In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media  Rebecca L. Kelley, David K. Gardner  Reproductive BioMedicine Online  Volume 34, Issue 5, Pages 441-454 (May 2017) DOI: 10.1016/j.rbmo.2017.02.001 Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Microwells in (A) well-of-the-well (Primo Vision) and (B) single microwell (EmbryoScope) format. Not to scale. The Primo Vision dish is shown here in 9-well format, but the 16-well format was also tested. Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Effect of single culture during precompaction or postcompaction stages. Between 90 and 120 embryos were cultured per treatment, from five independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). No significant differences were found between treatments. (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments in total cell number (Group versus Single/Group: P < 0.05; others: P < 0.01); trophectoderm cells (P < 0.05); inner cell mass (Group versus Single: P < 0.05; others P < 0.01). ICM, inner cell mass; TE, trophectoderm. Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Effect of embryo density and oxygen on single embryo development. Between 92 and 101 embryos were cultured per treatment, individually, from four independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). Different letters indicate significant differences between treatments within each time point (day 4: oxygen: P < 0.01; density: P > 0.05; interaction between oxygen and density: P < 0.05. Day 5: oxygen: P < 0.001; density: P > 0.05; interaction: P > 0.05). (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments in total cell number (a–b: P < 0.05; ab–c: P < 0.001), trophectoderm cells (d–e: P < 0.01; de–f: P < 0.001) or inner cell mass (g–h: P < 0.01). No significant differences in percent inner cell mass. ICM, inner cell mass; TE, trophectoderm. Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure  4 Effect of culture in microwell dishes on morphokinetics, relative to single culture in a 2 µl drop. Data from Table 2 are represented here as (mean cleavage time) – (mean control cleavage time) ± SEM. This is for graphical representation only, and statistics have are described in the Materials and methods section. Notation of cleavage events are described in the Materials and methods section. All cultures were performed in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric). Between 88 and 96% of embryos developed to the hatching blastocyst stage in all treatments. Between 23 and 48 embryos were cultured per treatment, from three independent biological replicates. *indicates significantly different to control 2 µl drop; *P < 0.05, **P < 0.01, ***P < 0.001. Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure  5 Effect of culture in microwell dishes on cell numbers. Bars represent cells per blastocyst (mean ± SEM); n = 71–116 embryos per treatment, from five independent biological replicates. Embryos were stained at 124 h after HCG. All cultures took place in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric). *indicates significantly different to control 2 µl drop; *P < 0.05; **P < 0.01. ICM, inner cell mass; TE, trophectoderm. Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Effect of embryo-conditioned media on individual embryos. Embryos were cultured individually in conditioned media (obtained from the culture of grouped embryos; n = 10 per group; total = 560) or individually or grouped in control media (media incubated without embryos); n = 56–57 per treatment, from three independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). *indicates significantly different to other treatments (P < 0.05). (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments (P < 0.01). Reproductive BioMedicine Online 2017 34, 441-454DOI: (10.1016/j.rbmo.2017.02.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions