Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media

Abstract

Objective: Validate single versus sequential culture media for murine embryo development.

Design: Prospective laboratory experiment.

Setting: Assisted Reproduction Laboratory.

Animals: Murine embryos.

Interventions: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization.

Main outcome measures: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality.

Results: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts.

Conclusions: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed.

Keywords: UCHL1; UCHL3; culture; embryo; media.

Figure 1.

Experimental design. Thawed murine zygotes were pooled in collection media modified Human Tubal Fluid (mHTF) + 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The zygotes were randomly assigned to single (n = 60) or sequential (n = 60) media types. On day 3 (d3), single medium was refreshed, whereas sequential media were changed from P-1 to multiblast medium. Embryos were collected on d3 or day 5 (d5) and processed for analyses.

Figure 2.

Embryo development on day 3 (d3) and day 5 (d5). A, On d3, the percentage of embryos ≥ 8 cell (8C) or < 8C developing in single culture media tended to be greater but did not reach statistical significance (P < .06). B, On d5, the percentage of embryos developing to the blastocyst stage in single medium was significantly greater compared to the sequential media (P < .005; n = number of embryos per media).

Figure 3.

Number of trophoblast (TE) and inner cell mass (ICM) nuclei per blastocyst. More TE nuclei (P < .0001) but similar numbers of ICM nuclei (P < .230) were found in single versus sequential media. There was a significantly higher ratio of TE to ICM nuclei in single medium (P < .0001; n = total number of blastocysts per media type.)

Figure 4.

Blastocyst hatching, nuclear quality, and ubiquitin C-terminal hydrolase L1 (UCHL1)/ubiquitin C-terminal hydrolase L3 (UCHL3) localization on developmental day 5 per media type. A, More blastocysts hatched or were hatching in single medium versus sequential media (P < .001; n = total number of blastocysts per media.). B, Hatching blastocyst from single medium (black oval indicating trophectoderm emerging from zona pellucida). C, Unhatched blastocyst with intact zona pellucida (black arrow) from sequential media. D, Pyknosis (shrinking/condensation) and karyorrhexis (disintegration/granulation; red arrows) are forms of nuclear damage in this blastocyst cultured in sequential media. E, High-quality hatching blastocyst cultured in single medium has a distinctive clustering of cells in the inner cell mass (ICM) (white circle). UCHL1, green fluorescence (green arrows), localized in the cytoplasm and at the cell boarders and UCHL3, red fluorescence (red arrows), localized in nuclei. F, Lower quality nonhatching blastocyst cultured in sequential media with small nuclei of differing sizes, some undergoing pyknosis and karyorrhexis (see D), ill-defined trophectoderm cell borders lacking distinct UCHL1 staining (white bracket area) and a poorly formed ICM with indistinct UCHL3 red fluorescence dispersed throughout the cytoplasm compared to the hatching blastocysts with distinct cytoplasmic UCHL1 and nuclear UCHL3 patterns (see E; original magnification of 600×). The combination of nuclear damage and diffuse UCHL1 and UCHL3 staining was deemed indicative of reduced blastocyst developmental capacity.