Elsevier

Theriogenology

Volume 103, November 2017, Pages 169-172
Theriogenology

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation

https://doi.org/10.1016/j.theriogenology.2017.07.010Get rights and content

Highlights

  • A noninvasive embryo assessment technique can detect cryodamage in mice blastocysts.

  • A noninvasive embryo assessment technique can detect cryodamage in ovine blastocysts.

  • Ovine blastocysts exposed to a noninvasive embryo assessment technique can establish pregnancies which survive to term.

Abstract

Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15–20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P < 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established pregnancies which survived to term (P < 0.05). These results suggest NEAT can identify which blastocysts survive cryopreservation, thus significantly reduce the transfer of non-viable embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT.

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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