, both supplemented with HSA. Embryos were cultured individually in an Embryo-Scope and were assessed for blastocyst development on D5 and D6. On D6, blastocysts were placed into fibronectin coated dishes for outgrowth culture in IVC1 (Cell Guidance Systems) medium, along with thawed D6 human blastocyst controls donated to research after culture under standard clinical conditions (Sage CM/BM with SPS; CBL). After 48h in outgrowth, attachment was assessed and media was replaced with IVC2. Media was replaced daily until 96h of outgrowth (D10), at which point embryos were fixed and imaged to measure outgrowth area. Embryos were then stained with F-actin, DAPI, and POUF51, and imaged using confocal microscopy to determine outgrowth volume, total cell number, and epiblast cell number, respectively.RESULTS: Blastocyst development was not different between 3PN embryos cultured in CON and RN medium on D5 (30.4% and 24.0%) or D6 (34.7% and 28.0%). All embryos placed into outgrowth were attached by 48h (CON n¼11; RN n¼7; CBL n¼7). There was no difference in the area of outgrowth between treatments, either at 48h (0.05AE0.02mm 2 , CON; 0.09AE0.04mm 2 , RN; 0.06AE0.02mm 2 , CBL) or 96h (0.12AE0.06mm 2 , CON; 0.23AE0.10mm 2 , RN; 0.20AE0.10mm 2 , CBL). Of embryos placed into outgrowth, 73% CON, 60% RN, and 100% CBL had a 3D volume that was assessed using confocal microscopy. Of these embryos, 50% of CON, 67% of RN, and 40% of CBL embryos contained a visible epiblast; these embryos had similar average numbers of epiblast cells (59, CON; 41, RN; 67, CBL).CONCLUSIONS: This data demonstrates that an environment of reduced nutrient concentration successfully supports the development of human zygotes to the blastocyst stage, with equal developmental potential to both those cultured in control medium and those cultured in standard clinical conditions. In addition, 3PN zygotes developed to the blastocyst stage and successfully organized peri-implantation embryonic development equivalent to normally fertilized embryos. This innovative approach to safely investigating novel culture conditions for human embryos could significantly enhance research in the development of more effective embryo culture media for human ART.

Fertilization, embryo culture, and clinical results using low lactate embryo culture medium for pre‐culture, insemination, and beyond

“…In fact, when VerMilyea et al 23 compared CSCM‐NX against other companies’ sequential mediums, cultured them to blastocysts, biopsied the trophectoderm, and performed PGT‐A to compare embryo ploidy, the euploid rate of embryos cultured in CSCM‐NX was significantly higher. VerMilyea et al reported the use of CSCM‐NX in pre‐ICSI pre‐culture, and again, it is possible that oocyte energy metabolism is improved.…”

Controversies in ART: can the IVF laboratory influence preimplantation embryo aneuploidy?

“…Another retrospective study examined outcomes from two laboratories over a 2-year period, comparing a sequential media system to a low lactate continuous single step in one laboratory and comparing the same low lactate continuous single-step medium to a different continuous single-step medium in a second laboratory (Vermilya et al, 2018). Incubators for all comparisons were kept at 6% CO 2 .…”

Controversies in ART: considerations and risks for uninterrupted embryo culture

“…No well-designed, prospective randomized controlled trials have compared outcomes between modern single-step medium, designed to culture embryos to the blastocyst stage, with sequential culture media. Evidence on whether singlestep media may affect fidelity of mitotic chromosome dynamics, compared with sequential media in preimplantation embryos, is conflicting (Werner et al, 2016;Fragouli et al, 2017;Vermilya et al, 2018). On the basis of emerging data about the possible effect of culture media on offspring weight and cardiovascular health (Kleijkers et al, 2014;Zandstra et al, 2015;2018), it is prudent that this remains an active area of research.…”