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In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media

Reproductive BioMedicine Online, Volume 34, Issue 5, May 2017, Pages 441 - 454


  • Individual culture of preimplantation mouse embryos is detrimental during both the precompaction and postcompaction stages, demonstrated by reduced hatching rate and blastocyst cell numbers.
  • The effect of embryo density on singly cultured embryos is dependent on the incubator oxygen concentration.
  • Culture in microwell dishes increased the percentage of inner cell mass per blastocyst, and altered timing of developmental events.
  • Addition of embryo-conditioned media to single embryos improves hatching rates and cell numbers.


Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture.

Keywords: Group culture, IVF, Osmolality, Single culture, Stress, Time-lapse.


School of BioSciences, The University of Melbourne, Parkville, Victoria 3010, Australia

* Corresponding author.