Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries.

Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified-warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.

Different Origin, Different Response: Gene Expression Pattern in Collapsed Vitrified Blastocyst.

There is a large body of animal experimental data about assisted reproductive techniques that could be applied to improve clinical outcomes. The great part of this information was obtained from research on in vivo-derived embryos. But whether these results are always similar with those we expect from embryos having in vitro origin in the clinical cases is a critical question. The present study was designed to compare the effects of vitrification (VIT) and artificial collapse (AC) as two commonly used techniques on in vivo- and in vitro-derived mouse embryos. In this regard, both origins of blastocysts were produced and randomly divided into three experimental groups, including control (non-vitrified), VIT, and AC-VIT. The survival and hatching rates and the expression of development-related genes were assessed in all groups and compared with their control counterpart. According to our results, although in vivo and in vitro origins followed the same pattern in the hatching rate, the real-time PCR data showed two distinct patterns of gene expression. Compared to the control, vitrification increased the expression of pluripotency genes in in vivo group. While in vitro vitrified blastocysts showed a significant reduction in the transcripts of these genes. More interestingly, although AC resulted in a sharp decrease of Gata6 and Grb2 in post warmed in vivo blastocysts, it could not affect the vitrified IVP ones. In conclusion, it seems that vitrification and artificial collapse techniques have different effects on embryo fate depending on in vivo or in vitro origins of the embryos.