Impact of oxygen tension according to embryo stage of development: a prospective randomized study

Human embryo culture under 2–8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional» conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B’ (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B’(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B’ and C. Furthermore, blastocyst quality was statistically equivalent between groups B’ and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B’ and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B’ and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

P–769 FTIR spectroscopy analysis reveals differences between human embryo culture media composition by type of formulation and by manufacturer

Study question Can we detect the variation in different commercial human embryo culture media composition by Fourier-transform infrared (FTIR) spectroscopic analysis? Summary answer The spectra reveals distint features that allow distinguishing between continuous and sequential media, as well as between manufacturers of the same media type. What is known already: Manufacturers do not fully disclose commercially available culture media formulations. For this reason, it is important to gain insight into de differences between the available formulations, in order to understand how they might be linked with efficacy and safety ART parameters. Fourier Transform Infra-Red (FTIR) can be used for this purpose. Study design, size, duration Culture media samples (1 mL) were collected from local IVF laboratories, and stored frozen at –20 °C. Five repeats of 25 µL aliquots from the samples were analysed using FTIR spectroscopy to acquire the whole molecular fingerprint of each culture media. Participants/materials, setting, methods Three continuous (SAGE 1-STEP, Origio, G-TL, Vitrolife, and GERI, Genea) and four sequential (G1 PLUS, Vitrolife; Sequential Cleav with phenolred, Origio; G2 PLUS, Vitrolife and Sequential Blast with phenolred, Origio) media were analysed. Different pre-processing methods (atmospheric and baseline correction, normalizations, and derivatives) were carried out to minimize physical artefacts while highlighting chemical features. To compare the spectra of different media, multivariate analysis, as principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. Main results and the role of chance The whole molecular fingerprint of all media analysed showed a similar pattern, revealing that, overall, the composition is very similar. However, PCA and HCA analysis revealed that significant differences exist, both between media type (continuous vs. sequential), and between different manufacturers within the same media type. Average linkage clustering using Spearman’s rank correlation confirms the similarities between the continuous and the sequential formulations. An analysis focusing the fingerprint region of the spectra (900 – 1700 cm^–1), also revealed variability between manufacturer, between media type (continuous vs. sequential) and of stage-specific media (cleavage vs. blastocyst). For instance, GERI media visually appeared to have distinct peaks compared to all other media, which was confirmed later through multivariate statistical analysis. Limitations, reasons for caution FTIR spectroscopy does not allow for a direct identification of the analytes present in the culture media, we can only infer the functional groups, but that are common on diverse biomolecules. Wider implications of the findings: FTIR analysis reveals differences between different media, such as cleavage and blastocyst specific, sequential and continuous, or manufacturer’s formulations. The increased resolution of the FTIR profile proves to be a powerful tool for analysing human embryo media, and could be used to establish correlations with media clinical performance and safety. Trial registration number Not applicable