Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status.

Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic-uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription-quantitative polymerase chain reaction-based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.

Extracellular vesicles, microRNA and the preimplantation embryo: non-invasive clues of embryo well-being.

Elective single embryo transfer is rapidly becoming the standard of care in assisted reproductive technology for patients under the age of 35 years with a good prognosis. Clinical pregnancy rates have become increasingly dependent on the selection of a single viable embryo for transfer, and diagnostic techniques facilitating this selection continue to develop. Current progress in elucidating the extracellular vesicle and microRNA components of the embryonic secretome is reviewed, and the potential for these findings to improve clinical embryo selection discussed. Key results have shown that extracellular vesicles and microRNAs are rapidly detectable constituents of the embryonic secretome. Evidence suggests that the vesicular population is largely exosomal in nature, secreted at all stages of preimplantation development and capable of traversing the zona pellucida. Both extracellular vesicle and microRNA concentrations within the secretome are elevated for blastocysts with diminished developmental competence, as indicated either by degeneracy or implantation failure, whereas studies have yet to firmly correlate individual microRNA sequences with pregnancy outcome. These emerging correlations support the viability of extracellular vesicles and microRNAs as the basis for a new diagnostic test to supplement or replace morphokinetic assessment.

Microenvironmental regulation of telomerase isoforms in human embryonic stem cells.

Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δß hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.