In vitro-produced equine blastocysts exhibit dispersed ICM cell allocation

Although in vitro production (IVP) of horse embryos has increased markedly in clinical practice, pregnancy rates after transfer are lower, and the incidence of pregnancy loss and monozygotic twins, higher than for in vivo-derived (IVD) embryos. Cell lineage specification is critical to normal early embryo development, and starts with differentiation of trophectoderm (TE) from inner cell mass (ICM) cells, followed by division of the ICM into primitive endoderm (PE) and epiblast (EPI). We examined the influence of developmental stage and speed, IVP and culture (in vivo versus in vitro) on expression of markers for the 3 early cell lineages, namely CDX-2 (TE), GATA-6 (PE) and SOX-2 (EPI). Day 7 IVD blastocysts (n=3 early; n=3 expanded), and IVP embryos identified as blastocysts on day 7 (n=5) or day 9 (n=9) were examined using confocal microscopy to compare total cell number, proportions and distribution of cells expressing CDX-2, GATA-6 and SOX-2. Additional day 7 IVP blastocysts were cultured for 2 days, either in vitro (n=5) or in vivo (after transfer into recipient mares; n=3), to examine the impact on subsequent development. Numerical data were analyzed using T- or Mann-Whitney U-tests. In IVD embryos, CDX-2 expression was exclusive to the TE, whereas SOX-2 expression was detected in both ICM and TE cells in early blastocysts but only thepresumed EPI in expanded blastocysts. GATA-6 was co-expressed with SOX-2 in outer ICM cells in early blastocysts but specific to PE cells lining the EPI and TE in expanded blastocysts. In IVP blastocysts, CDX-2 was also specific to the TE. However, SOX-2 and GATA-6 positive cells were intermingled in the ICM, and both SOX-2 and GATA-6 were co-expressed in TE cells. Strikingly, the EPI cells of IVP blastocysts were more dispersed, with larger mean inter-EPI cell distances (day 7 blastocysts, 52±6µm; day 9, 68±9µm), than in IVD embryos (35±3µm). Total cell number was higher in day 7 IVD (486±81) than IVP (day 7, 317±21; day 9, 377±104) blastocysts, but proportions of the different lineages didn't differ. Cell distribution in day 7 IVP embryos didn't change during an extra 2-day in vitro culture whereas, after 2 days in a mare's uterus, the SOX-2 (EPI) cells had aggregated and compacted, and segregated from PE cells. Overall, with respect to cell lineage allocation, IVP embryos resemble early IVD blastocysts in co-expressing SOX-2 and GATA-6 in TE cells, but differ by having a more dispersed ICM/EPI. While the EPI cells do compact following transfer to a recipient mare, the initial dispersal might contribute to both lower developmental competence (inadequate EPI aggregation) and a higher incidence of monozygotic twins (separated EPIs).