Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation
Introduction
Cryopreservation, or storage of viable cells at low temperatures in liquid nitrogen, has improved Advanced Reproductive Technologies (ARTs) by simplifying the embryo transfer process, allowing genetics to be preserved over time, promote single embryo transfer, reduce cost, allow superior genetics to be shipped globally and promote the use of preimplantation genetic screening. Since the first birth of a frozen mouse embryo in 1971, cryopreservation has become a mainstream procedure with over 500,000 bovine frozen-thawed embryos transferred annually [1], [2], [3]. However, cryopreservation does not guarantee embryo viability, as the technique can induce cryodamage due to the formation of intracellular ice crystals. Youngs reports cryopreserved embryo transfer success rates to typically be at least 20% lower than seen with fresh embryos [4]. It is generally believed such decreases are the result of cryodamage to the embryos during storage. However, there is currently no method to detect embryo survival of cryopreservation beyond simple morphological assessment.
Previous research from our laboratory has suggested a noninvasive embryo assessment technique (NEAT) can be used to determine embryo viability in mice zygotes based on embryo buoyancy [5], [6], [7]. NEAT was designed from experimentation with a specific gravity system assembled in our laboratory (Fig. 1) [5]. The objective of the current study was to determine if NEAT can be used to detect embryo survival of cryopreservation in mice and ovine blastocysts, allowing a more objective determination of embryo viability after cryopreservation.
Section snippets
Materials and methods
Experimental protocols were approved by the Texas Tech University Animal Care and Use Committee.
Statistical analysis
All data were analyzed using the Statistical Package for the Social Sciences (SPSS ver. 12; Chicago, IL). The basic analysis was a two-way analysis of variance of treatment by time using a P-value of 0.05 for significance. In case of significance by the original analysis, the differences within time or treatment were reanalyzed with either Student's t-test or one-way analysis of variance with Tukey's means separation as appropriate.
Results
In the mouse model, 169 mice blastocysts were exposed to NEAT before freezing. There was no difference in pre-freeze drop times between viable and non-viable blastocyst, as determined by embryo hatching from zona pellucida (Fig. 2; P = 0.10). However, when blastocysts were re-exposed to NEAT after being frozen for a minimum of two weeks and thawed, those embryos which descended more slowly hatched from zona pellucida at a higher rate than the blastocysts with more rapid descent times (Fig. 2; P
Discussion
It is well documented cryopreservation decreases embryo viability due to two main causes: 1) intracellular ice crystal formation causing freeze-facture and 2) toxicity and osmotic shock from cryoprotectants necessary to cryopreserve embryos [8], [9]. Only procedures which do not allow the survival of the embryo, such as X-ray diffraction, calorimetry, freeze-fracture ultramicroscopy, and staining are current methods to detect for cryodamage because ice crystals are smaller than the wavelength
Conclusion
NEAT is an inexpensive, non-invasive and quantitative method to detect embryo viability in frozen and thawed blastocysts. NEAT can be incorporated into clinical settings to allow only the transfer of viable blastocysts into recipients. Further studies are currently performing a larger scale pregnancy trial in cattle.
Author's roles
All four authors were significant contributors to the work and manuscript preparation on this project. SP developed the original concept and initial design of NEAT. CW and LP refined the design prior to embryo testing. CW performed testing with mouse embryos. CW and KA performed testing with ovine embryos. While CW developed the original manuscript, all four were involved in editing for final content.
Conflict of interest
None Declared.
Acknowledgments
This work was supported by the South Plains Foundation and the Laura W. Bush Institute for Women's Health. We would also like to thank Aaron and Jessica Jennings for their generous support and interest in this project. Thank you to Dr. Michael Orth, Peter Cook, Ilan Arvelo, Gabriela Arteaga, and Alejandra Ramirez-Hernandez for their help with this paper.
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