IVF culture media refresh in a reduced volume on day4 aimed at improving non-invasive embryo selection does not affect embryo competence: a prospective analysis of 2605 embryos

Background and Aim: Omic analyses on spent-culture-media are emerging as non-invasive schemes for embryo selection. Media refresh and reduced culture volume are critical to improve their specificity and sensitivity. However, limited evidence exists to support embryo culture in reduced volumes and some authors claimed this practice might influence developmental and reproductive potential altering the concentration of autocrine factors and/or of potentially-toxic metabolites. It is therefore critical to outline suitable protocols to preserve embryonic competence while improving non-invasive embryo-selection strategies.Methods: Between March2018 and December2019, 1357 oocyte-pick-ups for PGT-A were conducted. All couples could choose to participate to a pilot non-invasive PGT-A study (ClinicalTrials.gov:NCT03520933) whose design involved culture media refresh the morning of day4 from 30 µl single drops of continuous-single-culture-media (CSCM) to 10 µl for ≥40hr until blastocyst full-expansion or developmental arrest. If not consenting to the study, no refresh was performed, and embryo culture was concluded in 30 µl of CSCM. ICSI, blastocyst-culture under mineral-oil (5%O2/6%CO2), grading according to Capalbo et al (2014), trophectoderm-biopsy, NGS-based analysis and vitrified-warmed euploid single-embryo-transfer (SET) were performed. Overall, 389 and 151 cycles met the inclusion criteria (maternal age <44yr, own gametes, ejaculated sperm, no severe-oligoasthenoteratozoospermia) in the control and study group. Only viable embryos in day4 were included: 1873 and 732 in the control and study group. The primary outcome was the blastulation-rate per day4 viable embryos. Secondary outcomes were blastocyst quality, euploidy, clinical-pregnancy-, miscarriage- and sustained-implantation-rate per euploid single-embryo-transfer. All transfers performed up to August 2021, and issuing from these cycles, were included (N=378 and 137, respectively). Fisher's exact tests and logistic-regression-analyses adjusted for confounders were conducted.Results: The blastulation-rate per day4 viable embryo was similar in the control and study group (n=915/1873,49% versus 365/732, 50%). Blastocyst quality was also similar (excellent: 47% versus 50%, good:20% versus 15%, average:12% versus 14%, poor: 21% versus 21%). The euploidy-rates were 36% (n=330/915) and 37% (n=136/365). No difference was reported in clinical-pregnancy- (n=232/378,61% versus n=83/137,61%), miscarriage- (n=34/220,15% versus n=10/76,13%) and sustained-implantation-rates (n=186/378,49% versus n=66/137,48%). Media refresh in 10 µl for ≥40hr from day4 did not impact blastocyst development (multivariate-OR 0.99,95%CI:0.83-1.2, p=0.97) and sustained-implantation (multivariate-OR 0.96,95%CI:0.64-1.42, p=0.83).Conclusions: This study supports single embryo culture in a reduced volume of media from day4. This strategy is critical to minimize the risk for external contamination and to increase the concentration of embryonic-molecules (cell-free-DNA, mtDNA, miRNAs, metabolites) for non-invasive embryo-selection purposes. Background and Aim: Omic analyses on spent-culture-media are emerging as non-invasive schemes for embryo selection. Media refresh and reduced culture volume are critical to improve their specificity and sensitivity. However, limited evidence exists to support embryo culture in reduced volumes and some authors claimed this practice might influence developmental and reproductive potential altering the concentration of autocrine factors and/or of potentially-toxic metabolites. It is therefore critical to outline suitable protocols to preserve embryonic competence while improving non-invasive embryo-selection strategies. Methods: Between March2018 and December2019, 1357 oocyte-pick-ups for PGT-A were conducted. All couples could choose to participate to a pilot non-invasive PGT-A study (ClinicalTrials.gov:NCT03520933) whose design involved culture media refresh the morning of day4 from 30 µl single drops of continuous-single-culture-media (CSCM) to 10 µl for ≥40hr until blastocyst full-expansion or developmental arrest. If not consenting to the study, no refresh was performed, and embryo culture was concluded in 30 µl of CSCM. ICSI, blastocyst-culture under mineral-oil (5%O2/6%CO2), grading according to Capalbo et al (2014), trophectoderm-biopsy, NGS-based analysis and vitrified-warmed euploid single-embryo-transfer (SET) were performed. Overall, 389 and 151 cycles met the inclusion criteria (maternal age <44yr, own gametes, ejaculated sperm, no severe-oligoasthenoteratozoospermia) in the control and study group. Only viable embryos in day4 were included: 1873 and 732 in the control and study group. The primary outcome was the blastulation-rate per day4 viable embryos. Secondary outcomes were blastocyst quality, euploidy, clinical-pregnancy-, miscarriage- and sustained-implantation-rate per euploid single-embryo-transfer. All transfers performed up to August 2021, and issuing from these cycles, were included (N=378 and 137, respectively). Fisher's exact tests and logistic-regression-analyses adjusted for confounders were conducted. Results: The blastulation-rate per day4 viable embryo was similar in the control and study group (n=915/1873,49% versus 365/732, 50%). Blastocyst quality was also similar (excellent: 47% versus 50%, good:20% versus 15%, average:12% versus 14%, poor: 21% versus 21%). The euploidy-rates were 36% (n=330/915) and 37% (n=136/365). No difference was reported in clinical-pregnancy- (n=232/378,61% versus n=83/137,61%), miscarriage- (n=34/220,15% versus n=10/76,13%) and sustained-implantation-rates (n=186/378,49% versus n=66/137,48%). Media refresh in 10 µl for ≥40hr from day4 did not impact blastocyst development (multivariate-OR 0.99,95%CI:0.83-1.2, p=0.97) and sustained-implantation (multivariate-OR 0.96,95%CI:0.64-1.42, p=0.83). Conclusions: This study supports single embryo culture in a reduced volume of media from day4. This strategy is critical to minimize the risk for external contamination and to increase the concentration of embryonic-molecules (cell-free-DNA, mtDNA, miRNAs, metabolites) for non-invasive embryo-selection purposes.