Amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microRNA expression versus in vivo controls

Abstract

Embryo development and implantation are dynamic processes, responsive to external signals, and can potentially be influenced by many environmental factors. The aims of this study were to evaluate the effects of a culture medium supplemented with amniotic-derived microvesicles (MVs) on in vitro embryo hatching after cryopreservation, and pregnancy rate following embryo transfer. In addition, miRNA profiling of blastocysts produced in vitro, with or without (control; CTR) amniotic MV supplementation, was also evaluated using blastocysts produced in vivo. In vitro embryos were cultured with and without amniotic MV supplementation. In vivo blastocysts were obtained from superovulated cows. Samples for RNA isolation were obtained from three pools of 10 embryos each (in vivo, in vitro-ctR and in vitro + MVs). Our results show that the hatching percentage of cryopreserved in vitro + MVs embryos is higher (P < 0.05) than in vitro-CTR embryos and the pregnancy rate with fresh and cryopreserved in vitro + MVs embryos is higher than in vitro-CTR embryos. In addition, the analysis of differently expressed (DE) microRNAs showed that embryos produced in vivo are clearly different from those produced in vitro. Moreover, in vitro-ctR and in vitro + MVs embryos differ significantly for expression of two miRNAs that were found in higher concentrations in in vitro-CTR embryos. Interestingly, these two miRNAs were also reported in degenerated bovine embryos compared to good quality blastocysts. In conclusion, MV addition during in vitro production of embryos seems to counteract the adverse effect of in vitro culture and partially modulate the expression of specific miRNAs involved in successful embryo implantation.In the mammalian reproductive tract, the oviduct secretes a variety of growth factors and cytokines, which may play an essential role in the development of initial stages of pre-implantation embryos 1 . The absence of these maternal-embryo signals could be an important cause of the poor quality of in vitro produced embryos (IVP), compared to those collected in vivo 2,3 . To mimic the in vivo crosstalk between oviduct and embryo and to improve the quality of in vitro produced embryos, co-culture systems with somatic cells have been widely used to increase blastocyst percentage and quality of the resulting embryos, and the induction of specific transcriptomic changes 4 .Unfortunately, concern regarding viral transmission has resulted in the elimination of co-culture and the development of improved culture methods characterized by definite medium as synthetic oviductal fluid with amino acids (SOFaa 5 ). However, the maternal-embryo communication in vivo and the communication between monolayer ('helper') cells and embryos in vitro, take place not only through soluble factors (growth factors, receptors and binding proteins) secreted by cells and embryos in the medium 6,7 but also from insoluble factors. Recent studies have demonstrated that some molecules, including mRNA fragments and microRNAs (miRNAs), cannot

Expression of IL-1β and implantation serine proteases is required for mouse blastocyst hatching

“…We observed that the supplementation of IL-1β to cultured embryos increased blastocyst hatching percentage, accompanied by accelerated hatching by 24 h. This hatching accelerating effect observed with the IL-1β-supplemented embryos is very significant, since in vitro cultured mammalian embryos normally show a substantial delay in blastocyst development, compared to their in vivo counterparts (Harlow & Quinn 1982, Roth et al 1994, Stokes et al 2005, Lange-Consiglio et al 2020. Of relevance, here, is the recent report which showed an increased IL-1β in cultured human embryos and it is considered as an early predictor of a successful pregnancy (Salama et al 2020).…”

Seasonal effects on miRNA and transcriptomic profile of oocytes and follicular cells in buffalo (Bubalus bubalis)

“…Samples for RNA isolation were obtained from pools (n = 20) of OOs and FCs for both conditions (BS and NBS). The methods described below have been reproduced in part from Lange-Consiglio et al 69 . Total RNA was isolated by NucleoSpin miRNA kit (Macherey-Nagel, Germany), following the protocol in combination with TRIzol (Invitrogen, Carlsbad, CA, USA) lysis with small and large RNA in one fraction (total RNA).…”

“…sample: 1.8 Vol. beads) twice 69 . The methods described below have been reproduced in part from Frattini et al 2017 70 .…”

The Role of Extracellular Vesicles in Embryo Implantation

“…More importantly, EVs-treated embryos show a significantly higher implantation rates in in vivo embryo transfer, which proves that EVs derived from outgrowth embryoconditioned media indeed increase the implantation ability of mice embryos (24). Similarly, amniotic microvesicles show positive effect on bovine embryos hatching and subsequent pregnancy (30).…”