Group culture of preimplantation embryos creates a microenvironment that determines development, viability, and response to external stressors

Over 400,000 babies worldwide are born following in vitro fertilisation (IVF) treatment every year and millions of babies have been born from IVF over the last 40 years (Adamson et al. 2018, Niederberger et al. 2018). Despite this, approximately half of couples undergoing IVF treatment do not achieve a successful pregnancy, even after multiple treatment cycles (Chambers et al. 2017, Fitzgerald et al. 2017). The period of in vitro culture is a crucial stage in determining not only the success of IVF treatment but also the long-term health of these babies (Feuer and Rinaudo 2016). Due to new embryo selection and tracking technologies, clinical embryologists increasingly culture embryos individually rather than in groups. However, individual culture can cause stress to the embryo (Reed 2012)because embryos are deprived of paracrine signals, which promote the development of embryos cultured in groups (Wydooghe et al. 2017). The identities and actions of most of these embryo-secreted molecules remain poorly understood. Using a mouse model, this study compares the development of embryos cultured individually or in groups, and how other culture conditions influence the development of individually cultured embryos. Firstly, it was found that any period of individual culture was detrimental to development rates, cell numbers and percentage of inner cell mass (ICM), and the precompaction stage was no more sensitive to this stress than the post-compaction embryo. Reducing the volume of culture media was beneficial to individually cultured embryos, but only under 5% oxygen, not atmospheric oxygen, indicating that there is an interaction between optimal embryo density and oxygen concentration. Reducing the media volume further by culturing embryos in microwells increased the % ICM and influenced the speed of cleavage and hatching. Addition of embryo-conditioned media to individually cultured embryos also increased hatching rate and cell numbers. The interaction between individual culture and atmospheric oxygen was investigated in more detail. Compared to group culture under 5% oxygen, either individual culture (at 5% oxygen) or atmospheric oxygen (group culture) caused slower cleavages, lower development rates, and fewer cells, plus changes to glucose and amino acid metabolism. Embryos cultured individually under 20% oxygen were affected further, so that combining the two detrimental conditions resulted in earlier and longer delays to development, fewer and smaller blastocysts, and more exaggerated changes to metabolism compared to those cultured under 5% oxygen in groups. There was very little effect of individual culture under 5% oxygen on fetal and placental development compared to group cultured embryos. However, individual culture under 20% oxygen resulted in smaller and skinnier fetuses, and lighter placentas compared to embryos cultured in groups. This may be because the further the culture conditions are from optimal, the more resources an embryo uses to adapt and survive, or there is an accumulation of trauma, and the manifestations of stress become more exaggerated. Embryos in culture secrete high levels of IL-6, and supplementing culture media with IL-6 increased hatching rate and cell numbers, showing IL-6 to be an embryotrophic cytokine. However, it is just one of hundreds of embryo-secreted molecules that may be involved in paracrine signalling during group culture. This study demonstrates that individual culture is detrimental to mouse embryos, especially in combination with atmospheric oxygen. This raises concerns regarding the increasing prevalence of single embryo culture in human IVF. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimising each element of the culture system.